Chemoprevention of Cancer by Targeting Pro inflammatory

Chemoprevention of Cancer by Targeting

Pro inflammatory Cytokines

By

Touseef Rehan

Reg. No. 03031313002

PhD Biochemistry/Molecular Biology

Faculty of Biological Sciences

Quaid-i-Azam University Islamabad

(2020)

Chemoprevention of Cancer by Targeting

Pro inflammatory Cytokines

A thesis submitted in partial fulfilment of the requirement for the degree of PhD in

Biochemistry/Molecular Biology

By

Touseef Rehan

PhD Biochemistry/Molecular Biology

Faculty of Biological Sciences

Quaid-i-Azam University Islamabad

(2020)

Certificate

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page i

Certificate

This thesis submitted by Mrs Touseef Rehan to the Department of Biochemistry, Faculty of

Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan, is accepted in its present

form as satisfying the thesis requirement for the degree of Doctor of Philosophy in

Biochemistry/Molecular Biology.

Supervisor: Dr. Aneesa Sultan

External Examiner:

Chairperson: Prof. Dr. Rashid Khan

Dated

Dedication

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page ii

Dedication

Dedicated to

My Father (Late)

For being my first teacher and who always taught me to believe in myself. His affection,

love, encouragement and support make me able to achieve so much success and honour.

My Mother

A strong and gentle soul who taught me to trust Allah, believe in hard work and that so

much could be done with little.

My Sweet and Innocent Kids, Zohan and Uhud

Whose charming acts give me a beam of joy and filled my life with love that encouraged

me to write this thesis.

Acknowledgments

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page iii

Acknowledgments

All the praises and glories to all mighty Allah for the strength, courage and His blessings in

completing this thesis, on whom ultimately we depend for sustenance and guidance.

This PhD thesis is the culmination of life-long interest in cancer biology and has turned into

as much a labour of love as a scientific study. Numerous people over the years have helped

me get here, so there are many people I need to thank.

Firstly, I appreciate the special and valuable efforts of my supervisor Dr. Aneesa Sultan

Associate Professort of Biochemistry, Quaid-i-Azam University, Islamabad. She supported

and encouraged me at all stages of completion of this thesis. Her constructive comments and

creative mind, made it possible for me to design this project and her suggestions at every step

made it possible to pursue this study.

I pay special debt of gratitude to Dr. Riffat Tahira Senior Scientific Officer at National

Agriculture Research Centre, Islamabad. She always assisted me and guided me. She has

given me valuable suggestions at all times from the very beginning up to the end of this

project.

I feel deep privilege to thank my foreign mentor Professor Dr. David MacEwan at the

Department of Translational Medicines, University of Liverpool for guiding and partially

financing my project during 06 months HEC-Pakistan IRSIP program.

I would like to acknowledge the special efforts of my husband Dr. Nasrullah Shah, Associate

Professor at AWKUM. He always encouraged me and supported me whole heartedly for

completing this study. He always paved the path before me and walked alongside me. He

guided me, placed opportunities in front of me and showed me the doors that might be useful

to open.

After whole hearted thanks to highly cooperative faculty and staff members of Biochemistry

department, I am really indebted to QAU, HEC-Pakistan, NARC and University of Liverpool

for supporting me academically and financially.

Last but not the least without any hesitation I am very thankful to my brothers and sisters for

their continuous support, motivation and encouragement to achieve this goal of completing

my study.

Touseef Rehan

Table of Contents

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Table of Contents

1. Introduction ....................................................................................................................... 1

1.1 Cancer ............................................................................................................................. 1

1.2 Leukemia ........................................................................................................................ 1

1.3.1 Cytogenetic changes associated with leukemia induced by benzene .............................. 8

1.5 Cytokines and their role in cancer .................................................................................... 9

1.5.1 Tumor necrosis factor-alpha (TNF-α) ........................................................................... 9

1.5.2 TNF-α induced apoptosis via TNFR-1 ..................................................................... 10

1.4 Molecular pathways involved in Leukemia .................................................................... 11

1.4.1 JAK-STAT pathway ................................................................................................... 12

1.4.2 Ras signalling ............................................................................................................. 13

1.4.3 Raf/MEK/ERK cascade .............................................................................................. 14

1.6 Chemoprevention: cancer prevention strategy................................................................ 17

1.6.1Classification of chemopreventive agents .................................................................... 18

1.6.1.1 Blocking agents ....................................................................................................... 19

1.6.2 Chemopreventive agents and associated risk factors ................................................... 20

1.6.2.1 Natural therapies in chemoprevention of Cancer ...................................................... 20

1.7 Medicinal plants of the family Lamiaceae as functional foods ....................................... 21

1.7.1 O. basilicum ............................................................................................................... 21

1.7.2 R. officinalis ............................................................................................................... 23

1.7.3 T. vulgaris .................................................................................................................. 25

1.8 Immunomodulatory effects of medicinal plants ............................................................. 27

1.10. Aims and objectives ................................................................................................... 28

Materials and Methods ........................................................................................................ 30

2.1 Plant material collection ................................................................................................ 30

2.2 Phytochemical Screening............................................................................................... 30

2.3.1Total phenolic content measurement ............................................................................ 30

2.3.2 Total flavonoid assay .................................................................................................. 30

2.4 Determination of mics ................................................................................................... 31

2.5 Determination of the in vitro effects of combinations of antimicrobial agents ................ 31

2.6 Experimental animals .................................................................................................... 32

2.6.1 Subacute toxicity assay ............................................................................................... 32

2.6.2 Histopathology analysis .............................................................................................. 33

Table of Contents

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2.6.3 Hematological assays ................................................................................................. 33

2.7 Statistical analysis ......................................................................................................... 33

2.8 Isolation and purity confirmation of isolated compounds ............................................... 33

2.8.1Column chromatography ............................................................................................. 33

2.8.2 Thin layer chromatography (TLC) .............................................................................. 35

2.9 In Vitro Assays ............................................................................................................ 35

2.9.1 Cell culture preparation and treatment with stimuli ..................................................... 35

2.9.2 Cytotoxicity assay ...................................................................................................... 35

2.9.3 Flow Cytometry ......................................................................................................... 35

2.9.4 Cell death in the presence of caspase inhibitor z-VAD-fmk ........................................ 36

2.9.5 Westernimmunoblotting ............................................................................................. 36

2.9.6 RNA extraction and real-time PCR ............................................................................. 36

2.10.1 Experimental animals ............................................................................................... 37

2.10.2 Experimental design ................................................................................................. 37

2.11 Blood smear preparation and Giemsastaining:. ............................................................. 40

2.11 Characterization of plants fractions .............................................................................. 40

2.11.1 Fourier transform infrared spectroscopy (FTIR) ........................................................ 40

2.11.2 Sample preparation for UV spectroscopy .................................................................. 40

2.11.3 Liquid chromatography-mass spectrometry (LC–MS) ............................................... 40

Results ................................................................................................................................ 42

3.1Total phenolics and flavonoids ....................................................................................... 42

3.2 Synergistic effects of three medicinal herbs with antibiotics .......................................... 43

3.3 Effects of selected medicinal plants extracts on rats organs ............................................ 44

3.4 Effects of administration of selected plants extracts on haematological parameters ........ 46

3.5 Results of in vitro assays ............................................................................................... 49

3.5.1 O. basilicum, R. officinalis and T. vulgarisfractions and crude extracts inhibited

leukemic cells proliferation .......................................................................................... 49

3.5.2 O. basilicum, R. officinalis and T. vulgaris fractions induced apoptosis of Leukemic cell

lines ............................................................................................................................. 49

3.5.3 O. basilicum, R. officinalis and T. vulgarisfractions induce apoptosis via activation of

JNK and cleavage of caspase 3 ..................................................................................... 50

3.5.4. Alterations in the level of TNF-α, CXCL2 and CXCL8 induced byO. basilicum, R.

officinalis and T. vulgaris fractions inu937 cell line ..................................................... 50

3.6 Results of in vivo Assays ............................................................................................... 72

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3.6.1 Effects of plant (O. basilicum, R. officinalis and T. vulgaris) crude extract and fractions

on organs of leukemic rats ............................................................................................ 72

3.6.2 Histopathological analysis: .......................................................................................... 72

3.6.3 Differential leukocytes count: ...................................................................................... 75

3.7 Characterization of Plant Fractions ................................................................................ 80

3.7.1 FTIR spectroscopy ..................................................................................................... 80

3.7.2 UV Spectroscopy and LC-MS Study .......................................................................... 80

References ........................................................................................................................ 101

List of Figures

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List of Figures

Figure 1.1. Contribution of environment and genes in cancer development. ................ 24

Figure 1.2. Chemical structures of various toxic chemicals responsible for liver cancer..

................................................................................................................................. 25

Figure 1.3. Schematic diagram showing metabolism of benzene from liver to bone

marrow.. .................................................................................................................... 27

Figure 1.4. TNF-α induced apoptosis via TNFR-1. ..................................................... 30

Figure 1.5. Mediation of signalling pathways by cytokines through jaks. ..................... 33

Figure 1.6. Ras proteins are activated and transmit signals when transmembrane

receptors are bound by their ligands including cytokines. ............................................ 33

Figure 1.7. Raf/MEK/ERK cascade is involved in regulation of certain apoptotic

regulatory genes. Targeting this pathway can provide an important therapeutic strateg for

the prevention of cancer.. ........................................................................................... 34

Figure 1.8 Various environmental factors and proinflamory cytokines are involved in

activation of p38 MAPK and JNK/SAPK pathway. .................................................... 35

Figure 1.9. (A) Apoptosis can be induced through ctivation of caspases, sustained

activation of JNK induce apoptosis through inactivation of the suppressors of

mitochondrial death patway.. ...................................................................................... 36

Figure 1.10. Phases of carcinogenesis include initiation, promotion, progression, and

metastasis.

Figure 1.11. Classification of Chemopreventive agents based on the stage at which they

restrain carcinogenesis. .............................................................................................. 38

Figure 1.12. O. Bacilicum. ......................................................................................... 42

Figure 1.13. R. officinalis. .......................................................................................... 43

Figure 1.14. T. vulgaris (Prasanth Reddy, Ravi Vital, Varsha, & Satyam, 2014)............ 44

Figure 2.1. Flow sheet diagram of rats grouping. ........................................................ 56

Table.2.2. Dose regimen of scheme for experimental groups of study. ......................... 57

Figure 3.1. Total Phenolics and flavonoids of O. basilicum, R. officinalis and T. vulgaris.

................................................................................................................................. 60

Figure 3.2. Histological sections of Liver, kidney, heart and spleen tissue stained with H

and E×400 (A1, A2, A3, A4) . .................................................................................... 65

Figure 3.3. Cytotoxic effects of O. basilicum crude extract and fraction against leukemic

cell lines. ................................................................................................................... 70

List of Figures

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Figure 3.4. BF2 and BF3 induces apoptosis in human leukemic cells.. ......................... 73

Figure 3.5. Effects of O. basilicum fractions on caspase 3. .......................................... 74

Figure 3.6. Effects of O. basilicum fractions on activation of JNK in leukemic cells. ... 75

Figure 3.7. Observation of apoptosis in leukemic cells via treatment with O. basilicum

fractions alone and in combination with zvad-fmk. ..................................................... 76

Figure 3.8. Cytotoxic effects of R. officinalis crude extract and fraction against leukemic

cell lines. ................................................................................................................... 77

Figure 3.9. RF1 and RF2 induces apoptosis in human leukemic cells.. ......................... 80

Figure 3.10. Effects of R. officinalis fractions on caspase 3.. ....................................... 81

Figure 3.11. Effects of R. officinalis fractions on activation of JNK in leukemic cells. . 82

Figure 3.12. Observation of apoptosis in leukemic cells via treatment with R. officinalis


Figure 3.13. Cytotoxic effects of T. vulgaris crude extract and fractions against leukemic

cell lines.. .................................................................................................................. 84

Figure 3.14. TF2, TF7 and TF8 induces apoptosis in human leukemic cells.). .............. 87

Figure 3.15. Effects of T. vulgaris fractions on activation of JNK in leukemic cells. .... 89

Figure 3.16. Observation of apoptosis in leukemic cells via treatment with T. vulgaris


Figure 3.17. Effect of administration of methanolic extracts and fracions of O. basilicum,

on organ body weight ratio of liver, heart, kidneys and spleen of Sprague Dawley rats . 92

Figure 3.18. Effect of administration of methanolic extracts and fracions of R. officinalis,

on organ body weight ratio of liver, heart, kidneys and spleen of Sprague Dawley rats. 92

Figure 3.19. Effect of administration of methanolic extracts and fracions of T. vulgaris,

on organ body weight ratio of liver, heart, kidneys and spleen of Sprague Dawley rats. 93

Figure 3.20. Histological examination of spleen.......................................................... 94

Figure 3.21. Blood smear examination........................................................................ 96

Figure 3.22. Effect of administration of methanolic extracts and fracions of O. basilicum,

on haematological parameters of Sprague Dawley rats. ............................................... 96

Figure 3.23. Effect of administration of methanolic extracts and fracions of R. officinalis,

on haematological parameters of Sprague Dawley rats ................................................ 96

Figure 3.24. Effect of administration of methanolic extracts and fracions of T. vulgaris,

on haematological parameters of Sprague Dawley rats. ............................................... 97

Figure 3.25. LC-DAD chromatograms of different fractions of methanolic extracts. ...106

Figure 3.26. FTIR spectrum of BF3 fraction of methanolic extract of O. basilicum. ....106

List of Figures

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Figure 3.27. FTIR spectrum of BF2 fraction of methanolic extract of O. basilicum. ....107

List of Tables

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List of Tables

Table 2.1. Different fractions of O. basilicum, R. officinalis and T. vulgaris obtained after

column chromatography using silica as a stationary phase and mixture of n-Hexane and

ethyl acetate with different ratio as mobile phase. ....................................................... 34

Table 2.2. Dose regimen of scheme for experimental groups of study. ......................... 38

Table 3.1. Results of the combination of O. basilicum plant extract and antibiotics

against three bacterial strains. .............................................................................. 43

Table 3.2 Results of the combination of T. vulgaris plant extract and antibiotics against three

bacterial strains.................................................................................................................44

Table 3.3. Results of the combination of R. officinalis plant extract and antibiotics agains

three bacterial strains................................................................................................................44

Table3.4. Effect of administration of methanolic extracts of O. basilicum, T. vulgaris and R.

officinalis on body weight and some organs weight of Sprague Dawley rats..........................45

Table 3.5. Effect of administration of methanolic extracts of O. basilicum, T. vulgaris and R.

officinalis on some hematological parameters of spraguedawley rats..............................47

Table 3.6. Total yield of fraction of O. basilicum, R. officinalis and T. vulgaris....................48

Table 3.7. Effects of O. basilicum fractions on the level of TNF-α, CXCL2 and CXCL8 in

U937 cell line....................................................................................................................57

Table 3.8. Effects ofR. officinalis fractions on the level of TNF-α, CXCL2 and CXCL8 in

U937 cell line...................................................................................................................64

Table 3.9. Effects of R. officinalis fractions on the level of TNF-α, CXCL2 and CXCL8 in

U937 cell line...................................................................................................................71

Table 3.10. Effect of administration of methanolic extracts and fractions of O. basilicum, T.

vulgaris and R. officinalis on TNF α level of Sprague Dawley rats.......................................79

Table 3.11. LC-MS results of the selected fractions BF2 and BF3 of O. basilicum.............82

Table 3.12. Interpretation of characteristic peaks appeared in FTIR spectrum of B3 fraction of

methanolic extract of O. basilicum.........................................................................................88

Table3.13. Interpretation of characteristic peaks appeared in FTIR spectrum of BF2 fraction

of methanolic extract of O. basilicum....................................................................................89

List of Abbreviations

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List of Abbreviation

DMSO: Dimethylsufoxide

FAS-L: Fas ligand;

FBS: fetal bovine serum;

IC50: Inhibitory concentration at which 50% cells die;

JNK: Jun N-terminal kinase;

NSCLC: Non-small cell lung cancer;

PI: Propedium Iodide;

SDS-PAGE: Sodium dodicylsulphate;

Str: stretching;

TNF-α: Tumour necrosis factor alpha;

Zvad-fmk: Z-Val-Ala-Asp-fluoromethylketone.

ALL: Acute lymphoblastic leukemia

AML: Acute myeloid leukemia

CML: chronic myeloid leukemia

CLL: chronic lymphoblastic leukemia

ROS: Reactive oxygen species

PPM: Parts per million

GST: glutathione-S-transferase

Meh: microsomal epoxide hydrolase

MPO: myeloperoxidase

PST: phenol sulfotransferase

UDPGT: uridine diphosphate glucuronosyltransferase

BQ: 1,4-benzoquinone

HQ: hydroquinone

NQO1 NADPH: quinoneoxidoreductase 1

IFN: Interferon

TNFR: TNF Receptor

TRADD: TNFR1-associated death domain protein

RIP1: receptor-interacting protein 1 ()

FADD: Fas-associated death domain protein

TRAF2: TNF-receptor-associated factor 2

MEK1/2: mitogen-activated protein kinase/ERK kinase

ERK1/2: extracellular signal regulated kinase


Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page xii

Janus family kinases jaks

Signal transducer and activator of transcription stats

Phosphatidylinositol 3-kinases PI3K

PKC: protein kinase C

Tyk: Tyrosine Kinase

G-CSF: Ganulocytes colony simulating factor

Gtpas: guanidine triphospphatase

IL: Inerleukene

Nsaids: non-steroidal anti-inflammatory drugs

FDA: Food and drug agency

Cams: complementary and alternative medicines ().

EF: Euphorbia formosana

GC Gas chromatography

LC-MS: Liquid chromatography mass specrophotometry

Teo: thymus vulgaris essential oil

CVL: carvacrol

Eos: Essential oils

NARC: National Agriculture Research Centre

GAE: Gallic acid equivalent

CE: Catechin equivalent

DW: Dry weight

MIC: Minimum inhibitory concentration

NB: nutrient broth

FIC: fractional inhibitory concentration

FICI: FIC index

EDTA: Ethylenediamine tetra acetic acid

H&E: Hamatoxylin and Eosin

PCV: Packed cell volume

HMCV: hemoglobin mean corpuscular volume

MCH: mean corpuscular hemoglobin

MCHC: mean corpuscular hemoglobin concentration

TLC: Thin layer chromatography

ELISA: Enzyme linked immunoorbant assay

PBS: phosphate buffer saline


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FTIR: Fourier Transform Infrared Spectrophotometer

PE: Plant extract

BW: Body weight

PI: proprdium iodide

DD: Death domain

Abstract

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ABSTRACT

Leukemia, one of the major cancers, affects a large proportion of people around the world.

Known causes of leukemia are ionizing radiations, certain cancer drugs and chemicals like

benzene. Due to high mortality and morbidity rate of leukemia, its chemoprevention is

desirable. Better treatment options for leukemia are required due to large number of side

effects associated with current therapeutic regimens. Epidemiologic studies have

demonstrated that consumption of medicinal herbs in diet may reduce the risk of various

cancers. Due to these observations cancer research focus shifts towards isolation and

characterization of chemopreventive agents present in medicinal herbs.

Medicinal plants of family lamiaceae Ocimum basilicum (O. basilicum), Rosemarinus

Officinalis (R. Officinalis) and Thymus Vulgaris (T. vulgaris) have been found to provide a

good array of cytotoxic compounds. Current study was carried out to characterize the

functional role of O. basilicum, R. officinalis and T. vulgaris in JNK and other apoptotic

pathways. Apoptosis was induced in human leukemia cells and leukemia induced rats by screening

out the pharmacologic and genetic approaches followed by these three plants.

To elucidate the safety of the selected plants subacute toxicity profiling was carried out in

Sprague dawley rats. Plant material of O. basilicum, R. officinalis and T. vulgaris was

collected, dried and subjected to methanolic extract preparation. Crude extract was

fractionated using column chromatography. N-hexane and ethyl acetate were used in different

ratios as eluting solvent while silica gel was used as stationary phase. In plants safety study

O. basilicum extract showed highest total phenols, flavonoids, antioxidant and antibacterial

activities. T. vulgaris extract caused hypertrophy of liver while R. officinalis caused atrophy

of spleen at both doses showing no significant histomorphological changes. T. vulgaris and

O. basilicum extract significantly increased red blood cells, packed cell volume, haemoglobin

and mean corpuscular volume at 1500 mg/kg body weight.

To investigate the in vitro anticancer activity of these plants, extracts and fractions were

applied on four types of leukemic cell lines U937, OCI-AML3, MV4-11and THP-1. The cell

viability was assessed via Cell titer GLO assay and apoptosis was measured through Annexin

V/ PI staining. Major Apoptotic markers like JNK and Caspase were analysed through

western blotting while pro-inflammatory cytokines like TNFα, CCL2 and CXCL8 were

analysed using QPCR. The most potent with lowest IC50 values among the fractions were

BF2 (2:8 n-hexane:ethyl acetate) and BF3 (3:7 n-hexane:ethyl acetate), RF1(1:9 n-

hexane:ethyl acetate) , RF2 (2:8 n-hexane:ethyl acetate), TF2 (2:8 n-hexane:ethyl acetate),

TF7 (3:7 n-hexane:ethyl acetate) and TF8 (8:2 n-hexane:ethyl acetate). Cytotoxicity was

Abstract

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page xv

associated with apoptosis. Apoptosis was found caspase dependent and P-JNK activation was

detected sustained. Significant increase in the level of TNF α and decrease in the level of

CXCL8 was observed in BF2, BF3, RF1, RF2, TF2, TF7 and TF8 treated cells.

To explore the in vivo anticancer activity of these plants, extracts and fractions of these plants

were applied on leukemia induced Sprague Dawley rats. Leukemia induced Sprague Dawley

rats were treated with each fraction and crude extract of each plant. Weight of liver, heart,

kidneys and spleen was carried out after dissection. A differential white blood cell counting

was also performed. No significant differences in weights of four organs and differential

count of white blood cells were recorded in rats treated with crude extracts, DMSO, and

extract fractions viz., BF2, BF3, RF1, RF2, TF2, TF7 and TF8, while in rats treated with

other fractions weight of liver and spleen increased significantly. Significant increase was

found in the level of TNF-α in serum samples of rats treated with fractions BF2, BF3, RF1,

RF2, TF2, TF7 and TF8 and highest TNF-α concentration (199.85±4.6µg/ml) was recorded

in serum samples of rats treated with basil extract fraction BF3.

Selected fractions were subjected to Fourier-transform infrared spectroscopy analysis for

functional group identification. The main functional groups were O-H (carboxylic/phenolic),

C-H (SP2, aromatic), C-H (SP3, alkane), -C=C- (alkene), -C=O (carbonyl) and C-X (halides)

which appeared in the range of 3300-3400, 3000-3100, 2720-2920, 1600-1630, 1710-1730

and 720-1300 cm-1, respectively. Predominantly Epicatechin and cinnamic acid derivatives

were found in these fractions as depicted from LC-MS data.

Overall the study concluded that O. basilicum, R. officinalis and T. vulgaris were found to be

rich sources of phenols and flavonoids and kill most cancer cells within very short time of

treatment. Mechanism followed by O. basilicum for execution of apoptosis in cancer cells

was found that TNF alpha is involved in activation of p-JNK that was found to be activated in

a time dependent manner which in turn leads to the activation of caspases and cause cell

death. Similarly enhanced expression of TNF alpha was also found in In vivo studies due to

treatment with these plants.

In this project an attempt has been made to characterize the plants and their extract that may

be utilized in cancer treatment in Pakistan and in other countries as well. These plants can be

used in diet and can be used for the treatment of cancer as recommended from this study.

The research work presented in this thesis contributed in publication of the following articles.

Publications

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page xvi

Publications

1- Touseef Rehan, Riffat T, Hanif U, Usman T, Tabassum R, Mariam A, Iram M, Aneesa

S*, In Vitro Bioactivities and Subacute Toxicity Study of O. basilicum, T. vulgaris and R.

officinalis, Turkish Journal of Biochemistry, 2018, 43 (4), 447-455.

2- Touseef Rehan, David MacEwan, Nasrullah Shah, Tabassum Rehan, Riffat Tahira,

Sheeba Murad, Mariam Anees, Iram Murtaza, Muhammad Farman, Aneesa Sultan*,

Apoptosis of Leukemia Cells by Ocimum basilicum Fractions Following TNF alpha Induced

Activation of JNK and Caspase 3, Current Pharmaceutical Design, 2019, 25(34), 3681-3691.

3- Touseef Rehan, Nasrullah Shah, Rabia Ghafoor, Hadiya Batool Tabassum Rehan, Riffat

Tahira, Sheeba Murad, Mariam Anees, Iram Murtaza, Muhammad Farman, Aneesa Sultan*,

Tumor necrosis factor α (tnf-α) up-regulation through selected lamiaceae plant extracts in

sprague dawley leukemia induced rats, Pakistan Journal of Agricultural Sciences., 2019,

(under review).

4- Touseef Rehan, David MacEwan, Nasrullah Shah, Tabassum Rehan, Riffat Tahira,

Sheeba Murad, Mariam Anees, Iram Murtaza, Muhammad Farman, Aneesa

Sultan*,Antileukemic effects of R. officinalis and T. vulgaris in U937, OVI-AML3, MV4-

Iiand THP1 , International Journal of Agricultural Biology 2019, (Submitted).

Introduction

Chemoprevention of Cancer by Targeting Pro inflammatory Cytokines Page 1

1. Introduction

1.1 Cancer

Cancer is a dreadful genetic disease representing an abnormal pattern of cell growth caused

by a variety of dramatic changes in the expression regulation of multiple genes leading to an

uncontrolled cell proliferation and eventually results in such a group of cells that can further

invade the surrounding tissues and metastasize to other locations, causing significant

morbidity and, ultimately death of the host (Ruddon, 2007). Globally cancer is considered the

second most frequent cause of mortality after cardiovascular disease and any experimentation

in fighting the disease is significantly important to public health (Rao et al., 2004). In 1996

there were about 10 million new prevalent cancer cases internationally and approximately six

million deaths were attributed to cancer. While expected rate in 2020 predicted to be 20

million new cancer cases and chances of 12 million deaths (Bray and Møller, 2006). Cancer

is basically caused by external factors; including infectious organisms present in

environmental resources like food, water, air, radiations, smoke, and chemicals and

significantly in the sunlight that people are daily exposed to (Parsa, 2012). Internal factors

responsible mainly include genetic mutations e.g. Inherited mutations and mutations caused

by endogenous cellular reactions (Pecorino, 2012).

There are different kinds of cancer and each one is named with respect to tissue or the organ

in which it arises. Tumours may be ‘malignant’ or ‘benign’. Malignant tumours are rapidly

growing, not only effect neighbouring tissues, but are also able to metastasize other locations,

more often essential organs and interfere with the, nervous, digestive and circulatory systems

affecting their activities and also release the hormones that can change the body functions, a

process known as metastasis.

Benign tumours are generally slow-growing masses that remain fixed at one place and exhibit

limited growth. Cancer has been subdivided into different categories: carcinoma refer to the

malignant tumours of epithelial and sarcoma refer to connective tissues respectively,

leukemia and lymphoma are malignant tumours of the bone marrow and the lymphoid tissue

respectively, and myeloma refers to cancer of skin cells that produce melanin (Malcolm,

2002).

1.2 Leukemia

The word leukemia (loo-ke´me-ah) originates from the Greek word leukos meaning "white"

and aima which means "blood"(Pokharel, 2012). In 1868, Neumann used the term first

“myelogenous” to imply that leukaemia arise from the bone marrow (Thomas et al., 2013).

Introduction


Leukemia is a progressive and malignant disorder of the blood forming tissues, which

characteristically begins in bone marrow and marked by distorted growth and high number of

white blood cells and their precursors in the blood as well as bone marrow (Pokharel, 2012).

Risk factors for leukemia covers age (>50 years), exposure to chemicals such as benzene,

ethylene oxides, dioxins, butadiene and styrene. Along with these factors some viral

infections like human T cell leukemia virus I (HTLV-I), smoking, exposure to radiation for

longer period, growth hormones, some kind of medicines like chloramphenicol and

phenylbutazone. According to the International Classification of Diseases (ICD-9-ICM,

1980), leukemias come and fit in to the general class known as neoplasms of lymphopoietic

and hematopoietic tissues (ICD numbers 2000 to 2008).

The hematopoietic tissues found mainly in the bone marrow, are significant precursors of the

blood cells. One primary cell line called lymphoid cell line, originates at specific sites such as

the lymph nodes and results in formation of different cancers known as lymphomas (solid

tumors),or various myelomas (cancer of the plasma cell), and lymphoid leukemias, often with

the invasion of proliferating cells in blood or in the dividing bone marrow. Second line is the

myeloid line (originate from myelos, means marrow) generates the leukemias known as

myelogenous (originating from the marrow cells) or granulocytic leukemias (As granules

appeared in their cytoplasm when after fixation and staining) (Lamm et al., 1989).

1.2.1 Incidence of leukemia

The incidence of leukaemia worldwide is approximately 8 to 9 /100,000 people each year.

About 250,000 new cases occur each year worldwide, and about 28,000 of these cases in the

United States (Schiffer and Schimpff, 1991).

1.2.2 Classification of leukemia

There are four major types of leukemia:

Acute lymphocytic leukemia (ALL)

Chronic lymphocytic leukemia (CLL)

Acute myelogenous leukemia (AML)

Chronic myelogenous leukemia (CML)

1.2.2.1 Acute lymphocytic leukemia (ALL)

Introduction


Hematopoietic stem cells are destined to lead to two pathways either T-cell or B-cell

pathway. It is believed that acute lymphocytic leukaemia originates from certain genetic

abnormalities in these stem cells. Chromosomal translocations activate certain genes, are a

specificity of human leukemia in general and of acute lymphocytic leukemia in particular

(Pui et al., 2004, Armstrong and Look, 2005).

1.2.2.2 Chronic lymphocytic leukemia (CLL)

B-cell lymphomas are frequently caused by chromosomal translocations in which oncogenes

are involved (Dalla-Favera and Gaidano, 2001). CLL was known to be a homogenous disease

of immature B-cells that lack the ability to elicit an immune response. Because of defective

mechanism of apoptosis, these B cells which do not have the capacity to renew themselves,

accumulate ruthlessly (Dameshek, 1967, Rozman and Montserrat, 1995). But now, CLL is

regarded as two associated entities, both emerging from mature B lymphocytes which either

escape death through intervention of outer signals or face apoptosis (Chiorazzi et al., 2005).

1.2.2.3 Acute Myeloid Leukemia

A rapid growth of immature and abnormal white blood cells, accumulated in bone marrow is

the main characteristic of acute myeloid leukemia (AML). These non functional immature

cells disturb the function of normal cells. About 80% of leukemic patients have been

diagnosed with this type of leukemia. Leaving the patients without any treatment may lead to

death within weeks or months.(Dohner et al., 2010). In the United States, the annual

incidence of AML is approximately 2.4 cases per100,000 adults, though the incidence

increases progressively with age and peaks at 12.6 cases per 100,000 adults of age 65 or

older. Leukemic cells occupy the space in bone marrow causes reduction in the count of red

blood cells, platelets and normal WBCs. Major Symptoms of acute myeloid leukemia are

shortness of breath, fatigue, easy bleeding, and high risk of getting an infection. Previous

studies showed the involvement of several chromosomal abnormalities but specifically what

causes it, is not clear. Acute myeloid leukemia is also treated primarily through

chemotherapy that is mostly started with Idarubicin which may cause apoptosis of cancer

cells which induces cell death through apoptosis that may follow either intrinsic or extrinsic

pathway. Mostly AML patients get relapse of the disease when develop resistance. So some

more developments needed in treatment that may have no side effects and will not allow

relapse of the disease. (Döhner et al., 2010).

Introduction


1.2.2.4 Chronic mylogenous leukemia (CML)

Chronic myeloid leukemia is a dysfunction of clonal haematopoietic stem cell. The

specificity associated with this type of leukemia is a reciprocal translocation between the long

arms of chromosomes 9 and22 resulting in the formation of Philadelphia (Ph) chromosome

(Rowley, 1973).Chronic myelocytic leukemia is caused by the rearrangement of c-abl which

is a proto-oncogene from chromosome 9 to chromosome 22, which leads to formation of

tyrosine kinase which is oncogenic (Stam et al., 1985).

1.2.3 Factors responsible for leukemia

Factors that are responsible for leukemia are diverse including heredity or genetic

abnormalities, environmental exposure and dietary factors. Genetic predisposition basically

involves inherited mutations, hormones alterations, the altered immune conditions or

immunoglobulin abnormalities, polymorphism and chromosomal abnormal modifications

(Boveri, 1914). Most importantly, these hereditary factors cannot be modified. Although

leukemia is not originated hereditarily but environmental factors and life style have a deep

effect on its development and become major cause of leukemia. These important external

factors that affect the occurrence and fatality of leukemia include tobacco, alcohol, benzene

infectious agents, cigarette smoke, diet, radiation, environmental pollutants, infectious

organisms and pesticides significantly (Irigaray et al., 2007). Air pollutants like organic

compounds that are volatile in nature as well as pestticides; enhance the danger of leukemia

and lymphoma in children.

For Long-term exposure to chlorinated water used for drinking purpose having nitrates and

derivatives in it, which can be modified rapidly into mutagenic N-nitroso compounds (DNA

damaging agents) that increase the danger of lymphoma or leukemia. Moreover,

electromagnetic induction and radiations exposure (ionizing, and nonionizing) are linked with

an elevated risk of leukemia in children mainly. Leukemia in adults actively correlates with

occasional exposure to the ionizing radiations. Chemicals consist of Benzene, Petrochemicals

and organic solvents with their fractions, Pesticides and other chemotherapeutic agents are

probable leukemia risk factors in adults (Greaves, 1997).

Predominantly, exposure to benzene which is an established short listed chemical causes

AML (also called the acute non-lymphocytic leukemia), and supportive evidence of benzene

is also present that it may cause acute as well as chronic lymphocytic leukemia, non-

Hodgkin's lymphoma and multiple myeloma (IARC, 2000).

Introduction


1.2.3.1 Effects of environment on cancer development

Our environment and life style are responsible for 90-95% of our most persistent and deep-

rooted ailments rather than our genes (Fig.1) (Anand et al., 2008).Instead of immense heap of

research and expeditious advancements in the course of previous decade, cancer is still

known to be a global killer. Recent demographic data show that almost 23% of overall deaths

in the USA are due to cancer, and is the second leading cause of mortality after cardiac

diseases (Jemal et al., 2007).

Figure 1.1. Contribution of environment and genes in cancer development. A. Environmental

and genetic factors percent contribution to cancer B. Selected family risk ratios for cancer. C.

Percentage contribution of each environmental factor. The percentages depict the explicable-

fraction of cancer deaths due to the particular environmental risk factor (Anand et al., 2008).

1.2.3.2 Carcinogens involved in cancer development

Several carcinogens have been identified by different studies carried out on animals and also

high frequency of certain substances causing cancer such as cigarette involvement in lung

cancer induction in human populations. Malignancy is induced in several steps and too many

factors are involved in the likelihood to develop cancer and it cannot be concluded for any

Introduction


cancer that it may be developed due to single cause. Radiations, chemicals and viruses are the

most recorded causes of cancers in humans and animals.

The mechanism that may be followed by most of the radiations and carcinogens is to damage

DNA and induce mutations. Carcinogens are generally considered to initiate the events of

mutation in key target genes that may further induce certain other events and finally lead to

induction of cancer. Carcinogens mainly reported in development of human cancers are solar

ultraviolet rays that cause skin cancer tobacco smokes a major cause of lung cancer, aflatoxin

a major carcinogen for liver cancer. Some of the structures of carcinogens are given in Fig.

1.2.

Figure 1.2. Chemical structures of various toxic chemicals responsible for liver cancer.

(Cooper and Hausman, 2000).

1.3 Benzene: A leukemogen

Benzene is one of the carcinogen which triggers toxicity of blood through a number of

noxious metabolites (McHale et al., 2011).It has been revealed by a recent study that when

high benzene concentration was added to HL60 cells, it can cause cytotoxicity by increasing

level of reactive oxygen species (ROS) (Nishikawa et al., 2011). Benzene metabolizes into

toxic metabolites which is a crucial factor in toxicity of benzene (Ross, 2000). Metabolism of

benzene is intuit ively complex (Snyder and Hedli, 1996). It occurs substantially in

liver (Sammett et al., 1979), in the lung (Powley and Carlson, 2002), while secondary

metabolism occurs in bone marrow (Subrahmanyam et al., 1991). Rats subjected to partial

hepatectomy exhibited reduced toxicity and metabolism of benzene (Sammett et al., 1979).

Introduction


Same effects were observed in transgenic cytochrome- P450 2E1 (CYP2E1) knockout mice

(Valentine et al., 1996). Toulene competitively inhibits metabolism of benzene. Mice which

were administered with high toluene concentration, exhibited reduced toxicity and

metabolism of benzene (Andrews et al., 1977). It has been suggested by a recent study that

lower doses of toluene in mice enhances the DNA damaging effects of benzene (Wetmore et

al., 2008).

Fig 3 illustrates a simplified scheme of benzene metabolism. The preliminary step is

primarily catalyzed in liver at elevated exposure and involves benzene conversion to benzene

oxide by cytochrome- P450 (Valentine et al., 1996).

Recently it has been proposed that CYP2F1 and CYP2A13 have metabolic activity at 1 p.p.m

benzene levels (Rappaport et al., 2009) and both of them are immensely active in the human

lung (Powley and Carlson, 2000). So, lung may be the prime site for benzene metabolism at

low dose (McHale et al., 2011).

Figure 1.3. Schematic diagram showing metabolism of benzene from liver to bone marrow.

Major pathways and enzymes involved in metabolism and toxicity. GST, glutathione-S-

transferase; meh, microsomal epoxide hydrolase; MPO, myeloperoxidase; PST, phenol

sulfotransferase; UDPGT, uridine diphosphate glucuronosyl transferase (McHale et al.,

2011).

Introduction


A large bulk of benzene oxide mantains equilibrium with its tautomer oxepin, which

immediately converts to phenol (PH), and the remaining via benzene dihydrodiol either

produces 1,2-benzoquinone and catechol or produce S-phenylmercapturic acid by reacting

with glutathione. Ph is eliminated or metabolized to 1,4-benzoquinone (BQ) and

hydroquinone (HQ). HQ undergoes through CYP2E1 catalysis to convert into labile

metabolite 1,2,4-benzenetriol (Inoue et al., 1989). Oxepin metabolism open up aromatic ring

and yields reactive muconaldehydes which leads to hematotoxicity in vulnerable mice (Witz

et al., 1996). Muconaldehydes, Benzene oxide and benzoquinones are electrophiles which

immediately react with proteins and hinderes normal cellular function (Smith,

1996).Quinones and Semiquinone radicals are extremely toxic. Initially, they are produced in

bone marrow through phenolic metabolites and peroxidases. They directly bind to proteins

and nucleic acids in the cell and generates oxygen radicals through repeated redox reactions

(Subrahmanyam et al., 1991). In bone marrow, formation of BQ from HQ via

myeloperoxidase may be responsible for benzene carcinogenicity (Smith, 1996). It is

supported by the evidence which showed that when mice were induced for myelodysplasia

via benzene, BQ-detoxifying enzyme NADPH:quinoneoxidoreductase 1 (NQO1) showed

protection in these mice (Iskander and Jaiswal, 2005) and also protected humans against

hematoxicity induced by benzene (Rothman et al., 1996). HQ Glutathione conjugates may be

responsible for benzene toxicity (Irons and Stillman, 1993).

1.3.1 Cytogenetic changes associated with leukemia induced by benzene

Chromosomal aberrations in lymphocytes circulating in blood of humans who are chronically

exposed to benzene support this fact that metabolites of benzene are involved in onset of

leukemia (Bogadi-Šare et al., 1997). Chromosomal changes which are frequently noticed in

AML have been related with benzene exposure. These changes include 5q-/-5or7q-/-7,þ8 and

t(8;21) in blood circulating lymphocytes (Zhang et al., 2002, Zhang et al., 1999).

Chromosomal changes associated with AML are produced in human cell and CD34+

progenitor cell cultures through benzene metabolites (Smith et al., 2000). All these evidences

support that benzene is charged with AML induction via genetic pathways that were

previously proposed (Pedersen-Bjergaard et al., 2006). Primitive cytogenetic methods were

used to reveal the induction of chromosomal aneuploidy by benzene (Sasiadek, 1992, Li and

Ding, 1990). Aneuploidy associated with benzene may be due to microtubule assembly

inhibition (Smith, 1996). It is supported by the fact that 1,2,4-benzenetriol and HQ also cause

Introduction


same effect (Irons, 1985, Zhang et al., 1994). Most possible mechanism of benzene inducing

leukemia and lymphoma associated translocations like t(8;21) and t(14;18) is Topo II

inhibition (Zhao et al., 2007).Mice exposed to benzene showed increased DNA cleavage and

repression of topo II function (Eastmond et al., 2001). Same effects were observed due to

invitro exposure of BQ and HQ (Lindsey et al., 2005). Inhibition of topo II was found to be

enhanced by metabolic activation of HQ to BQ (Eastmond et al., 2001). Humans exposed to

benzene were found to be induced by gene amplification mutations at glycophorina locus in

circulating red blood cells (Rothman et al., 1996). This causes a phenotype which is related to

topo II inhibitors (Boyse et al., 1996).

1.5 Cytokines and their role in cancer

Cytokines are released or they are membrane-bound proteins that functions to maintain a

homeostatic immune system as they act as arbitrator of intercellular signalling. In response to

tumour antigens and microbes, cells of adaptive and innate immunity produce cytokines.

Local concentration and sequence of receptor expression of cytokines, including

incorporation of various signalling pathways involved in immune response are the factors

which are related to the effect of particular cytokine on immunity. The higher recurrence of

spontaneous cancers in mice genetically deficient in type I or II interferon (IFN) receptors or

IFN receptor signal transduction downstream elements is an evidence of importance of

cytokines in tumor immune surveillance (Picaud et al., 2002, Shankaran et al., 2001).

Transformations occurring at cellular level that lead to cancer metastasis evoke changes in

local expression of cytokines (Paul, 1989). In some conditions, immune cells make up an

additional, distinguished element of the host reaction to cancer (Clark Jr et al., 1989).

1.5.1 Tumor necrosis factor-alpha (TNF-α)

Tumor necrosis factor-alpha (TNF-α), characterized as one of the major cytokines, was

identified during endotoxemia in the mouse serum for its anti proliferative activity (Bruce-

Keller et al., 1999). TNF is primarily produced by natural killer (NK) cells, T cells and

macrophages. It has also been reported that nonimmune cells like smooth muscle cells,

fibroblasts and tumour cells have the ability to produce low quantity of cytokine (Tse et al.,

2012). TNF-α is an exceptional pleiotropic cytokine which is involved in inflammation,

homeostasis and anti-tumor responses via involvement of two single-pass transmembrane

glycoprotein receptors: TNF-R1 and TNF-R2. (Tian et al., 2014). TNF-R2 signalling effects

a large number of pro-inflammatory responses such as triggering T cells (Liu, 2005)

myofibroblasts (Frankel et al., 2006) hindrance of angiogenesis and tumor repression (Zhao

Introduction


et al., 2007). TNFR1 accounts for apoptotic and TNFR2 for any purpose associated with T-

cell survival functions of TNF (Tian et al., 2014). Transmembrane TNF-α plays a crucial part

in local inflammation (Mitoma et al., 2004). Transmembrane TNF-α also acts as a ligand and

expresses on several cell types, thus plays its part in pathological and physiological response

in healthy and diseased condition. Acting as a receptor, transmembrane TNF-α enables itself

to function as a pleiotropic cytokine and thus responsible for optimal performance of immune

response (Eissner et al., 2004).TNF expresses both tumor-inhibitory and tumor-promoting

properties, depending on circumstances of experiment according to which the conclusions are

formulated (Tse et al., 2012). It has been reported that persistent synthesis of low quantity of

TNF inside a tumor microenvironment encourages tumour growth and promotes

angiogenesis, while elevated amount can incite tumor cell necrosis, provoke antitumor

immunity and stimulate vascular collapse (Szlosarek et al., 2006).

1.5.2 TNF-α induced apoptosis via TNFR-1

Trimeric TNF-alpha binding to TNFR1 results in trimerization of receptor which leads to

recruitment of TNFR1-associated death domain protein (TRADD). This recruitment plays its

part in bringing three other mediators in the vicinity of this trimerized receptor: receptor-

interacting protein 1 (RIP1), Fas-associated death domain protein (FADD) and TNF-receptor-

associated factor 2 (TRAF2). A signal is transmitted via these adaptor proteins from TNFR1

to other cascades which leads to apoptosis (Baud & Karin, 2001). A caspase cascade is

activated by RIPK and FADD followed by apoptosis. Initiator caspases 2, 8, and 10 are

activated by adaptor proteins RAIDD and FADD. These initiator caspases are involved in

activation of effector caspases 3, 6 and 7 via their cleavage, which is the path leading to cell

death (Baud and Karin, 2001, Shearwin-Whyatt et al., 2000).

Introduction


Figure 1.4. TNF-α induced apoptosis via TNFR-1.

In response to tumor growth, particular T-cells are activated to obstruct tumor growth. These

T-cells which are tumor-specific secrete cytokine in response to tumor. Also cells which

express CD4 produce TNF-α which particularly attacks cancerous cells (Yee et al., 2002).

Since hematopoietic and non-hematopoietic cells secrete cytokines which are crucial in

innate and adaptive immunity. Cytokine expression by cells may be altered in inflammatory,

immunological, neoplastic and infectious disease states. As a result, cytokines influence

growth, differentiation, gene activation, expression of cell surface molecules and triggering

antibody responses to promote tumor destruction (Dranoff, 2004). Therefore, the present

study was aimed to suggest a possible mechanism of O. basillum anti cancerous properties by

investigating its modulator effect on TNF- α in benzene-induced leukemic rats.

1.4 Molecular pathways involved in Leukemia

Hematopoitic cytokines induce differentiation of the myeloid progenitor cells into

granulocytes and monocytes/macrophages. Mature myeloid cells that are differentiated play a

major role in host defence. Any pathological condition that may lead to deficiency in the

number of mature myeloid cells or to improper functioning of these cells can have some

drastic effects. In patients suffering from myeloid leukemias myeloid progenitor cells cannot

Introduction


differentiate normally those results in accumulation of immature proliferative blasts which in

turn leads to reduce a space for the production of normal mature cells (Miranda and Johnson,

2007). Cytokines such as granulocyte-colony stimulating factor (G-CSF) is normally

administered clinically to patients of AML for the enhancement of mature differentiated

myeloid cells during chemotherapy or radiation therapy. Specific receptors are present on the

surface of myeloid progenitor cells for certain hematopoietic cytokines that proceed the

differentiation of myeloid progenitor cells (Barreda et al., 2004). In bone marrow when

receptors for specific cytokines are bound by their legands, a number of downstream signal

transduction pathways get activated including Raf, Ras, MEK1/2 (mitogen-activated protein

kinase/ERK kinase), ERK1/2 (extracellular signal regulated kinase), Janus family kinases

(jaks), signal transducer and activator of transcription (stats), phosphatidylinositol 3-kinases

(PI3K), Akt, protein kinase C (PKC) and Smads (Chang et al., 2003, Steelman et al., 2004).

Historically knowledge regarding the function of these proteins involved in signalling inside

myeloid cells was provided by studies in leukemic cells. A better knowledge of the signalling

pathways that are involved in induction of cytokines mediated differentiation of myeloid

progenitor cells may provide best clues for designing new therapeutic strategies for the

treatment of myeloid leukemia.

1.4.1 JAK-STAT pathway

STAT proteins that are activated by hormones, growth factors and cytokines are mainly

involved in proliferation, differentiation and survival (Imada and Leonard, 2000, Williams,

2000). Cytokines that induce differentiation of myeloid cells, when bind to their cognat

receptors leads to phosphorylation of STAT proteins via activation of tyrosine kinase.

Activated stats in phosphorylated form dimerize and an additional phosphorylation occurs at

serine residues. In dimerized form stats translocate to nucleus where play a role in

transcription of genes involved in differentiation, cell cycle arrest and cell survival during

differentiation (Miranda and Johnson, 2007).

Several cytokines use JAK pahway and activate it.which in turn leads to activation of stats.

Four members Tyk, JAK1, JAK2, and JAK3 comprise Janus Kinas family of proteins that

are involved in differentiation of myeloid cells(Rane and Reddy, 1994). Over expression of

JAK3 as found to be involoved in G-CSF induced granulocytic differentiation and cell cycle

arrest (Mangan et al., 2004).

Introduction


Figure 1.5. Mediation of signalling pathways by cytokines through jaks. When cytokines bind

to their receptors they get dimerize and jaks get phosphorylated which in turn leads to

phosphorylation of stats. Stats in activated form translocat to the nucleus and activate or

suppress promoters of target genes.(Baker et al., 2007)

1.4.2 Ras signalling

Ras belong to gtpas family of proteins that play a role as intermediates in transfer of signals

from cell surface receptors to downstream signalling pathways(Magee and Marshall, 1999).

A controversial role of Ras proteins in myeloid differentiation has been explained in various

studies. In some studies Ras has been reported to inhibit the differentiation of

erythroleukemic cells (Zaker et al., 1997). In contrast in another study Ras has been observed

for is role in enhancement of granulocytes and monocytes differentiation of human leukemic

cells(Gallagher et al., 1998).

Introduction


Figure 1.6. Ras proteins are activated and transmit signals when transmembrane receptors are

bound by their ligands including cytokines. An exchange of GDP for GTP occurs on Ras and

this makes the confirmation of Ras suitable for switching various downstream effectors

involved in differentiation, proliferation and survival. (Ward et al., 2012).

1.4.3 Raf/MEK/ERK cascade

Raf is downstream to Ras and its activation is followed by interaction with Ras and

phosphorylation of some amino acids. Over expression of Raf in HL-60 cells was found to

be in differentiation of monocytic cells. Previous studies showed that two stages are involved

in activation of Raf signalling: in early differentiation MEK/Erk cascade is involved whereas

in late differentiation P90Rsk is found to be involved (Hong et al., 2001). Activated Ras

further activate MEK which in turn activate ERK (Wang and Studzinski, 2001).

Raf/MEK/ERK cascade has been reported to be involved in differentiation of myeloid cells

via the mechanism of activation of myeloid transcription factors.

Introduction


Figure 1.7. Raf/MEK/ERK cascade is involved in regulation of certain apoptotic regulatory

genes. Targeting this pathway can provide an important therapeutic strateg for the prevention

of cancer. Downregulation of his signalling may provide better results in treating cancer.

(McCubrey et al., 2007).

1.4.4 p38 MAPK and JNK/SAPK

Effects of stress and apoptosis are mediated by p38 MAPK and JNK/SAPK (c-Jun N-

terminal kinase/stress-activated protein kinase) pathways (Chang et al., 2003). Discussion

about the role of p38/MAPK pathway in myeloid differentiation is controversial. In some

studies it has been suggested that it is not a necessary factor for differentiation of myeloid

cells but is activated when differentiation is induced by some other means(Hong et al., 2001,

Wang and Studzinski, 2006).

Introduction


Figure 1.8. Various environmental factors and proinflamory cytokines are involved in

activation of p38 MAPK and JNK/SAPK pathway. This pathway is major therapeutic

gateway towards prevention of cancer.(Cuenda and Rousseau, 2007).

JNK/SAPK pathway has been reported very rarely for its role in myeloid differentiation. One

study on U937 cells has shown JNK pathway activation in differentiation of monocytic cells

induced by TPA followed by release of cytochrome that trigger apoptosis related

differentiation(Ito et al., 2001). IL-3 and G-CSF are also reported for activation of JNK

pathway in NF-60 and baf3 cells (Miranda and Johnson, 2007). JNK pathway was also found

to be activated in differentiation of AML cells induced by 1,25-dihydroxyvitamin D3 (Wang

et al., 2005a).

Introduction


Figure 1.9. (A) Apoptosis can be induced through activation of caspases, sustained

activation of JNK induce apoptosis through inactivation of the suppressors of

mitochondrial death patway. TNF-α activates NF-κb that blockes caspase activation and

also prolong activation of JNK, so that inhibits TNF-α induced apoptosis. (B) while

blocking NF-κb removes inhibition on caspases and JNK, and thus allow TNF-α to induce

apoptosis.(Jing and Anning, 2005).

1.6 Chemoprevention: cancer prevention strategy

Due to considerable elevation of worldwide cancer prevalence in correspondence with its

morbidity and lethality, with a rapid increase in costs of healthcare treatments, there is an

escalating interest for disease prevention. Chemoprevention is one such approach with

immense potential. It involves use of synthetic, biological or natural agents to revoke,

repress, or halt either the early stages of carcinogenesis or the promotion of cells from

premalignant phase to invasive phase. (Sporn, 1976). Chemoprevention may include

disruption of a number of stepsin tumour initiation, development and progression (De Flora

and Ferguson, 2005). Several success stories of chemoprevention of breast, prostate and

colon cancer has prompted the interest of scientists towards this field of research. More

importantly now FDA has approved 10 chemopreventive agents for precancerous lesions and

cancer risk. (Wu et al., 2011).

Use of chemopreventive agents with three vast approaches has been reported. To administer

chemopreventive agent to healthy population or to those who do not have certain risk factors

of disease is termed as primary chemoprevention. Oltipraz is such an example which

modifies metabolism of carcinogen via phase I or II enzymes. To identify individuals with

premalignant lesions and to administer these agents to inhibit the development of invasive

cancer comes under secondary level of chemoprevention. Examples include the

Introduction


administration of non-steroidal anti-inflammatory drugs (nsaids) in colorectal adenoma

patients. Patients who have sustained after successful treatment of disease pass through

tertiary level of chemoprevention to prevent recurrence of cancer via administration of agents

(Steward and Brown, 2013).

Figure 1.10. Phases of carcinogenesis include initiation, promotion, progression, and

metastasis. (A) In initiation phase genes get changed, mutated or altered raised spontaneously

or due to some carcinogen. These alterations lead to deregulated biochemical signalling

pathways that are involved in cellular differentiation and survival. These changes depend on

the type and rate of metabolism of carcinogen and also the response of function of DNA

repairing. (B)The lengthy and reversible phase of carcinogenesis is promotion that involves

development of various benign tumors made up of proliferating preneoplastic cells. At this

stage chemopreventice agents can alter the process. Conversion of premalignant cells to

malignant ones is progression. (C) In final stage of transformation that is progression, genetic

and phenotypic changes occur and finally cells proliferation. Chemopreventive agents should

be selected on their ability of acting between initiation and promotion phases. (D) In

Metastasis cancer get spread through blood stream and lymph system from primary site to

other parts. Chemopreventive agents are promised for inhibiting matastasis of cancer.

(Siddiqui et al., 2015).

1.6.1Classification of chemopreventive agents

Chemopreventive agents could be classified as blocking or suppressing agents by the phase in

the carcinogenesis at which they show their inhibitory effects (Arnold et al., 1995).

Introduction


Figure 1.11. Classification of Chemopreventive agents based on the stage at which they

restrain carcinogenesis.(Greenwald, 1996).

1.6.1.1 Blocking agents

Compounds that restrain beginning of cancer are commonly termed as ‘blocking agents’.

Their mechanism of action is prevention of interaction between internal free radicals and

chemical carcinogens and DNA. It minimizes the rate of damage and mutations, thus

minimizing the rate of cancer initiation, genomic instability and transformation of a normal

tissue into neoplastic one. Protection against cancer may be attained as a result of reduced

cellular uptake and bioactivation of pro-carcinogens and/or upregulated scavenging of free

radicals and eradication of susceptible electrophiles, as well as onset of repair pathways (Yu

and Kong, 2007).

Inhibition of cancer initiation may also be the consequence of generation of nitrogen and

reactive oxygen species and down regulation of pathways which cause chronic inflammation.

Other preventive strategies include activation of tumour suppressor genes by reversing their

hypermethylation pattern via DNA methyl transferases. Among other effects of blocking

agents on gene expression pattern in cancer include suppression of histone deacetylases

(Hauser and Jung, 2008).

1.6.1.2 Suppressing agents

Introduction


Suppressing agents may affect the progression of already initiated cancerous cells. These

agents act by inhibiting the pathways leading to cell proliferation like nuclear factor (NF)-kb

signal transduction pathway (Karin, 2006).It has also been found that some inhibitors have

the quality of a blocking as well as suppressing agent (Wattenberg, 1990).

In some examples, anti-oestrogens e.g., tamoxifen can block tumor advancement effect of

hormones (Yager and Davidson, 2006). Mtor signalling and AMPK pathways may serve as

striking goals for chemopreventive agents as they can interfere with energy homoeostasis and

metabolism of cancer cell according to recent reports (Din et al., 2012). Initiation of

apoptosis and perturbation of angiogenesis are also the mechanisms which are used by

chemopreventive agents (Noonan et al., 2007).

1.6.2 Chemopreventive agents and associated risk factors

Tamoxifen was the first chemopreventive agent approved by FDA, for it reduces breast

cancer risk (Anand et al., 2008). Women at high risk of breast cancer showed minimized

incidence of breast cancer by 50% (Waters et al., 2010). But there are serious side effects

associated like blood clots, hypercalcemia, ocular disturbances, uterine cancer and stroke

(Anand et al., 2008).

Another agent approved to refrain familial adenomatous polyposis (FAP) is Celecoxib which

is a COX-2 inhibitor. However, it has been reported by several studies that cyclooxygenase

(COX) inhibition lead to therapeutic side effects of non-steroidal anti-inflammatory drugs

(nsaids) (Warner et al., 1999). Efficacy of celecoxib in inflammatory diseases is no much less

than other nsaids (Simon, 2001), but recent researches have shown that administration of

these drugs may cause thrombotic cardiovascular problems (Mukherjee, 2002).

The critical side effects of chemopreventive drugs approved by FDA are a crucial issue. This

reveals the demand for safe and effective agents to prevent cancer. Natural products derived

from diet may be suitable for this purpose (Anand et al., 2008).

1.6.2.1 Natural therapies in chemoprevention of Cancer

Natural products that have strong anticancer potential are derived mainly from plants. (Ait-

Mohamed et al., 2011). These have advantages over synthetic drugs as they are more

economic, plentiful and are having no side effects. Several studies have reported the use of

Introduction


crude extracts of plants more strong agents as compared to their isolated constituents.

(Mijatovic et al., 2011).

A large number of studies have been executed worldwide to isolate the agile and innovative

compounds from plants to treat cancer. Secondary metabolites like flavonoids, alkaloids,

triterpenes and polyphenols were isolated from medicinal plants (Rates, 2001).

Approximately 30–35% of cancer deaths are associated with obesity, diet and metabolic

syndromes,which is a favourable indication that by modification of dietary habits, fairly

acceptable chunk of cancer deaths can be avoided. It has been proclaimed by a major amount

of research that a diet consisting of spices, vegetables, fruits, and grains has the competency

to prevent cancer (Anand et al., 2008). The distinct substances in these nutritional foods that

are liable to hamper the production of cancer and the mechanisms by which this prevention is

made possible have also been studied significantly. Numerous phytochemicals have been

ascertained in grains, vegetables, spices and fruits that express chemopreventive potential and

a major research has indicated that protection against cancer can be made possible by proper

diet (Vainio and Weiderpass, 2006).

A plentiful number of anticancer drugs which are diverse in variation and mode of action

have been contributed by medicinal plants. It has been proposed by extensive number of

reports that many substances which occur naturally show cancer chemotherapeutic effects

(Khan et al., 2013).

1.7 Medicinal plants of the family Lamiaceae as functional foods

Lamiaceae is considered a very important family of plants from medicinal point of view. It

contains about 236 genera and species are more than 6000. Members of this family are

widely ued to cure certain diseases(Carović-StanKo et al., 2016). Phenolic compound of

family lamiacea plants include euginol, tannins, quinines, lignin, terpenoids and flavonoids.

Flavonoids got attraction due to their versatile biological activities like antitumor,

antimicrobial, antioxidant and antiinflamatory (Rai et al., 2013).

1.7.1 O. basilicum

The plant used in this study was O. basilicum commonly known as Basil (family Lamiaceae).

It is a very famous herb in the Mediterranean diets, as it is commonly used in lots of food

recipes. Basil belongs to genus Ocimum and is among considerable amount of essential oils

producing species. (Grayer et al., 2002). Its extracts are also used in the preparation of

Introduction


cosmetic and medicinal products (Umerie et al., 1998). Basil has phenolic acids and aromatic

compounds due to which it shows antimicrobial, antioxidant and anti-tumor activities

(Gutierrez et al., 2008). Flavonol-glycosides and phenolic acids are major phenolics that have

been reported in basil (Javanmardi et al., 2002). Moreover, another antioxidant phenolic

compound such as rosmarinic acid is also present in basil (Tada et al., 1996).The ability to

absorb and neutralize free radicals, to quench singlet and triplet oxygen and to decompose

peroxides is responsible for the antioxidant activity of these phenolic compounds (Asami et

al., 2003).

Several therapeutic effects for several conditions such as appetizing and digestive systems

have been associated with basil. Asimgil in 1997, Kosekia et al in 2002 and Umerie et al in

1998 reported that tonic, antiseptic and insecticidal properties are found in the leafy parts of

basil. Leaves of basil are also known for the cure of cough and pain (Basilico and Basilico,

1999). Moreover, basil is used for ailment of acid indigestion, inflammation and aches

(McClatchey, 1996).

1.7.1.1 Chemical constituents

O. basilicum has been found as rich source of beta carotene. Beta carotene has a strong

potential to prevent the effects of free radicals causing damage to the cells. Important mineral

magnesium has also been found in bail that plays an important role in relaxing blood vessels

and improvement of blood flow. Calcium, iron, potassium and vitamin C have also been

found in basil (Gebrehiwot et al., 2015). Several important chemical constituents of O.

basilicum extract recorded are tannins, flavonoids, saponins and volatile terpenes like

linalool, eugenol, thymol, camphor and 1-8- cineol. Estragole, 1-isopropyl-4-

methylenecyclohex-1-ene, 2-propenoic acid-3-phenyl-methyl ester ((E)-methyl cinnamate),

p-mentha-1(7), 8-diene, L-Fenchone, α-caryophyllene, eucalyptol, α-pinene, α-cubebene,

isocaryophillene, methyl-2-phenyl-prop-2-enolate, eugenol, eudesma-3,7(11)-diene, beta-

bisabolene, and trans-α-bergamotene were found as the major compounds (Gebrehiwot et al.,

2015).

Introduction


Figure 1.12. O. Bacilicum.

1.7.1.2. Anticancer Properties

Considering the results of the previous studies carried out on essential oil of O. basilicum on

murine leukemic cell line which showed its anti proliferative potential, chemopreventive

effects of hydroalcoholic extract of leaves of O. basilicum on skin cancer induced by

carcinogen and antiproliferative effects of O. basilicum extract and its fractions on human

breast cancer cell lines has prompted us to evaluate the effects of O. basilicum crude

methanolic extract and its sub fractions on human leukemic cell lines. Four acute myeloid

leukemia derived cell lines were used in the present investigation. An attempt was also made

to investigate the mechanism by which O. basilicum fractions were causing apoptosis.

1.7.2 R. officinalis

Commonly R. officinalis is known as rosemary. This perennial culinery herb is very popular

and is cultivated all over the world. Both fresh and dried leaves of rosemary have a good

application in food cooking due to its strong aroma and it is also popular for making tea in

small amounts. It has a strong antioxidant potential and due to its properties it is used for

increasing the shelf life of foods. Use of rosemary due to its antioxidant potential for

preservation of foods has been recommended safe by European Union (Food standard

agency). Traditionally rosemary is useful for the treatment of cancer (Cheung and Tai, 2007),

bacterial infection(Bozin et al., 2007), as an antioxidant (Bakırel et al., 2008), antiinflamatory

(González-Trujano et al., 2007), antidiabetic (Bakırel et al., 2008) and hepatoprotective

(Abdel-Wahhab et al., 2011).

Introduction


Figure 1.13. R. officinalis.

1.7.2.1. Chemical constituents

Rosemary has a vast array of secondary metabolites contributing to its medicinal uses. Of

these chemical constituents one small molecular weight group is of essential oils that majorly

contain 1,8-cineole, α-pinene, camphene, α-terpineol, and borneol (Atti-Santos et al., 2005).

Other group of secondary metabolites that contribute to medicinal properties of this culinary

herb are polyphenolics that include homoplantaginin, cirsimaritin, genkwanin, gallocatechin,

nepetrin, hesperidin, and luteolin derivatives) and phenolic acid derivatives (e.g., rosmarinic

acid) (Bai et al., 2010). Now a days the most important group of compounds found in

rosemary are polyphenolic diaterpenes (Habtemariam, 2016).

1.7.2.2 Anticancer studies

In several previous studies anticancer effects of rosemary have been reported. Growth of

colon cancer cell lines was found to be reduced significantly due to treatment with rosemary

extract (Yi and Wetzstein, 2011). In rat rinm5f insulinoma cells, cell proliferation was

reduced significantly due to treatment with rosemary extract and induction of apoptosis was

recorded (Kontogianni et al., 2013). Pancreatic cell lines PANC-1 and MIA-paca-2 when

exposed to rosemary extract have shown a significant inhibition of cell viability (González-

Vallinas et al., 2014). Cell growth was inhibited in breast cancer lines MCF-7 (ER+) and

MDA-MB-468 (TN) cell lines by rosemary extract (Cheung and Tai, 2007). Cell viability

was inhibited in prostate cancer cell lines DU145 and PC3 by rosemary extract (Yesil-

Celiktas et al., 2010). Another study has shown results in agreement with this data on lncap

and 22RV1 prostate cancer cell lines (Petiwala et al., 2014).Similar results have been

Introduction


observed in bladder carcinoma cells (Mothana et al., 2011), in cervical cancer cells

(Berrington and Lall, 2012), in liver cancer cells (Peng et al., 2007).

1.7.3 T. vulgaris

Figure 1.14. T. vulgaris (Prasanth Reddy et al., 2014)

T. vulgaris L. Belongs to family Lamiaceae and it comprises of about 215 to 350 species as

inferred by various data obtained from literature. They are generally herbaceous perennials,

short shrubs basically originating from the Mediterranean zone, which is pivotal origin of the

whole genus, and are additionally trademark for Asia, Southern Europe and North Africa

(Zaidi and Crow, 2005).

Thymus genus in Romania consists of one species characteristically classified as aromatic

plant (T. vulgaris) and other 18 wild species (Grigoreet al., 2010). T. vulgaris L. (thyme), is

well-understood species of genus Thymus, in local language is known as “zaatar”, and an

individual member from the family Lamiaceae, which is a wonderful aromatic herb,

cultivated in different areas of the world (Güner et al., 2000) and greatly studied for its

chemical and also biological properties (Simandi et al., 2001, Soković et al., 2008). It also

has been well documented that its essential oil has various biological actions such as

antiseptic or, antispasmodic, antimicrobial (Marino et al., 1999) and antioxidant activities

(Miura et al., 2002).

1.7.3.1 Chemical constituents and medicinal uses of thyme

The pharmaceutical characteristics of aromatic plants are mainly due to the essential oils

(Edris, 2007). Essential oils are characteristically volatile, natural, and complex chemically

Introduction


identified by a very strong fragrance and produced by aromatic plants significantly as

secondary metabolites (Bakkali et al., 2008). Interpretation of compounds present in plants

have become more available and more worthless due to the establishment of Thin layer

chromatography (TLC) and other major chromatographic techniques such as GC, LC-MS and

other spectroscopic techniques for example Fourier transform infrared (FTIR) spectroscopy

and UV Spectrophotometry (Sahaya et al., 2012). Phytochemical analysis showed that

thymol, linalool, myrcene, γ-terpinene, ρ-cymene α-pinene, carvacrol, eugenol and α-

thujeneare the prominent characteristically found compounds in thymus. According to

European Pharmacopoeia the lesser part of essential oil (EO) in T. vulgaris is 12 ml/kg, but

chemically it shows, various six chemotypes being largely documented , are geraniol,

linalool, gamma-terpineol, carvacrol, thymol and trans-thujan-4-ol/terpinen-4-ol (Rota et al.,

2008, Thompson et al., 2003). FTIR functional group interpretation of T. vulgaris showed the

presence of different functional groups for example Alkyl groups, Aromatic compounds,

Phenolic and alcoholic groups, amines, Nitro compounds, Aldehydes , as well as α and β

aliphatic compounds having unsaturation (Kulkarni et al., 2013).

Volatile oil of T. vulgaris was taken from Yemen and analyzed to check the composition. So,

the major components analyzed contained thymol and p-cymene followed via caryophyllene,

α-pinene, β-myrcene, thymol methyl ether and carvacrol (Al–Maqtari et al., 2011).

The fundamental mixture of T. vulgaris essential oil (TEO) has a composite mixture of

monoterpenes largely and the main compounds of this EO oil are natural terpenoid, thymol

and also its phenol isomer carvacrol (CVL) (Amiri, 2012, Nickavar et al., 2005), which have

biological activities such as antioxidative, antimicrobial, expectorant, antispasmodic, and

antibacterial properties (Höferl et al., 2009). Terpenoids, phenolic acids flavonoid glycones

and flavonoids glycosides were additionally present in Thymus spp. (Vila, 2002).

Isolation yield and chemical composition of essential oils depend on various elements, for

example, the environment, growth area and cultivation expertise (Hudaib and Aburjai, 2007).

Moreover, other than the flavouring characteristic that is highly possessed by eos dynamic

constituents, essential oil of thyme also possesses highly antimicrobial action (Rota et al.,

2008, Dorman and Deans, 2000) and strong antioxidant activity (Grigore et al., 2010).

Recently, the use of the essential oils as antioxidant agent and natural products is extensively

documented (Milhau et al., 1997). Researchers have presented antibacterial as well as

antifungal properties of essential oils (Pandey et al., 1996). Thyme oil is world’s one of the

top 10 essential oils and also used in food preservation (Sáez and Stahl-Biskup, 2002).

Introduction


T. vulgaris demonstrate polymorphism in monoterpene formation, the vicinity of intraspecific

chemotype alterations being usual in the genus Thymus. Each of the six chemotypes, geraniol

(G), α-terpineol (A), thuyanol-4 (U), linalool (L), carvacrol (C), and thymol (T), is named

due to its predominant monoterpene. Various factors, for example, genetic, climatic and

geographic fluctuations as well as ontogeny of gathered plants might seriously influence

essential oil chemical composition and their biological characteristics (Zouari et al., 2012).

1.7.3.2 T. vulgaris anticancerous and immunomodulatory activities

T. vulgaris plant extracts are generally utilized for several purposes, for example, anti-

asthmatic, as bronchodilator, antitussive, antispasmodic (Meister et al., 1999), antibacterial as

well as antifungal (Pina‐Vaz et al., 2004). Additionally, these extracts have also

immunomodulatory effects (Ocaña and Reglero, 2012). Various studies reported that T.

vulgaris extracts may have potential anticancerous effects (Berdowska et al., 2013). T.

vulgaris eos were previously appeared to interrupt the transcription of some genes that play

an important role in cell cycle, apoptosis and cancer, involved also in, interferon signaling, N-

glycan biosynthesis as well as extracellular signal-regulated kinase 5 (ERK5) signaling

(Sertel et al., 2011).

A significant activation of leucopoiesis, an increase in number of thrombocyte in blood, anti-

inflammatory activities against acute as well as chronic inflammation in the rats

specifically.(Von Ardenne and Reitnauer, 1981).

Cytotoxicity of Thyme essential oil might be because of its compounds that have lipophilic

nature; aggregate in the cancer cell membranes and stimulate their further permeability,

bringing about leakage of enzymes and other metabolites (Sikkema et al., 1995).

Interestingly, beside its anticancerous activity, thyme extract has also indicated to stimulate

immune response including leukocytopoiesis as well as thrombocytopoesis. Thymol has

significant cytotoxic property against several human tumor models (Medina-Holguín et al.,

2008, Horvathova et al., 2007).

1.8 Immunomodulatory effects of medicinal plants

The term immunomodulation implies the modification of immune system response which

might enhance or reduce the immune system response. Enhancement of the immune system

response is known as immunostimulation and reduction in the immune response is known as

immunosuppression (Sell and Max, 2001). An immunomodulator may be characterized as an

agent that cause modulation of the immune system and its components, for example, altering

Introduction


intensity of responses by induction of maturation of development of actively competent

immune cells, epitopes exposing on the surface of cell membranes, differentiating response to

particular antigens and regulation of cellular metabolism.

In this way an immunomodulator are the medications influencing the activity of immune

system and are useful when the immune response become dysfunctional. So, to keep up a

healthy state, immune response alteration is necessary by either its enhancement or reduction,

as a useful therapy. Immunomodulator can give strong treatment impact to the chemotherapy

(Kokate et al., 2006). There are specific medicinal plant that are known to possess

immunomodulatory effects (Agrawala et al., 2001) as well as antioxidant activities for

anticancerous effects.

In spite of the fact that various engineered drugs such as cyclosporine, corticosteroids,

Azathioprine etc. Are utilized as a part of treating immune disorder however they have

harmful effects also as Anemia, Thrombocytopenia, Bone Marrow suppression etc. Today

natural therapeutic plants and their derivatives have demonstrated promising

immunomodulatory characteristics, antioxidant properties and anti-inflammatory reactions.

Various bioactive compounds are also reported that have been separated from the plants such

as cordifolioside A, syringing, curcumin, flavopiridol, lycopene and some polysaccharides as

well , show potent immunomodulatory effects (Sharma et al., 2012, Pan et al., 2012).

1.10. Aims and objectives

To assess the safety of the selected three medicinal herbs (O. basilicum, R. officinalis

and T. vulgaris).

To evaluate a mechanistic approach for the treatment of cancer using common

medicinal herbs O. basilicum, R. officinalis and T. vulgaris.

To explore anticancereous effects of these medicinal herbs in vivo and in vitro.

To explore active fractions and ingredients of the selected herbs having high anti

cancer property.

To provide a clue to scientists worldwide of the treatment of cancer via low cost

natural products having no side effects.

To characterize the functional role of JNK and other apoptotic pathways in O.

basilicum, R. officinalis and T. vulgaris apoptosis in human leukemia cells by using

pharmacologic and genetic approaches.

Materials and Methods



2.1. Collection of Plant Material

Leaves of the three selected medicinal herbs Ocimum basilicum (O. basilicum), Thymus vulgaris

(T. vulgaris) and Rosmarinus officinalis (R. officinalis) were collected from the clonal

repository of National agriculture research centre Islamabad, Pakistan having Latitude:

33.6982 and Longitude: 73.0393.

2.2. Crude Methanolic Extract

The collected plant material was dried in shadow under conditions of glass house. Coarse

powder of these leaves was made and their extracts were prepared using methanol. 70grams

of this coarse powder of each plant leaves was soaked in 300 ml of methanol using air tight

bottles for 48 hrs. The extract was filtered through Whatman filter paper 1. Using rotary

evaporator (Bibby, RE200B) methanol of each plant filterate was evaporated. This pure form

of extract was collected in flasks and and were measured for their weight. Each plant material

was saved in air tight bottles and were labelled and stored at 4˚C.

2.3. Total Phenolic and Flavonoid assay

Minor modifications were made in the protocol followed by Atanassova et al for total

phenolics and flavonoid measurement. Standard folin Cicalteu reagent assay was used for

total phenolics and Aluminium chloride colourimetric assay was used for total flavonoids.

Gallic acid and catechin were used as standards for total phenolics and flavonoids

respectively. (Atanassova et al., 2011). Experiment was conducted in duplicate.

2.4. Minimum inhibitory concentration (MIC) of selected herbs

MIC of the methanolic extracts of three selected medicinal herbs was measured through

standard broth microdilution method against three types of bacterial strains Escherichia coli,

Staphylococcus aureus and Streptococcus mutans following the procedure of clinical and

laboratory standard institute (Wikler, 2006). DMSO was used as a diluent for making serial

dilutions of the plants extracts.

2.5. Synergistic effects of plants extracts with antibiotics

To study the antimicrobial potential of the selected three plants extracts and antibiotics

combinations Checkerboard test was done. The combination was consisted of amoxicillin and

ciprofoxacine and methanolic extract of the selected plants. The nutrients broth (NB) was

prepared and serial dilutions of upper mentioned drugs and methanolic extracts of plants were



mixed in it. Three types of bacterial strains were grown on the NB and their inocula were

prepared. MIC was determined while keeping the inocula in incunator at 37 oC for 24 h. for

the drug alone as well as for drug mixed with plants extracts. Triplicate experiments were

performed. Fractional inhibitory concentration (FIC) was calculated to find out interaction of

drug with plant extracts. Following equation was used to calculate the FIC:

FIC index = FICA + FICB = [A] MICA + [B] MICB ..............................................ii

Where FIC[A] and FIC[B] are the FIC of molar concentration of drug A and B, respectively;

MIC[A] and MIC[A] are the MIC of drug A and B for the organism, respectively. The FIC

index found was deduced as follows: for <0.5 representing synergy; 0.5–0.75 representing

partial synergy; 0.76–1 showing an additive effect; 1–4 showing indifference; and >4

showing antagonism (Mun et al., 2013).

2.6. Subacute toxicity assay

Sprague dawley rats were selected for this experiment. Separate cages were used for every

group of rats that have a good ventilation system diet of water ad labitum and rodent pellets.

For toxicity study seven groups A, B, C, D, E, F and G of rats were designed for rats each

having 8 rats. Two different concentrations of each plant extract were prepared that is

1000mg/kg body weight and 1500 mg/kg body weight. Rats in group A were kept healthy

and were maintained on normal diet. Rats of group B, C and D were treated with O.

basilicum, R. Officinalis and T, vulgaris at 100mg/kg BW and same pattern was repeated

with E, F and G groups but the dose was 1500mg/kg BW. All the rats were treated for one

month with plants extracts and their weight was measured every week. Rats were kept in

fasting conditions overnight on 29th day of the treatment and were dissected on 30th day after

weight measurement. Blood samples were blood samples were taken in EDTA tubes while

four organs liver, heart, kidneys and spleen were taken and preserved in 10% buffered

formalin.

2.6.1. Histopathology analysis

Thin fine sections of each of the four organs were made and paraffin was used for their

fixation. Their staining was carried out via hamatoxylin and Eosin (H and E). Light



microscope was used for tissue section examination. Histology of the organs was compared

with the control through representative images.

2.6.2. Haematological assays

Blood from EDTA tubes was used for determination of haematological parameters.

Parameters total leukocytes count, red blood cells count, Packed cell volume (PCV),

haemoglobin mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH),

mean corpuscular haemoglobin concentration (MCHC) and platelets using Sysmex KX21 , a

product of SYSMEX Corporation, Japan (Dacie and Lewis, 1991).

2.7. Statistical analysis

All the experiments were repeated three times. For phenolics, flavonoids and antioxidant

activity standard curves were drawn. Data has been represented in the form of bar graph. To

find out the difference of organ body weight ratio and haematological parameters from

control LSD ranking was applied using Statistix 8.1. Data expressed was actually mean of 8

replicates. Statistical significance was measured at P<0.05.

2.8. Fractionation and affirmation of compounds in selected fractions

2.8.1. Column chromatography

The fractionation of methanolic crude extract was done using column chromatography. In

these experiments a glass column (length=450mm, bore=30 mm) packed with 60-120 mesh

sized silica gel was used. The mobile was consisted of the mixture of n-hexane and ethyl

acetate. Initially, the column was rinsed with n-hexane and then fully dried before use. The

packing of the column was done in such a way that the column was filled with n-hexane up to

3/4 of its total length and then the silica. The n-hexane was drained out leaving the wet silica

packed column. Before applying to column packing the silica was activated by treating it up

to 100 oC for 1 h. The mobile phase used during these experiments was the mixture of n-

hexane and ethyl acetate combine in the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 4:6, 3:7, 2:8, 1:9 of n-

hexane and ethyl acetate, respectively. The fractions collected were run on TLC for further

purification and fractionation. Fractions with matching spots and RF values were mixed

together and headed for further analyses. Table 2.1 is showing the details of fractions and the

solvent composition.



Table 2.1. Fractions of O. basilicum, R. officinalis and T. vulgaris methanolic crude was done

by column chromatography using silica as a stationary phase. The mobile phase used was the

mixture of n-Hexane and ethyl acetate in different ratio.

2.8.2. Thin layer chromatography (TLC)

To fractionate the methanolic crude extract of the selected plants by column chromatography,

TLC analysis was done for optimizing the suitable solvents which were then used as a mobile

phase. TLC analysis was done using TLC plates (Silica-gel 60 F254, Merck, Germany).

Different solvent mixture such as ethyl acetate:n-hexane; chloroform:ethyl acetate;

chloroform:methanol and water: methanol with varying ratios to obtain solvent of different

polarities were evaluated for better separation and good Rf value. After development of TLC

plates and calculating their Rf values best solvent mixture was selected for use in column

chromatography.

All these obtained fractions were dissolved in DMSO for further in vitro and in vivo studies.

DMSO is a strong aprotic solvent which has the ability to dissolve almost all the polar and

non-polar organic compounds. It is widely used in biological studies due to its beneficial

nature. It has shown analgesic, antibacterial, antioxidant and anticancer activities (Wang et

al., 2012; Brayton, 1986; Jacob & Herschler 1986; Jacob et al., 1964). Hence, the use of

DMSO as a solvent to dissolve the obtained fractions as well as its use a control was made in

the present study.



2.9. In Vitro Assays

2.9.1. Cell culture preparation and treatment with stimuli

Four types of acute myeloid leukemic cells U937, OCI-AML3, MV4-11and THP-1 were

collected from European Collection of Cell Cultures. Cytogenetic analysis was performed for

the identification of cell lines. RPMI-1640 (Invitrogen) media was used for culturing, where

it was supplemented with 10% fetal bovine serum (FBS) and 1% glutamine. The atmosphere

of incubator for cell culturing was humid and was infused with 5% CO2.

2.9.2. Cytotoxicity assay

Six different concentrations (0, 1, 2, 5, 10 and 20µg/ml of DMSO) of each fraction and crude

extract of O. basilicum, R. officinalis and T. vulgaris were used to treat cells for 24 hrs. After

which cells were treated with cell titre GLO (Promega) according to manufacturer’s

instructions to asses cell viability. IC50was calculated for DMSO, all fractions and crude plant

extract. All experiments were performed in triplicate. Seven fractions B2 (2:8 n-

hexane:ethyle acetate) , B3 (3:7 n-hexane:ethyle acetate) from O. basilicum, R1 (1:9 n-

hexane:ethyle acetate), (2:8 N hexane:ethyle acetate) frpm R. Officinalis, T2 (2:8 n-

hexane:ethyle acetate), T7 (7:3 n-hexane:ethyle acetate) and T8 (8:2 n-hexane:ethyle acetate)

from T. vulgaris showed good activity as compared to others, so these were chosen for

further experiments.

2.9.3. Flow cytometry

Apoptosis of four types of AML derived cell lines (U937, OCI-AML3, MV4II and THP-1)

induced by selected seven fractions of three medicinal herbs at 24hrs, 4 hrs, and 2hrs

treatment was measured. Each cell line was treated with 20µg/ml of stimuli for three time

intervals. Samples were collected and centrifuged at 500 ×g for 3 minutes. Cell palates were

resuspended and stained with annexin-V and PI dye. Apoptosis was detected via Accuri C6

flow cytometer.

2.9.4. Cell death in the presence of caspase inhibitor z-VAD-fmk

To study the association of caspase in induction of apoptosis induced by selected fractions of

O. basilicum, R. officinalis and T. vulgaris, U937 and OCI-AML3 cells were treated with z-

VAD-fmk, a general caspase inhibitor before treatment with B2 and B3. In one plate

pretreated cell with z-VAD-fmk for 1 hr were treated with selected fractions for 24hrs and in

the other plate cells were treated only with plant fractions. After 2 hrs flow cytometric

analysis was carried out as described above.



2.9.5. Western immunoblotting

Western immunoblotting for P-JNK and caspase was performed by the protocol followed by

Rushworthet al,(Rushworth et al., 2012). Briefly four types of AML derived cells were

treated with seven fractions of O. basilicum, R. officinalis and T. vulgaris and DMSO as a

control for two time intervals (15 and 60 minutes). Whole cell lysates were extracted from all

the treated cells and SDS-PAGE was performed. Protein was transferred to nitrocellulose

membrane and was probed for JNK and cleaved caspase-3 (Cell Signaling Technology,

Danvers, USA, 1 : 200 dilution), following manufacturer’s instructions. Protein bands were

detected using enhanced chemiluminescence detection.

2.9.6. RNA extraction and real-time PCR

U937 cells were treated with selected seven fractions for 24 hrs at 20µg/ml concentration.

TNF alpha was used as a positive control and DMSO as a negative control. RNA lysis

solution and a nucleic acid Prepstation purchased from applied biosystems were used for the

extraction of total RNA according to the instructions of manufacturer. RNA PCR core kit was

used to produce cdna via reverse transcription.cdna was further used in Light cycler 480

cyber green technology (Roche). After pre-amplification (95 °C for 2 min) the reactions were

amplified for 45 cycles (95°C for 15 s, then 60°C for 10 s, followed by 72°C for 10 s) on a

Lightcycler 480 (Roche) as described (Cao et al., 2010). For Real time PCR reactions a total

volume of 10 µl was made using 1X quantitect SYBER green PCR master mix (Qiagen), 1X

quantitect primer assay (CXCL2 QT00212730, TNF QT0107956, CXCL8 QT00000322) and

20ng of cdna. mRNA expression was standardised against GAPDH (QT00079247)

expression using the standard curve analysis method.

2.10. In Vivo Assays

2.10.1. Experimental animals

Sprague dawley rats weighing 150-300 g, were purchased from experimental animal house of

NIH (National Institute of Health) Islamabad, were used for this study. All these animals

were kept in stainless steel wire cages inside a well aired room and were retained under

standard laboratory conditions of temperature 24-28°C, relative humidity 60-70% and 12

hour light/dark cycle at animal house of Quaid-e-Azam University, Islamabad. Standard

chow diet and drinking water was provided to them. Experimental procedure was performed

according to the rules set by the ethical committee of the university.



2.10.2. Experimental design

210 rats were randomly selected and were divided into six groups. In the first three groups

G1, G2 and G3 seven healthy, leukemic and DMSO treated rats were allocated in each one

respectively. The next three groups G4, G5 and G6 have 63 rats each treated with O.

basilicum extract and fractions, R. officinalis extract and fractions and T. Vulgaris extract and

fractions respectively as depicted below. Later three groups were subdivided into 9 subgroups

each having 7 rats.

Figure 2.1. Flow sheet diagram of rats grouping.

Weights of rats were recorded before and after treatment and rats were physically examined

throughout the experiment. Healthy rats were given distilled water and normal saline

(0.90% w/v) subcutaneously for three consecutive weeks. Leukemia was successfully

induced in rats of group L. Benzene injection for induction of leukemia was prepared in the

following way.

Benzene: 1.5 ml

Isopropanol: 2.0 ml

Distilled water: 1.5 ml

Rats were given benzene (0.2ml) injected with disposable syringe (1 ml) intravenously via

tail on alternative days for three consecutive weeks. Precautionary measures regarding the

use of benzene were taken in to account and untoward contact with benzene was avoided

http://en.wikipedia.org/wiki/Mass_concentration_(chemistry)



during the experiment as benzene is toxic and can cause several serious problems for human

health also.

Rats in G4-G6 were pretreated with crude extract (0.5ml) and different fractions of O.

basilicum, R. officinalis and T. vulgaris, respectively for 2 weeks prior to the administration

of benzene (0.2ml) via gastric gavage. After 2 weeks these pretreated rats were given

intravenous benzene injections (0.2ml) via tail vein along with plants fractions on alternating

days for 3 consecutive weeks. The animals were settled under standard laboratory conditions

of temperature, light/dark cycle and relative humidity as mentioned above. They were fed a

balanced chow diet and water. Behavioural changes of rats were observed i.e change of fur,

increased urination, irregular heartbeat, body weight changes, serum biochemical parameters,

hematological parameters and mortality throughout the experimentation to analyze subacute

toxicity. Benzene treated rats showed low locomotion and weakness after the completion of

the benzene injection treatment.

Table 2.2. Dose regimen scheme for experimental groups of study.

Groups Dose regimen

Healthy (G1) Normal saline

Leukemic group (G2) Benzene 0.2ml

DMSO treated (G3)

Benzene+ plant extract

and fractions group

(G4, G5, G6)

(Subgroups)

= benzene+F1 Pretreated with F1 (0.5ml) and then simultaneous

treatment with benzene (0.2 ml) and F1 (0.5ml)

C2 = benzene+F2 Pretreated with F2 (0.5ml) and then simultaneous




















C11= benzene+CE Pretreated with CE (0.5ml) and then simultaneous


On 30th day of benzene treatment rats were dissected and blood samples were taken via

cardiac puncture using disposable sterile 3-5ml syringes in EDTA tubes. Weights of four

organs (Liver, heart, kidney and spleen) were recorded and were preserved for further

histopathology examination.

Relative organ weight = Absolute organ weight (g)×100/ Body weight of rat on sacrifice day

2.10.3. Histopathology analysis: Histopathological examination of paraffin embedded liver,

heart, kidneys and spleen of all the rats was carried out by taking 5 micron thick section of

each organ. Hematoxylin and Eosin (H& E) stain was used for tissues staining.

Histopathological profile of each group was compared with that of control via representative

images.

Blood smear preparation and Giemsa staining: Leukocytes were differentially counted (200

cells) on a blood film prepared manually and were stained with Wright-Giemsa stain

(MATSUMOTO, 1984).

2.10.4. TNF-α quantification through enzyme linked immunosorbant assay (ELISA)

Blood samples of all rats were centrifuged and stored at -70oC. Rat standard ELISA kit

(Abcame, UK) was used for TNF-α measurement in serum samples. Plates were pre coated



with TNF-α antibody, standard, control and testing samples were added. All other procedures

like washing of plates, addition of antibodies, substrates and stop solution and analysis was

done according to the instructions of manufacturer. Plates were read at 450nm via microplate

reader (BIO-RAD Model 680).

2.11. Characterization of plants fractions

2.11.1 Fourier transform infrared spectroscopy (FTIR)

Fourier Transform Infrared Spectroscopy (FTIR) is one of the most reliable analytical method

for determination of functional groups present in molecules. It gives information about major

class of compounds on the basis of bond stretching and bending frequencies. The peak

position for functional groups is the characteristic of bond that absorb the electromagnetic

radiation in IR range. Hence, by interpreting the infrared absorption spectrum, the chemical

bonds in a molecule can be determined. In the present study, samples from fractions obtained

from column chromatography of crude extracts of each plant materials were analysed by

FTIR. Samples were loaded in FTIR spectrophotometer (Shimadzu, IR Affinity 1, Japan), in

ATR mode with a scan range from 400 to 4000 cm-1 with a resolution of 4 cm-1.

2.11.2. Sample preparation for UV spectroscopy

In order to know the basic structural characteristics of the constituents of each selected

fraction UV spectra was recorded with UV-Visible spectrophotometer (UV-1700 (E) 23

OCE, UV 1700 Pharmaspec, Shimadzu Corp). The measurement was made in the range 200-

400 nm and 200-700 nm for colourless and colour compounds, respectively.

2.11.3. Liquid chromatography-mass spectrometry (LC–MS)

LC-MS analysis of the selected fractions was performed by Ion Trap LC/MS system (serial

No. DE6220680) consisted of a Liquid Chromatograph (1200 Series, Agilent Tech) a vacuum

degasser, autosampler, column 4.6 x 12.5 mm (XDB-C18, Agilent Tech) and a diode array

detector. MS detector with electrospray ion source and time of flight analyzer (Liquid

Chromatograph Mass Spectrometer, Agilent Tech) was used for getting MS data. The

gradient elution was performed using starting mobile phase consisting of 10% CH3CN

organic solvent in 90% double distilled H2O aqueous solvent. During the analysis a

composition was varied as 10% (0 min), 10% (15 min), 40% (40 min), 80% (50 min) and

10% (60 min). The set flow rate was 0.5 ml/min and column temperature was 25oc. The

injection volume was 5µl. The MS spectra were recorded in the range of 100-1000 m/z

whereas the scan time was 0.5 s.



2.12. Statistical analysis

All experiments were carried out in triplicates. Results represent means + standard deviation.

To compare the statistical significance of plant fractions treated parameters with that of

control t test was applied on data. Results showing P≤ 0.05 were considered to have

statistically significant differences from control and are marked with * in figures.

Results


Results

3.1Total phenolics, flavonoids and antioxidant activity O. basilicum has shown highest total phenolics 138.65mg GE/100g DW and total flavonoids

102.45mg CE/100g DW) as depicted in figure 3.1.

Figure 3.1. Total Phenolics and flavonoids of O. basilicum, R. officinalis and T. vulgaris.

3.3 Synergisim of three plants extracts with antibiotics

Combination effects of three selected herbs extract with two antibiotics, ciprofloxacin and

amoxacilin against three strains of bacteria, E.coli, S. Aurius and S.mutans. Plants extracts

lowered the MICs of antibiotics markedly. O. basilicum was found to show best synergistic

effects with antibiotics as compared to R. officinalis and T. vulgaris. as recorded in Tables

3.1-3.3.

Table 3.1. Antibacterial activity results of the combination of O. basilicum plant extract

against three bacterial strains.

Results


Table 3.2. Antibacterial activity results of the combination of T. vulgaris plant extract against

three bacterial strains.

Table 3.3. Antibacterial activity results of the combination of R. officinalis plant extract

against three bacterial strains.

3.4 Effects of selected plants extracts on organs of rats

Weights of liver, heart, kidneys and spleen of plants extracts treated rats were compared with

control and also organ body weight ratio results are recorded in Table 3.4. T. vulgaris caused

Results


a increase in liver body weight ratio while R. officinalis caused a decrease in spleen body

weight ratio at both doses. For other treatment no such differences were observed from

control.

Histomorphological examination of these organs showed no injuries or lesions and did not

confirm any toxicity. Representative images of plants treated organs were comparable with

the control. Results are depicted in figure 3.3.

Table 3.4. Effect of O. basilicum, T. vulgaris and R. officinalis extracts on organ-body weight

ratio of rats.

Mean + SD values carrying different superscripts from the control for each parameter are significantly different (P<0.05).

Results


Figure 3.2. Histomorphological examination of Liver, heart, kiney and spleen

Results


3.5 Effects of selected plants extracts on haematological parameters

An increase in RBCs, PCV, MCV and Hb was observed in rats treated with O. basilicum and T. vulgaris at 1500mg/kg BW. Table 3.5 shows the

results. While no significant differences were recorded in other haematological parameters of rats treated with plants extracts from control.

Table 3.5. Effect of O. basilicum, T. vulgaris and R. officinalis extracts on some hematological parameters of Sprague dawley rats (n=8).

Mean + SD values carrying different superscripts from the control for each parameter are significantly different (P<0.05).

Results


3.6 Fractions yield

The total yield of each fraction obtained after column chromatography of methanolic extract of O.

basilicum, R. officinalis and T. vulgaris was also measured. Varying amount was obtained for each

fraction from each plants extract. The Table 3.6 represents the total yield for each fraction.

Table 3.6. Total yield of fraction of O. basilicum, R. officinalis and T. vulgaris.

S.No % Composition Total yield (g)

O.basilicum

Total yield (g)

R.officinalis

Total yield (g)

T. vulgaris

1 10% n-hexane,

90% ethyl acetate

0.82 4.10 1.00

2 20% n-hexane,

80% ethyl acetate

0.32 0.55 0.46

3 30% n-hexane,

70% ethyl acetate

0.38 0.38 0.23

4 40% n-hexane,

60% ethyl acetate

1.00 0.20 0.39

5 50% n-hexane,

50% ethyl acetate

0.80 0.37 0.30

6 60% n-hexane,

40% ethyl acetate

0.40 0.22 0.24

7 70% n-hexane,

30% ethyl acetate

0.90 0.62 0.32

8 80% n-hexane,

20% ethyl acetate

0.50 0.44 0.32

9 90% n-hexane,

10% ethyl acetate

0.70 0.19 0.39

3.7 Results of in vitro assays

3.7.1 O. basilicum, R. officinalis and T. vulgaris fractions and crude extracts

inhibited leukemic cells proliferation

To delineate the fractions these three medicinal herbs obtained through column

chromatography for further cellular and molecular activities, eight fractions and crude extract

of O. basilicum were first tested for inhibition of cell proliferation of four leukemic cell lines

(U937, OCI-AML3, MV4-11 and THP-1) via Cell titre GLO assay. DMSO was used as a

Results


control. Plant fractions inhibited cell proliferation in a dose dependent manner. IC50 was

calculated for all the fractions, crude extract and DMSO. Interestingly all the fractions and

crude extract have shown good anti-proliferative activity on all the four cell lines but the

most potent with lowest IC50 values among these were found BF2 ( IC50: 11.68, 12.84, 1.17

and 10.11) and BF3 (IC50: 11.95, 15.4, 12.21 and 10.55) of O. basilicum against U937, OCI-

aml3, MV4-11 and thp1s respectively. Similarly among the fractions of R. officinalis RF1

(IC50:9.95, 10.72, 10.7 and 10.8) and RF2 (IC50:10.008, 10.19, 10.65 and 10.88) have been

found with very good cytotoxic effects. In the same way three fractions of T. vulgaris TF2

(IC50:11.04, 10.8, 13.63 and 11.4), TF7 (IC50:22.39, 22.16, 17.85 and 17.89 ) and TF8

(IC50:18.44, 17.99, 15.35 and 19.95) have been found the most potent ones. Results have

been shown in fig 3.4, 3.9 and 3.14.

On the basis of these results these seven fractions of O. basilicum, R. officinalis and T.

vulgaris were selected for further experiments.

3.7.2 O. basilicum, R. officinalis and T. vulgaris fractions induced apoptosis

of Leukemic cell lines

To explore the apoptotic effects of the BF2, BF3, RF1, RF2, TF2, TF7 and TF8 annexinv/ PI

assay was used. Results indicated that cytotoxic activity of these fractions was associated

with apoptotic mechanisms. Apoptosis was measured at three different time points (24, 4 and

2 hrs) after the treatment as depicted in fig 3.5, 3.10 and 3.15. The apoptotic potential of the

fractions was found to be optimal after 24hrs treatment. Interestingly it was observed that the

plant fractions have a great potential of apoptosis even at 4 hrs treatment. At 2 hrs treatment

they have a shown less potency as compared to 4 and 24hrs treatments.

3.7.3 O. basilicum, R. officinalis and T. vulgarisfractions induce apoptosis

via activation of JNK and cleavage of caspase 3

Alteration in the level of JNK and cleavage of caspase 3 were studied by western blotting to

explore the possible signalling pathway involved in the induction of apoptosis induced by

selected seven fractions as shown in Fig 3.6, 3.11 and 3.16. Procaspase 3 was found in

cleaved form followed by treatment with fractions of O. basilicum BF2 and BF3, R.

officinalis RF1 and RF2 and T. vulgaris TF2, TF7 and TF8 as compared to DMSO treated

ones. Protein levels were shown relative to DMSO control ones that are down regulated after

treatment with BF2 and BF3 confirming the cleavage of caspase 3. Expression levels of JNK

were recorded at 0, 15 and 60 min treatment of four types of cell lines with stimuli. Proteins

Results


were quantified using densitometer. Time course study showed an increase in JNK

expression after treatment of cells with BF2, BF3, RF1, RF2, TF2, TF7 and TF8 for 15

minutes and the expression was reached to peak level when observed after 60 minutes

treatment. Results have been depicted in figure 3.7, 3.12 and 3.17.

3.7.4. Induction of caspase dependent apoptosis in AML derived cells by O.

basilicum, R. officinalis and T. vulgaris fractions

Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) a general caspase inhibitor was used in the

study to further reveal the role of caspase in programmed cell death of leukemic cells. U937

and OCI-AML-3 were treated with BF2, BF3, RF1, RF2, TF2, TF7 and TF8 alone and also in

combination with zvad-fmk. Results of annexin V/PI assay have depicted that caspase

inhibitor causes significant reduction in number of dead cells as depicted in figure 3.8, 3.13

and 3.18. This results leads us to the suggestion that apoptosis induced by these fractions was

suppressed by caspase inhibitor. The result implies that selected fractions induced apoptosis

is at least caspase dependent.

3.7.5. Alterations in the level of TNF-α, CXCL2 and CXCL8 induced by O.

basilicum, R. officinalis and T. vulgaris fractions in u937 cell line

U937 cells were selected for analysis of cytokines as plant fractions showed good results in

these cells as compared to other cell lines. Cells were treated with BF2, BF3, RF1, RF2, TF2,

TF7 and TF8 for 1 hour in order to explore the plant fraction-stimulated cytokines

production. DMSO and TNF were used as negative and positive controls, respectively.

Significant increase in the level of TNF α and decrease in CXCL8 was observed in BF3

treated cells as depicted in Table 3.7, 3.8 and 3.9. Values were expressed as relative units.

Results


Figure 3.3. Cytotoxic effects of O. basilicum crude extract and fraction in terms IC50 against

leukemic cell lines. U937, OCI-Aml3, MV4II and Thp1 cells were treated with DMSO, O.

basilicum fraction 1-8 (represented as BF1-8) and crude extract of O. basilicum (BCE) for 24

hours and IC50 values were recorded via cell titre GLO assay.

A

Results


B

Results


C

Results


Figure 3.4. BF2 and BF3 induces apoptosis in human leukemic cells.Whereas, (A)Bar chart is

showing collectively percentage of dead cells. That were either necrotic or apoptotic. Data

were shown as mean+ SD of three independent experiments (*P≤0.05). (annexin V-PI+)

cells. (B, C and D) Flow cytometric analysis of U937, OCI-Aml3, MV4II and Thp1 cells

treated with 20µg/ml of BF2 and BF3 fractions of O. basilicum for 24 hours. 4 hours and 2

hours. Representative figures showing population of viable (annexin V-PI-), early apopotic

(annexin V+PI-), Late apoptotic (annexinv+PI+) and necrotic (annexin V-PI+).

D

Results


Figure 3.5. Effects of O. basilicum fractions on caspase 3. U937, OCI-Aml3, MV4II and

Thp1 were treated with BF2 and BF3 fractions of O. basilicum and DMSO as a control for 24

hours and cleavage of caspase was observed.

Results


Figure 3.6. Effects of O. basilicum fractions on activation of JNK in leukemic cells.

Effects of O. basilicum fractions on activation of p-JNK in leukemic cells U937, OCI-Aml3,

MV4II and Thp1 cells were treated with BF2 and BF3 fractions of O. basilicum for 15

minutes and then for 60 minutes. The p-JNK intensities were normalised to GAPDH and the

results expressed as percentages of the corresponding values in the cells treated for 0 min

(0 m). Values are means ± SEM; n = 3. (A and B represents western blot results of BF2

treated cells whereas C and D shows BF3 treated cells).

Results


Figure 3.7. Observation of apoptosis in leukemic cells via treatment with O. basilicum

fractions alone and in combination with zvad-fmk.

Table 3.7. Effects of O. basilicum fractions on the level of TNF-α, CXCL2 and CXCL8 in

U937 cell line. Values with * have significant differences from the standardized values

(*P≤0.05).

Tnfα

CXCL2

CXCL8

DMSO 1+0.36 1+0.83 1+1.38

TNF 22.25**+6.8 11.43+8.4 273.92**+135.33

BF2 3.38*+0.65 0.99+0.37 0.61*+2.99

BF3 3.89*+1.18 0.97+0.36 0.54*+14.46

Results


R. Officinalis

Figure 3.8. Cytotoxic effects of R. officinalis crude extract and fraction in terms IC50 against

leukemic cell lines. U937, OCI-Aml3, MV4II and Thp1 cells were treated with DMSO, R.

officinalis fraction 1-8 (represented as RF1-8) and crude extract of R. officinalis (RCE) for 24


A

Results


B

Results


C

Results


Figure 3.9. RF1 and RF2 induces apoptosis in human leukemic cells.Whereas, (A) Bar chart

is showing collectively percentage of dead cells. That were either necrotic or apoptotic. Data

were shown as mean+ SD of three independent experiments (*P≤0.05). (annexin V-PI+)

cells. (B, C and D)Flow cytometric analysis of U937, OCI-Aml3, MV4II and Thp1 cells

treated with 20µg/ml of RF1 and RF2 fractions of R. officinalis for 24 hours. 4 hours and 2

hours. Representative figures showing population of viable (annexin V-PI-), early apoptotic


D

Results


Figure 3.10. Effects of R. officinalis fractions on caspase 3. U937, OCI-Aml3, MV4II and

Thp1 were treated with RF1 and RF2 fractions of R. officinalis and DMSO as a control for 24


Results


Figure 3.11. Effects of R. officinalis fractions on activation of JNK in leukemic cells.Effects

of R. Officinalis fractions on activation of p-JNK in leukemic cells U937, OCI-Aml3, MV4II

and Thp1 cells were treated with RF1 and RF2 fractions of R. Officinalis for 15 minutes and

then for 60 minutes. The p-JNK intensities were normalised to GAPDH and the results

expressed as percentages of the corresponding values in the cells treated for 0 min (0 m).

Values are means ± SEM; n = 3. (A and B represents western blot results of RF1 treated cells

whereas C and D shows RF2 treated cells).

Results


Figure 3.12. Observation of apoptosis in leukemic cells via treatment with R. officinalis


Table 3.8. Effects ofR. officinalis fractions on the level of TNF-α, CXCL2 and CXCL8 in


(*P≤0.05).

Tnfα

CXCL2

CXCL8

DMSO 1+0.36 1+0.83 1+1.38

TNF 22.25**+6.8 11.43+8.4 273.92**+135.33

RF1

3.01*+1.12

0.77+0.65

0.43*+0.33

RF2

4.39*+1.34

0.91+0.29

0.51*+0.43

Results


T. vulgaris

Figure 3.13. Cytotoxic effects of T. vulgaris crude extract and fractions in terms IC50 against

leukemic cell lines. U937, OCI-Aml3, MV4II and Thp1 cells were treated with DMSO, T.

vulgaris fraction 1-8 (represented as TF1-8) and crude extract of T. vulgaris (TCE) for 24


A

Results


B

Results


C

Results


Figure 3.14. TF2, TF7 and TF8 induce apoptosis in human leukemic cells. Whereas, (A)Bar

chart is showing collectively percentage of dead cells. That were either necrotic or apoptotic.

Data were shown as mean+ SD of three independent experiments (*P≤0.05). (annexin V-PI+)

cells. (B, C and D)Flow cytometric analysis of U937, OCI-Aml3, MV4II and Thp1 cells

treated with 20µg/ml of TF2, TF7 and TF8 fractions of T. vulgaris for 24 hours, 4 hours and

2 hours. Representative figures showing population of viable (annexin V-PI-), early apopotic


D

Results


Figure 3.15. Effects of T. vulgaris fractions on caspase 3. U937, OCI-Aml3, MV4II and Thp1

were treated with TF2, TF7 and TF8 fractions of T. vulgaris and DMSO as a control for 24


Results


Figure 3.16. Effects of T. vulgaris fractions on activation of JNK in leukemic cells.

Effects of T. vulgaris fractions on activation of p-JNK in leukemic cells U937, OCI-Aml3,

MV4II and Thp1 cells were treated with TF2, TF7 and TF8 fractions of T. vulgaris for 15

minutes and then for 60 minutes. The p-JNK intensities were normalised to GAPDH and the

results expressed as percentages of the corresponding values in the cells treated for 0 min

(0 m). Values are means ± SEM; n = 3. (A and B represents western blot results of TF2

treated cells, C and D shows TF7 treated cells. Whereas E and F represents TF8 treated cells).

Results


Figure 3.17. Observation of apoptosis in leukemic cells via treatment with T. vulgaris


Table 3.9. Effects of R. officinalis fractions on the level of TNF-α, CXCL2 and CXCL8 in


(*P≤0.05).

Tnfα

CXCL2

CXCL8

DMSO 1+0.36 1+0.83 1+1.38

TNF 22.25**+6.8 11.43+8.4 273.92**+135.33

TF2

4.61*+0.33

0.8+0.4

0.35*+1.12

TF7

6.05*+0.45

0.91+0.09

0.53*+0.55

TF8 3.62*+0.2

0.87+0.28

0.75+0.7

Results


3.8 Results of in vivo Assays

3.8.1 Effects of plant (O. basilicum, R. officinalis and T. vulgaris) crude

extract and fractions on organs of leukemic rats

Weight of four organs (liver, heart, kidneys and spleen) of benzene induced leukemic rats,

treated with plants extracts was measured. Healthy rats, benzene induced leukemic rats and

DMSO treated rats were used as control. Weight of liver and spleen were significantly

increased in leukemic rats from that of healthy and DMSO treated. In rats treated with O.

basilicum, no significant differences in weights of four organs were recorded in rats treated

with crude extract, BF1, BF2 and BF3 while in rats treated with other fractions weight of

liver and spleen increased significantly. In rats treated with R. Officinalis, no significant

differences were found in weights of the four mentioned organs of rats treated with RF1 and

RF2 while, liver and spleen weights were increased significantly in rats treated with other

fractions. Similarly in T. vulgaris treated rats no significant differences were found in

weights of four organs treated with fractions TF2, TF7 and TF8 while, spleen and liver

weights were significantly increased in rats treated with other fractions. Results have been

depicted in Fig 3.19-3.21.

Histopathological analysis: Histomorphological examination of liver, heart, kidneys and

spleen showed that liver and spleen morphology was changed in leukemic rats and large

lesions were seen in liver and spleen tissues but these changes were prevented in leukemic

rats treated with fractions BF2, BF3, RF1, RF2, TF2, TF7 and TF8 as shown in Fig 3.22 and

3,23. In heart and kidneys, no significant changes were observed.

Results


Figure 3.18. Effect of administration of methanolic extracts and fracions of O. basilicum, on

organ body weight ratio of liver, heart, kidneys and spleen of Sprague Dawley rats where H

represents healthy, L, leukemic DMSO, DMSO treated, BCE, crude extract of O. basilicum

and BF (1-8), different fractions of O. basilicum. The effects of each fraction of O. basilicum,

its crude extract and DMSO were analysed on four organs-body weight ratio and the results

were compared statistically with standards (n=7).

.

Figure 3.19. Effect of administration of methanolic extracts and fracions of R. officinalis, on


represents healthy, L, leukemic DMSO, DMSO treated, RCE, crude extract of R. officinalis

and RF (1-8), different fractions of R. officinalis. The effects of each fraction of R. officinalis,

its crude extract and DMSO were analysed on four organs-body weight ratio and the results


Results


Figure 3.20. Effect of administration of methanolic extracts and fracions of T. vulgaris, on


represents healthy, L, leukemic DMSO, DMSO treated, TCE, crude extract of T. vulgaris and

TF (1-8), different fractions of T. vulgaris. The effects of each fraction of T. vulgaris, its

crude extract and DMSO were analysed on four organs-body weight ratio and the results


Figure 3.21. Histological examination of liver (C: control, L: leukemia, B2: 2:8 and B3: 3:7

fraction of O. basilicum, R1: 1:9 and R2: 2:8 fraction of R. officinalis, T2: 2:8, T7, 7:3 and

Results


T8: 8:2 fraction of T. vulgaris).Significant morphological changes in liver tissue and lesions

have been observed in leukemic rats as compared to control and plants fractions treated ones.

Figure 3.22. Histological examination of spleen (C: control, L: leukemia, B2: 2:8 and B3: 3:7

fraction of O. basilicum, R1: 1:9 and R2: 2:8 fraction of R. officinalis, T2: 2:8, T7, 7:3 and

T8: 8:2 fraction of T. vulgaris) Significant morphological changes in spleen tissue and lesions

have been observed in leukemic rats as compared to control and plants fractions treated ones.

3.8.2 Hematological Assays

Differential leukocytes count:

Percentage of lymphocytes, monocytes, neutrophils, basophils and eosinophils was measured

in benzene induced leukemic, healthy, DMSO treated, crude extracts treated and fractions of

O. basilicum, R. officinalis and T. vulgaris treated rats. Results were comparable with

previous experiments. In O. basilicum treated rats, crude extract and BF1, BF2 and BF3

showed good potential of preventing leukemia showing no significant differences in %age of

cells from those of healthy rats. Same results were recorded for crude extract, RF1, and RF2

in R. officinalis treated rats and crude extract, TF2, TF7 and TF8 in T. vulgaris treated rats.

Results are shown in Fig 3.24-3.27 where representative images show difference in cell count

and morphology in blood and bone marrow of control and leukemic rats.

Results


Figure 3.23. Blood smear examination (A) Blood smear of Control group (B) Blood smear of

leukemic Rat showing reduced RBCs, leukocytosis (significantly increased number of

neutrophils and monocytes) (C) Blood smear view of benzene +plant fraction treated group

Increased RBCs and lymphocytes count and regulated neutrophils and basophils and normal

morphology.

A

B

C

Results


Figure 3.24. Effect of administration of methanolic extracts and fracions of O. basilicum, on

haematological parameters of Sprague Dawley rats where H represents healthy, L, leukemic

DMSO, DMSO treated, BCE, crude extract of O. basilicum and BF (1-8), different fractions

of O. basilicum.(n=7).

Figure 3.25. Effect of administration of methanolic extracts and fracions of R. officinalis, on


DMSO, DMSO treated, RCE, crude extract of R. officinalis and RF (1-8), different fractions

of R. officinalis (n=7).

Results


Figure 3.26. Effect of administration of methanolic extracts and fracions of T. vulgaris, on


DMSO, DMSO treated, TCE, crude extract of T. vulgaris and TF (1-8), different fractions of

T. vulgaris (n=7).

3.8.3. Cyokines

Alterations in the level of TNF-α

TNF alpha concentration was measured inbenzene induced leukemic, healthy, DMSO treated

and crude extracts and fractions of O. basilicum, R. officinalis and T. vulgaristreated rats

through ELISA. DMSO and TNF were used as negative and positive controls respectively.

Results are presented in Table 3.10.Significant increase was found in the level of TNF-α in

serum samples of rats treated with fractions BF2, BF3, RF1, RF2, TF2, TF7 and TF8.

Highest TNF-α concentration (199.85±4.6µg/ml) was recorded in serum samples of rats

treated with basil extract fraction BF3 followed by serum samples treated with RF1 fraction

of rosemary (199.71±4.57µg/ml). Three fractions of basil and thyme while, two fractions of

rosemary plant extracts were identified for which serum TNF-α concentration up surged from

190 µg/ml depicting up regulation of TNF- α gene.

Results


Table 3.10. Effect of administration of methanolic extracts and fractions of O. basilicum, T.

vulgaris and R. officinalis on TNF α level of Sprague Dawley rats (n=7).

S.No Fractions O. basilicum R. officinalis T. vulgaris

1 Healthy 181.00+6.23 - -

2 Leukemic 178.71+9.83 - -

3 Crude Extract 181.00+4.82 167.42+3.03 177.28+6.32

4 DMSO 170.14+6.85 175.71+8.99 176.71+5.80

5 F1 196.28+2.05* 199.71+4.57* 175.00+4.00

6 F2 198.71+1.77* 199.42+4.68* 196.71+6.41*

7 F3 199.85+4.6* 182.28+6.85 171.85+3.57

8 F4 176.28+5.15 168.57+3.35 181.00+4.22

9 F5 173.85+6.96 174.71+5.43 171.00+8.38

10 F6 170.71+7.68 176.71+35.16 171.14+7.11

11 F7 181.00+6.67 156.85+ 8.26 196.14+9.01*

12 F8 175.28+6.19 180.00+62.77 193.57+5.28*

Results


3.9 Characterization of Plant Fractions

3.9.1 FTIR spectroscopy

Promising fractions of basil, rosemary and thyme plant extracts which caused an upsurge in

TNF-α concentration i.e., BF2, BF3, RF1, RF2, TF2, TF7 and TF8) were subjected to FTIR

analysis and results are presented in Fig 7. In the FTIR spectra of fractions BF2 and BF3

given as Fig 6A and 6B respectively, the absorption bands in the range of 3300-3400 cm-1

correspond to the stretching vibration of O-H due to the presence of hydroxyl group of

organic compounds. Peaks at 3000-3100 cm-1 are in the range for C-H stretching in aromatic

compounds. The peaks in the range of 2720-2920 cm-1 appeared due to the stretching

vibrations of sp3 C–H bonds and peaks at 1610 cm-1 representing C=C stretching. Bands at

1719.28 and 1721.28 cm-1 are due to the C=O (carbonyl) stretch. Fig 6A and 6B show that

halides were also present in the extracted fractions. In case of BF2 the peaks at 1237.84 ,

1039.28 and 727.84 cm−1 while in case of B3 peaks at 1280.01, 1124.28 and 742.12 cm−1 are

representing the presence of C-X whereas the first two peaks in both the cases are due to C-F

and the third peak is due to C-Cl, respectively.

3.9.2 UV Spectroscopy and LC-MS Study

The UV range and LC-MS results of the selected fractions BF2 and BF3 of methanolic

extract of O. basilicum are given in Table 3.10. The UV spectroscopy study indicated that the

major compounds present in fractions showed maximum absorption in the range of 230-330

nm. The interpretation of LC-MS is made on the basis of comparing the LC-MS spectra with

that of MS data bank as well as by following fragmentation pattern method. From the

obtained results it was found that the major compounds found in BF2 were epicatechin

derivatives, caffiec acid-3-glucoside dimers, ester derivatives of D-Alanine and ester

derivatives of Cinnamic acid. In case of BF3 the derivatives of Kaempferol (flavonoid),

Carboxylic acid derivatives, ester derivative of Succinic acid and ester derivatives of

Cinnamic acid. Among the two selected fractions, the nature of the compounds was similar to

a greater extent; however the BF3 contains few additional compounds such as flavonoids and

additional ester derivatives. These results are also in agreement with FTIR results of the

respective fractions in which the main functional groups are correlated to the compounds

identified by using LC-MS.

Results


The results of selected fractions of R. officinalis and T. vulgaris were interpreted on the basis

of retention time and UV-vis data. It was found that these two fractions contained almost the

same compounds as given and discussed in case fractions BF2 and BF3 of methanolic extract

of O. basilicum.

Table 3.11. LC-MS results of the selected fractions BF2 and BF3 of O. basilicum.

Fraction Compounds Rtime

(min)

MS (m/z) Other

fragment

ions

UV

maxima

(nm)

BF2 (-) Epicatechin derivative 7.8 922.1 707.3, 365.6,

187.6, 129.1

240, 320

Caffiec acid-3-glucoside

(Dimer)

9.0 683.1 341.5 242, 330

D-Alanine, N- (2,5-

ditrifluoromethylbenzoyl),

nonadecyl ester

27.6 545.0 507.2, 425.9 280, 330

D-Alanine, N- (2,5-

ditrifluoromethylbenzoyl),

hexadecyl ester

29.7 553.2 187.8 265, 330

Trans-3-trifluoromethyl

cinamic acid, 3-4-

dichlorophenyl ester

(Dimer)

32.5 719.4 360 230, 322


cinamic acid, 3-4-


(Dimer) derivative

35.3 756.7 361.4, 856.7 280, 322

BF3 Kaempferol-3-o-alpha-L-

rhamopyranosyl-(1-6)-beta-

D-galactopyranosyl]7-o-

alpha-L-rhamanopyranoside

(flavonoid)

3.6 945.1 470.5, 407.5 262

(1b, 3a, 5b)-1,3-

Dihydroxycholan-24-oic

acid

7.2 956.0 391.1, 203.8 260, 290,

330

Results


Succinic acid, 3,5-

dichlorophenyl hexadecyl

ester

27.7 956.2 486.5, 263.0 230, 280


cinamic acid, 3-4-


(Dimer)

32.5 719.4 360 220, 320


cinamic acid, 3-4-


derivative

35.3 756.7 361.4, 856.7 270, 325

Results


Results


Results


Figure 3.27. MS spectra of different fractions of methanolic extracts.

Results


Results


Figure 3.28. LC-DAD chromatograms of different fractions of methanolic extracts.

Results


BF3

Wave number (cm-1)

1000200030004000

%T

20

40

60

80

Figure 3.29. FTIR spectrum of BF3 fraction of methanolic extract of O. basilicum.

Table 3.12. Interpretation of characteristic peaks appeared in FTIR spectrum of BF3 fraction

of methanolic extract of O. basilicum.

Results


Wave number (cm-1)

1000200030004000

%T

75

80

85

90

95

100

BF2

Figure 3.30. FTIR spectrum of BF2 fraction of methanolic extract of O. basilicum.

Table 3.13. Interpretation of characteristic peaks appeared in FTIR spectrum of BF2 fraction

of methanolic extract of O. basilicum.

Discussion


Discussion

Cancer is one of the most complex fatal disease characterized by uncontrolled cell division of

abnormal cells that may form solid tumors or metastasize to other body parts (Pecorino,

2012) and considered the second most leading cause of mortality mainly caused due to

carcinogens present in environment. Leukemia is a malignant disorder of blood forming

tissues, begins in bone marrow and marked by distorted growth and high number of white

blood cells and their precursors in the blood and bone marrow (Pokharel, 2012). Recently,

accumulating researches have shown that cancer chemotherapy because of drug resistance

phenomenon and other treatment procedures having side effects and are not very much

effective. So, researchers are interested in natural products of plant origin to treat cancer. The

present study was first time carried out to fulfil the aim of discovering new possible

mechanism by which methanolic crude extract (CE) of Ocimum basilicum (O. basilicum),

Rosemarinus officinalis (R. officinalis) and Thymus vulgaris (T. vulgaris) and their different

fractions play anti-leukemic role. Previous studies on in vitro anti-leukemic and in vivo

immunomodulatory effects of these plants were the scientific base of this study. Medicinal

plants contribute a plentiful number of anticancer drugs which are diverse in variation and

mode of action. It has been proposed by extensive number of reports that many substances

which occur naturally, show cancer chemotherapeutic effects, e.g., secondary metabolites

isolated from plants like alkaloids and polyphenols induce apoptosis via direct or indirect

modification of expression of genes e.g., bcl2, p53 and caspase-3. Owing to this property of

medicinal plants, the present study was conducted to access the chemopreventive and anti-

leukemic activity of crude extract and fractions of these plants eluted via column

chromatography.

To assure that these medicinal herbs are safe to be used and having no disturbing effects on

body themselves, a toxicity study was carried out. Generally total phenolics and flavonoids

have been measured. The results of this study showed highest amounts of total phenols and

flavonoids in O. basilicum.

O. basilicum extract lowered the MIC of antibiotics markedly as compared to other two

extracts followed by R. officinalis extract. This high antibacterial potential can be attributed

to presence of phytoconstituents in basil extract.

Medicinal plants have been reported to be providing promising tharapeutic against bacterial,

fungal and viral diseases. fungal and viral diseases (Saad et al., 2006). Certain phenolic and

aromatic compounds have been recorded in various plants that are best for the inhibition of

bacterial growth. (Holley and Patel, 2005). Ilhan and co-workers have reported the

Discussion


antibacterial potential of O. basilicum against E. Coli and S. Aurius and found a complete

damage of the cells under scanning electron microscope. Cell wall lysis of these bacteria was

comparable to antibiotics. In some other studies also the antibacterial potential of O.

basilicum has been reported such as candida albicans, Pseudomonas aeruginosa, E. Coli,

Salmonella paratyphi and Shigella dysenterea (Kaya et al., 2008). This high antibacterial

potential can be attributed to the camphor, eugenol and linalool. (Joshi, 2014). Similarly

rosemary has also been recorded as a good antibacterial and antioxidant agent. This

antibacterial activity can be attributed to the presence of camphor, α-pinene and cineole while

antioxidant activity to carnosol and carnosic acid. (Pintore et al., 2002). The antibacterial

activity of thyme recorded can be attributed to the presence of thymol and carvacrol. They

have shown to cause strong disintegration of the outer membrane that causes increased ATP

permeability from cytoplasmic membrane. (Nazzaro et al., 2013).

To explore the hypertrophic or atrophic effects of these plants extracts organ body weight

ratio was determined for liver, heart, kidneys and spleen. (Ashafa et al., 2012). Except the

atrophic effects of R. officinalis for spleen and hypertrophic effects of T. vulgaris on liver no

such significant differences were found in organ body weight ratio of plants treated ones from

control. But our histomorphological data is not supporting this atrophy of R. officinalis and

hypertrophy of liver by T. vulgaris because no injuries or lesions have been found in the

representative images obtained through microscope.

Haematological profiling always ansures the toxicity of the stimulant applied (Olson et al.,

2000). In this study extracts of three selected medicinal herbs were applied on rats so to

explore their toxicity these were applied on rats and than blood samples were examined for

various parameters. No significant differences were found in blood parameters of plants

treated rats as compared to control except O. basilicum and T. vulgaris have significantly

increases RBCs, PCV, Hb and MCV when applied at a dose of 1500mg/kg BW. As previous

studies showed that T. vulgaris has high iron content that may stimulate Hb synthesis. Certain

phenolics and flavonoids of this plant have also been shown to improve the haematological

picture. (Kaviarasan et al., 2004). The result of O. basilicum is in accordance with another

study carried out on O. Gratissmum where also an increase in RBCs count, PCV and Hb was

recorded. Normally erythropoietin production increases with anoxia which may cause

increase RBCs production. (Ofem et al., 2012). So it can also be estimated that constituents

of O. basilicum acts like erythropoietin that may lead to increased RBCs production. So it

can be concluded from this safety study that these plants are good sources of phenolics and

flavonoides due to which they have strong antioxidant and antibacterial activity and

Discussion


interstingly thay are safe upto a dose of 1500mg/kgBW and can be used for the treatment of

various ailments.

Medicinal plants contribute a plentiful number of anticancer drugs which are diverse in

variation and mode of action. It has been proposed by extensive number of reports that many

substances which occur naturally, show cancer chemotherapeutic effects, e.g, secondary

metabolites isolated from plants like alkaloids and polyphenols induce apoptosis via direct or

indirect modification of expression of genes e.g., bcl2, p53 and caspase-3. Owing to this

property of medicinal plants, this study was conducted to access the chemopreventive and

anti-leukemic activity of O. basillum, R. officinalis and T. vulgaris crude extract and its

fractions eluted via column chromatography.

To replace the anticancer drugs having toxic effects with natural products having minimum

toxicity is a major goal of scientists nowadays.47.1% of the clinically approved anticancer

drugs have been reported to be derived from plants (Qamar et al., 2010). This prompted us to

evaluate the application of three important herbs of family lamiaceae, O. basilicum, R.

officinalis, and R. vulgaris on human leukemic cell lines, having several important medicinal

values against other diseases.Our study is in agreement with earlier findings about the

essential oil of O. basilicum on murine leukemic cells (IC50: 36.2g/ml)(Manosroi et al.,

2006), its chemopreventive effects in carcinogen induced papilloma-genesis in mice

(Dasgupta et al., 2004) and growth inhibitory effects of O. basilicum extract and its fractions

on breast cancer cell line MCF-7 (Qamar et al., 2010).Our results suggest that two fractions

of O. basilicum have a very strong cytotoxicity on all the four cancer cell lines U937, OCI-

AML3, MV4II and THP I with IC50 values of (BF2 IC50: 11.68, 12.84, 1.17 and 10.11 and

BF3 IC50: 11.95, 15.4, 12.21 and 10.55) respectively as compared to other fractions and crude

plant extract. The growth inhibition of all the four types of AML derived leukemic cell lines

by O. basilicum highlights its broad spectrum cytotoxic effects on cancer cell lines. Ursolic

acid a major constituent of basil was reported to be involved in anti proliferative activities via

effects on cell cycle, apoptosis and differentiation (Qamar et al., 2010).

Similarly R. officinalis a common ornamental plant with its blue flowers has also been

reported for its cytotoxic and anticancer properties in several studies (Karimi et al., 2017). Its

constituents have strong anticancer and therapeutic effects in cancer cell lines (Horváthová et

al., 2010). The most important compounds reported in rosemary extract are rosemarinic acid

and cafeic acid which contribute to strong antioxidant potential of rosemary. The rosmarinic

acid, phenolic compound, obtains one of its phenolic rings from phenylalanine Literature indicates

Discussion


that intake of antioxidants can help to protect body against several threatenining diseases like

cancer (Karimi et al., 2017). In accordance with these studies our study also has shown strong

anticancereous effects on above mentioned four types of cell lines. Crude extract and 8

fractions of rosemary were tested for their cytotoxic effects and among these two fractions R1

(IC50:9.95, 10.72, 10.7 and 10.8) and R2 (IC50:10.008, 10.19, 10.65 and 10.88) have been

found with very good cytotoxic effects.

Likewise T. vulgaris, the most popular medicinal plant has also been reported for its various

pharmacological actions since thousands of years. It has been reported for its broad spectrum

antibacterial, antifungal and antioxidant activities (Thuille et al., 2003). Maintenance of

superoxide dismutase, glutathione peroxidas activities and total antioxidant status due to

thyme oil supplementation has also been reported (Tsai et al., 2007). Involvement of thyme

extract in DNA repair has also been reported. Thyme extract was shown to be involved in

leukopoiesis and increasing thrombocyte count in blood (Ait M'Barek et al., 2007). Our

results are in agreement wih the previous studies recording pharmacological actions of

thyme. This study recorded a great cytotoxic potential of thymus vulgaris crude extract and

different fractions obtained as mentioned above. Three of these fractions TF2 (IC50:11.04,

10.8, 13.63 and 11.4), TF7 (IC50:22.39, 22.16, 17.85 and17.89) and TF8 (IC50:18.44, 17.99,

15.35 and 19.95) have been found the most potent ones.

A highly structured process of programmed cell death or apoptosis is involved in removal of

damaged, unwanted and aged cells to maintain tissue homeostasis (Schafer and Kornbluth,

2006). In eukaryotic cells this homeostasis is maintained by a delicate balance between

survival and death signals. Cancer cells fail to respond to damage DNA using this apoptotic

machinery. Developments of therapies that can reinstall this machinery can be a promising

strategy for cancer control. Most of cancer drugs use neoplastic cells as target. A large

number of natural products have also been explored for the prevention and treatment of

cancer via induction of apoptosis (Mukhtar et al., 2012). One of the technique used to study

apoptosis involves binding of apoptotic cells to annexin V (Bhatia et al., 2015). Our results

from the experiment to assess apoptosis using annexin V/PI assay have indicated that BF2

and BF3 from O. basilicum, RF1 and RF2 from R. officinalis and TF2, TF7 and TF8 from T.

vulgaris have significantly committed the cells to apoptosis as early as at 2 hours treatment.

A time dependent apoptosis was observed in all the four treated cell lines. Various factors

may be involved in induction of apoptosis of cells by these fractions ofbasil, rosemary and

thyme. These results suggest that anti-cancerous effects of basil, rosemary and thyme

fractions may be promoted by apoptosis.

Discussion


A family of cysteine proteases known as caspases are divided into two classes. One is

composed of initiator caspases (caspase 8 and 9) and the other is executioner caspases

(caspase 3 and 7). Mainly two signaling pathways are involved in the process of apoptosis

induction. One is extrinsic that is using death receptors and the other one is using

mitochondria. Extrinsic pathway is reported to be involved in the activation of caspase 8 that

either leads to leakage of cyochrome c from mitochondria that is involved in the activation of

caspase 9 or direct activation of caspase 3(Looi et al., 2013). Activation of both of the

initiator caspases in turn leads to the activation of executioner caspases that trigger apoptosis

(Woo et al., 2011). Initiator caspases (8 and 9) activate effector caspases (3 and 7) either

directly or indirectly. These effector caspases are than involved in cleavage of poly (ADP-

ribose) polymerase (PARP) that is responsible for significant morphological changes of

apoptosis. Despite the fact that caspases may play a major role in triggering programmed cell

death, it is not the sole factor for execution of apoptosis. In some studies caspase independent

apoptosis is reported in jurkit cells, Hela cells and MCF-7(Chen and Wong, 2009).

Considering these results it was important to determine that whether these 7 fractions of three

plants follow a caspase dependent or independent pathway to induce apoptosis. Our western

blot analysis showed that caspase 3 was cleaved after treatment with plant fractions that

suggest caspase dependent pathway followed by these fractions. We further confirmed our

results by using caspase inhibitor zvad-fmk to know that whether these fractions can still

induce apoptosis in these leukemic cell lines in the presence of this inhibitor. The general

irreversible caspase inhibitor z-VAD-fmk contains aspartate and fmk group. Catalytic site of

caspases is irreversibly bound by z-VAD-fmk and mimick the cleavage site of caspase

(Wong et al., 2013). The results from this study have provided evidence that these 7

fractions followed a caspase dependent pathway for the induction of apoptosis because z-

VAD-fmk treatment suppressed the overall apoptosis triggered by these fractions. It is thus

suggested from this study that apoptosis induced by thesefractions is caspase dependent.

An important therapeutic strategy that can be used to treat leukemia is to trigger

differentiation of circulating leukemic cells and the first step towards achievement of this

goal is to execute apoptosis of these cells (Wang et al., 2005b, Zhou et al., 2007). Apoptosis

of cancer cell lines involves complex signaling pathways. To initiate apoptosis signaling

pathways are broadly classified into two types, one is intrinsic and second is extrinsic.

Intrinsic pathway is initiated by mitochondrial events while extrinsic pathway follows the

expression of TNF-α, TRAIL and FAS-L. JNK is reported to have a major role in the

activation of both of these pathways. Activated JNK in phosphorylated form is translocated to

Discussion


the nucleus where it leads to the activation of C-Jun which in turn leads to the formation of

activator protein (AP-1) which is involved in the transcription of a wide variety of pro-

apoptotic genes mainly TNF –α , Bak and FAS-L (Dhanasekaran and Reddy, 2008). JNK

has been reported to be the major molecule that has promoted differentiation of monocytic

cells that was induced by 1,25-dihydroxyvitamin D3, a derivative of vitamin D3 (Wang et al.,

2005b). Several drugs including vinblastin and taxol have been reported to trigger JNK

dependent apoptosis in breast cancer cell line (Kolomeichuk et al., 2008). Induction of

apoptosis in ovarian cancer cells by using cisplastin also followed JNK activation

(Dhanasekaran and Reddy, 2008). We provided a clear evidence that these fractions

committed the cells to apoptosis via activation of JNK signaling pathway. P-JNK expression

was increased in a time dependent manner and was sustained. JNK pathway is mainly

reported to be involved in various cellular events (Zhong et al., 2016) like apoptosis and

autophagy (Tsujimoto and Shimizu, 2005). JNK signalling pathway has been proved in

various studies as a promising drug target in therapy of cancer (Cao et al., 2010). JNK is

reported to be involved in modulation of the functions of mitochondrial proteins that are

regulating apoptosis which in turn leads to facilitate the release of apoptosis inducing factors

(Horbinski and Chu, 2005).

Our study suggests that treatment of cells with BF2, BF3, RF1, RF2, TF2, TF7 and TF8 has

resulted in a significant increasein TNF α.TNF receptor-1 (TNFR-1) when triggered by TNF

can lead to cell death in a classical way involving activation of JNK and in turn to caspases.

TNF α induced apoptosis may involve transient as well as prolonged activation of JNK.

There is a cross talk between NFκB and JNK signaling. Generally in viable cells TNF α

induced JNK activation is rapid and transient. The duration of JNK activation is considered

crucial in susceptibility of cells for apoptosis due to TNF α. NFκB inhibits the sustained

activation of JNK. Mainly prolonged JNK activation is involved in cell death. On the other

hand in NFκB inhibited cells JNK is persistently activated and the cells are primed to go for

apoptosis. So there may be a possibility that these seven fractions blocked NFκB and

resulted in TNF induced prolonged activation of JNK that lead to apoptosis of cells

(Ashkenazi, 2002). But these findings need to be explored further.

The proposed apoptotic pathway followed by these seven fractions, concluded from this

study is given in Fig 4.1. An additional route has also been reported for TNF α to commit

cells to apoptosis that involve acidic vesicles which can lead to production of ceramide as a

second messenger(Bröker et al., 2005).

Discussion


Discussion about the role of CXCL8 in tumour development and progression is controversial.

An up regulation of CXCL8 and its receptors in cancer was reported in previous studies

(Kargi et al., 2012, Varney et al., 2011, Oladipo et al., 2011, Chen et al., 2012). A correlation

of CXCL8 expression with distant metastasis was observed (Dimberg et al., 2012). We found

a significant decrease in level of CXCL8 in cells treated with these seven fractions that

provides an important clue for therapeutic strategy. So it can be suggested from the current

study that these fractions can increase the life expectancy of cancer patients with an end stage

disease but further investigations are required before applying the fractions practically. No

change in the level of CXCL2 was recorded after treatment of cells with these selected seven

fractions.

To elucidate the anti-cancerous effects of these fractions of the above mentioned selected

medicinal plants, study was performed on Sprague dawley rats. Benzene was used to induce

cancer of the myeloid cell as it has the potential to foster leukaemia by a number of suggested

mechanisms and via metabolites like phenol, catechol, hydroquinone and 1,2,4-benzenetriol

(Smith et al., 2000, Vaughan et al., 2005). Blood smears of rats from all the groups suggested

that leukemia was induced in all of them and it was established due to the presence of band

neutrophils and increased monocyte and neutrophil count. Our results showed that weightof

liver and spleen significantly increased in leukemic rats. When leukemic rats were treated

with plants extracts and fractions BF1 and BF2 fracions of O. basilicum, RF1 and RF2

fractions of R. officinalis and TF2, TF7 and TF8 fractions of T. vulgaris, these fractions

showed a good potential of prventing leukemia. These results are coinciding with those

reported by Yamamura et al in 1999 (Yamamura et al., 1999). This data suggests that

functional groups present in these fractions might have intervened with the changes caused

by leukemia. Similarly the above mentioned fractions have shown a good potential of

reversing the changes in white blood cells count induced by benzene.

By characterizing the immunological profile, role of each kind of immune cells to cancer

suppression or succession could be revealed. Serum samples from all groups were subjected

to ELISA to check TNF-α level and significant increase was recorded in rats treated with

above mentioned fractions. It has been reported that persistent synthesis of low quantity of

TNF inside a tumor microenvironment encourages tumour growth and promotes

angiogenesis, while elevated concentration can incite tumor cell necrosis, provoke antitumor

immunity and stimulate vascular collapse (Szlosarek et al., 2006). Apoptotic effects of TNF-

α via its DD have also been reported. In fact, TNF-α has anti-tumor properties alone or in

association with other molecules such as interferon (IFN) (Manusama et al., 1996). Since

Discussion


cytokines effect gene activation, growth and differentiation and functions of effector cells

related to tumor eradication (Kotla et al., 2009), this study was designed to provide a feasible

mechanism of selected three medicinal herbs anti-leukemic activity by investigating its

immunomodulatory effect on TNF-α in leukemic rats. Up-regulation of TNF-α in this study

may be a possible mechanism of action of these plants against uncontrolled growth of

hematopoietic cells.

Physical signs of toxicity e.g., increased urination, low body movement, irregular heartbeat,

weakness, less food and water intake, fur change and nose bleeding appeared in second to

third week of benzene-injection treatment compared to control group. Maronpot et al

(Maronpot et al., 2010) reported that liver enzymes stimulation is an adaptive response linked

with increased weight of liver. As such no previous studies have been reported for the in

vivoanti cancerous effects of the selected medicinal herbs.

Our results are in line with Saha et al (2012) who stated certain protective role of O.

basilicum in chemically induced cancers. They reported an enhanced expression of p21 and

p53 in mice with benzene induced hematotoxicity and lymphocyte count was found increased

in mice treated with plant extract after benzene treatment as compared to only benzene

treated mice (Saha et al., 2012).

Parmer et al. explored antitumor effects of rosemary extract by treating skin cancer in mice

induced by dimethylben(a) anthracine (DMBA) and a significant decrease was observed in

lipid peroxidation, GSH and total protein level in skin and liver of mice whereas expression

of antioxidant enzymes was found increased in skin and liver. This finding provided a

suggestion that rosemary plantextract has a potential to inhibit chemically induced cancers

similar to contemporary studies (Parmar et al., 2011).

Carvacrol and thymolwhich are major constituents of thyme plant extract were reported to

inhibit DMBA induced tumorigenesis and causing an increased expression of antioxidant

enzymes SOD and GPX in rats by Zeytinoglu et al., (1998) and Youdim & Deans (1999),

respectively. In another study, wild thyme (T. Surphyllum) has been instituted for induced

cytotoxicity and apoptosis in breast cancer cells (Bozkurt et al., 2012). Further it also has

inhibited growth of murine B16 melanomas (He et al., 1997) and also non-small lung cancer

cells, A549 (Koparal and Zeytinoğlu, 2003).

In order to evaluate the main composition of the fractions of plant extracts which caused an

elevation in TNF-α level, FTIR analysis was performed, which helped in deducing main

functional groups comprising the specific fractions. From the results, it is clear that the

fractions were rich in saturated and unsaturated hydrocarbons, hydrocarbon halides along

Discussion


with carbonyl and alcoholic group containing compounds. From the interpretation of FTIR

spectra of the selected seven fractions of these three medicinal herbs of family lamiaceae, it

was found that hydroxyl group, carbonyl group and halides as well as aromatic compounds

were present in these fractions. The FTIR study is in agreement with UV absorption data

which ranges from 230 to 330 nm as most of the polyunsaturated and aromatic compounds

including polyphenolic compounds (flavonoids, carboxylic acid derivatives etc) show

maximum absorption of electromagnetic radiations in this wave length range. The presence

of these compounds greatly supports the effective role of O. basilicum, R. officinalis and T.

vulgaris in the anticancer activities. The slight differences in the activities of the three plants

extracts might be due to the variation in the concentration of these active compounds in the

extracts. Bunghez et al suggested FTIR analysis as a reliable tool for evaluating and

characterizing formulas in plant extracts. They also reported the presence of flavonols,

flavanols, phenolic acids and volatile phenols in 1400-1760 cm-1 region in basil, thyme,

oregano, rosemary and clove methanolic extracts (Bunghez et al., 2013).

To further investigate the chemical nature of these fractions, LC-MS analyses was done

which indicated the existence of flavonoids, ester derivatives of amino acids and carboxylic

derivatives in the selected fractions. The presence of all the above mentioned compounds

strongly supports the role of these fractions against leukemia. Epicatechin derivatives have

been reported as anticancer agents. These have been used against human prostate cancer.

Epicatechin oligomers conceal the expression of the cancer-promoting gene. (−)-

Epicatechin is an aromatic compound which has oxygen scavenging ability and cells

signaling modulating property for MAP kinase pathway (Takanashi et al., 2017; Shay et al.,

2015). The derivatives of caffeic acid have also been reported as anticancer agents. There

mechanism of action as potential anticancer compounds has been extensively studied

(Tyszka-Czochara et al., 2017; Rao et al., 1992). The amino acid ester derivatives have been

studied as anticancer compounds. The presence of D-Alanine, n-(2,5-

ditrifluoromethylbenzoyl), nonadecyl ester and D-Alanine, n-(2,5-ditrifluoromethylbenzoyl),

hexadecyl ester in the studied fractions ensure the role of these compounds in as anti

leukemic property of the selected fractions (Song et al., 2005). The cinnamic acid derivatives

are using since long as anticancer agents due to presence of aromatic rings with polyphenolic

structure (De et al., 2011). Similarly the presence of carboxylic acid derivatives also

strengthens the potential anticancer activity of the selected fractions.

These compounds have been reported as nutritional antioxidants in previous study by Forman

et al. (Forman et al., 2014). The paradigm of a function activated in carcinogenesis is cell

https://www.ncbi.nlm.nih.gov/pubmed/?term=Tyszka-Czochara%20M%5BAuthor%5D&cauthor=true&cauthor_uid=28230778

Discussion


proliferation, angiogenesis and escape from apoptosis. Indeed these events commonly shared

a crucial motif in cell signalling and that is the formation of redox signalling molecules

H2O2, lipid hydroperoxides, α,β-unsaturated carbonyls, and possibly other electrophiles),

that activate a series of redox switches through oxidation of specific cysteine residues

(Brigelius-Flohe & Flohe, 2011; Hanahan & Weinberg 2011; Trachootham et al., 2008)

The measured antioxidant capacity of phytochemicals is simply an index of sensitivity to

oxidation rather than an index of protection that these agents can produce in vivo. The

principal manner by which nutritional antioxidants act is Nrf2(nuclear factor erythroid 2-

related factor 2) pathway. activation through modification of a specific cysteine in Keap1

(Kelch-like ECH-associated protein 1). This modification of Keap1 requires that the

antioxidant be, or be converted to, an electrophile. The consensus chemistry underlying

Keap1 modification is Michael addition, which is the reductive addition of a nucleophile (the

specifically reactive Keap1 cysteine) to a 3,3-unsaturated carbonyl compound (the active

form of the antioxidant). Addition to an isothiocyanate, such as sulforaphane is also a

reductive addition reaction. ‘Adequate’ nutritional intake of antioxidants provides an

adjustable level of nucleophiles that can be regulated to cope with increased electrophiles

including oxidants. These nutritional antioxidants are actually providing a stimulus for

regulation of protective defense and repair systems rather than scavenging free radicals.

(Forman et al., 2014)

In the present study, the analytical data of selected fractions discussed above are in support of

the presence of important organic constituents specially antioxidants which acted as

chemopreventive agents for leukemia.

Discussion


Figure 4.1. Proposed pathway for apoptosis caused by the selected fractions of the selected

plants deduced in the present study.

Conclusion and Recommendation


Conclusion and Future Perspectives

Over all our data provide evidence that O. basilicum, R. officinalis and T. vulgaris contain

strong anticancer agents that further need to be explored. Previously, we learned about a

variety of pathways that can be targeted for the treatment of cancer. From the present study

we concluded that these selected plants are causing cytotoxicty of four types of leukemic cell

lines via apoptosis. Further JNK pathway was also sought to be involved in induction of

apoptosis by fractions of these plants. In addition to that, these fractions were also observed

to be involved in alterations in the level of pro-inflammatory cytokines. Such considerations

are extremely important in the design of new therapies to treat cancer because these will be

more safe and productive as compared to synthetic drugs. From the FTIR and LC-MS data

and from the already reported literature study we can claim that the presence of ester

derivatives of amino acids, flavonoids and phenolic compounds in the selected fractions of

methanolic extracts resulted in the enhanced anticancer (anti leukemia) activities in the

present study.

These plants are often used in diet and it may prove better in prevention of cancer in a much

better and more economic way to fight cancer than treating an already advanced and often

intractable disease.

Recommendations for Research and Development

From the results of the present study, it is recommended that in future these three plants

extracts should be included in the diet of cancer patients and improvement in their cell count

and other related parameters should be monitored. Further their blood samples should be

taken and JNK pathway activation as well as cleavage of caspase 3 should be taken into

consideration. Apart from this the model screening pathway plays a critical role in cell fate,

being implicated in a multitude of diseases ranging from cancers to neurological,

immunological and certain inflammatory conditions. So in future these plants can be used to

study the screened pathway in the above conditions also. Further it can be recommended that

these plants can be harvested in different climatic conditions in order to improve the

secondary metabolites of these plants and then can be used for the chemoprevention studies.

More improvements in the study can be made by extracting pure compound from the selected

fractions used in the present study and then can be implicated for the treatment of cancer

through pharmaceutical industries. The less expensive herbal drug treatment may highly be

recommended to the rural and poor people to treat effectively the cancers of various types

Conclusion and Recommendation


will be an ideal choice. Nowadays the pharmaceutical industries are moving towards

development of herbal drugs, and hence these three studied plant extracts may also be

considered for development of a cheap and effective drug for cancer therapy.

References


References

Abdel-Wahhab, K. G. E.-D., El-Shamy, K. A., El-Beih, N. A. E.-Z., Morcy, F. A. Mannaa, F.

A. E. 2011. Protective effect of a natural herb (Rosmarinus officinalis) against hepatotoxicity

in male albino rats. Comunicata Scientiae, 2, 9-17.

Agrawala, S., Chatterjee, S. Misra, S. 2001. Immunepotentiation activity of a polyherbal

formulation ‘Immu-21’(research name). Phytomedica, 2, 1-22.

Ait-Mohamed, O., Battisti, V., Joliot, V., Fritsch, L., Pontis, J., Medjkane, S., Redeuilh, C.,

Lamouri, A., Fahy, C. Rholam, M. 2011. Acetonic extract of Buxus sempervirens induces

cell cycle arrest, apoptosis and autophagy in breast cancer cells. PloS one, 6, e24537.

Ait M'Barek, L., Ait Mouse, H., Jaâfari, A., Aboufatima, R., Benharref, A., Kamal, M.,

Bénard, J., El Abbadi, N., Bensalah, M. Gamouh, A. 2007. Cytotoxic effect of essential oil of

thyme (Thymus broussonettii) on the IGR-OV1 tumor cells resistant to chemotherapy.

Brazilian Journal of Medical and Biological Research, 40, 1537-1544.

Al–Maqtari, M., Alghalibi, S. M. Alhamzy, E. H. 2011. Chemical composition and

antimicrobial activity of essential oil of Thymus vulgaris from Yemen. Turkish J. Biochem,

36, 342-349.

Amiri, H. 2012. Essential oils composition and antioxidant properties of three thymus

species. Evidence-Based Complementary and Alternative Medicine, 2012.

Anand, P., Kunnumakara, A. B., Sundaram, C., Harikumar, K. B., Tharakan, S. T., Lai, O. S.,

Sung, B. Aggarwal, B. B. 2008. Cancer is a preventable disease that requires major lifestyle

changes. Pharmaceutical research, 25, 2097-2116.

Andrews, L. S., Lee, E. W., Witmer, C. M., Kocsis, J. J. Snyder, R. 1977. Effects of toluene

on the metabolism, disposition and hemopoietic toxicity of [3H] benzene. Biochemical

pharmacology, 26, 293-300.

Armstrong, S. A. Look, A. T. 2005. Molecular genetics of acute lymphoblastic leukemia.

Journal of Clinical Oncology, 23, 6306-6315.

Asami, D. K., Hong, Y.-J., Barrett, D. M. Mitchell, A. E. 2003. Comparison of the total

phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and

corn grown using conventional, organic, and sustainable agricultural practices. Journal of

agricultural and food chemistry, 51, 1237-1241.

Ashafa, A. O. T., Orekoya, L. O. Yakubu, M. T. 2012. Toxicity profile of ethanolic extract of

Azadirachta indica stem bark in male Wistar rats. Asian Pacific journal of tropical

biomedicine, 2, 811-817.

References


Ashkenazi, A. 2002. Targeting death and decoy receptors of the tumour-necrosis factor

superfamily. Nature Reviews Cancer, 2, 420.

Atanassova, M., Georgieva, S. Ivancheva, K. 2011. Total phenolic and total flavonoid

contents, antioxidant capacity and biological contaminants in medicinal herbs. Journal of the

University of Chemical Technology and Metallurgy, 46, 81-88.

Atti-Santos, A. C., Rossato, M., Pauletti, G. F., Rota, L. D., Rech, J. C., Pansera, M. R.,

Agostini, F., Serafini, L. A. Moyna, P. 2005. Physico-chemical evaluation of Rosmarinus

officinalis L. essential oils. Brazilian Archives of Biology and Technology, 48, 1035-1039.

Bai, N., He, K., Roller, M., Lai, C.-S., Shao, X., Pan, M.-H. Ho, C.-T. 2010. Flavonoids and

phenolic compounds from Rosmarinus officinalis. Journal of agricultural and food

chemistry, 58, 5363-5367.

Baker, S., Rane, S. Reddy, E. 2007. Hematopoietic cytokine receptor signaling. Oncogene,

26, 6724.

Bakırel, T., Bakırel, U., Keleş, O. Ü., Ülgen, S. G. Yardibi, H. 2008. In vivo assessment of

antidiabetic and antioxidant activities of rosemary (Rosmarinus officinalis) in alloxan-

diabetic rabbits. Journal of ethnopharmacology, 116, 64-73.

Bakkali, F., Averbeck, S., Averbeck, D. Idaomar, M. 2008. Biological effects of essential

oils–a review. Food and chemical toxicology, 46, 446-475.

Barreda, D. R., Hanington, P. C. Belosevic, M. 2004. Regulation of myeloid development

and function by colony stimulating factors. Developmental & Comparative Immunology, 28,

509-554.

Basilico, M. Basilico, J. 1999. Inhibitory effects of some spice essential oils on Aspergillus

ochraceus NRRL 3174 growth and ochratoxin A production. Letters in Applied

Microbiology, 29, 238-241.

Baud, V. Karin, M. 2001. Signal transduction by tumor necrosis factor and its relatives.

Trends in cell biology, 11, 372-377.

Berdowska, I., Zieliński, B., Fecka, I., Kulbacka, J., Saczko, J. Gamian, A. 2013. Cytotoxic

impact of phenolics from Lamiaceae species on human breast cancer cells. Food Chemistry,

141, 1313-1321.

Brigelius-Flohe, R., Flohe, L. 2011. Basic principles and emerging concepts in the redox

control of transcription factors. Antioxidants & redox signaling, 15(8), 2335-2381.

Berrington, D. Lall, N. 2012. Anticancer activity of certain herbs and spices on the cervical

epithelial carcinoma (HeLa) cell line. Evidence-Based Complementary and Alternative

Medicine, 2012.

References


Bhatia, D., Mandal, A., Nevo, E. Bishayee, A. 2015. Apoptosis-inducing effects of extracts

from desert plants in HepG2 human hepatocarcinoma cells. Asian Pacific journal of tropical

biomedicine, 5, 87-92.

Bogadi-Šare, A., Brumen, V., Turk, R., Karacic, V. Zavalic, M. 1997. Genotoxic effects in

workers exposed to benzene: with special reference to exposure biomarkers and confounding

factors. Industrial health, 35, 367-373.

Boveri, T. 1914. Zur Frage der Entstehung maligner Tumoren (The Origin of Malignant

Tumors)(Jena: Gustav Fischer).

Boyse, J., Hewitt, M. Mott, M. G. 1996. Glycophorin A mutations and risk of secondary

leukaemia in patients treated for childhood acute lymphoblastic leukaemia. British journal of

haematology, 93, 117-124.

Bozin, B., Mimica-Dukic, N., Samojlik, I. Jovin, E. 2007. Antimicrobial and antioxidant

properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L.,

Lamiaceae) essential oils. Journal of agricultural and food chemistry, 55, 7879-7885.

Bozkurt, E., Atmaca, H., Kisim, A., Uzunoglu, S., Uslu, R. Karaca, B. 2012. Effects of

Thymus serpyllum extract on cell proliferation, apoptosis and epigenetic events in human

breast cancer cells. Nutrition and cancer, 64, 1245-1250.

Bray, F. Møller, B. 2006. Predicting the future burden of cancer. Nature Reviews Cancer, 6,

63-74.

Brayton, C. F. 1986. Dimethyl sulfoxide (DMSO): a review. The Cornell Veterinarian, 76(1),

61-90.

Bröker, L. E., Kruyt, F. A. Giaccone, G. 2005. Cell death independent of caspases: a review.

Clinical Cancer Research, 11, 3155-3162.

Bruce-Keller, A. J., Geddes, J. W., Knapp, P. E., McFall, R. W., Keller, J. N., Holtsberg, F.

W., Parthasarathy, S., Steiner, S. M. Mattson, M. P. 1999. Anti-death properties of TNF

against metabolic poisoning: mitochondrial stabilization by MnSOD. Journal of

neuroimmunology, 93, 53-71.

Bunghez, F., Socaci, C., Zăgrean, F. Fetea, F. 2013. Application of FT-MIR Spectroscopy for

Fingerprinting Bioactive Molecules of Plant Ingredients and a New Formula with

Antimicrobial Effect. Bull. UASVM Food Sci. Technol, 70, 64-65.

Cao, X.-h., Wang, A.-h., Wang, C.-l., Mao, D.-z., Lu, M.-f., Cui, Y.-q. Jiao, R.-z. 2010.

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-

mediated mitochondrial/caspase pathway. Chemico-biological interactions, 183, 357-362.

References


Carović-StanKo, K., PeteK, M., Martina, G., Pintar, J., Bedeković, D., Ćustić, M. H. Satovic,

Z. 2016. Medicinal Plants of the Family Lamiaceaeas Functional Foods–a Review. Czech

journal of food sciences, 34, 377-390.

Chang, F., Steelman, L., Lee, J., Shelton, J., Navolanic, P., Blalock, W. L., Franklin, R.

McCubrey, J. 2003. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from

cytokine receptors to transcription factors: potential targeting for therapeutic intervention.

Leukemia, 17, 1263-1293.

Chen, T. Wong, Y.-S. 2009. Selenocystine induces caspase-independent apoptosis in MCF-7

human breast carcinoma cells with involvement of p53 phosphorylation and reactive oxygen

species generation. The international journal of biochemistry & cell biology, 41, 666-676.

Chen, Y., Shi, M., Yu, G.-Z., Qin, X.-R., Jin, G., Chen, P. Zhu, M.-H. 2012. Interleukin-8, a

promising predictor for prognosis of pancreatic cancer. World journal of gastroenterology:

WJG, 18, 1123.

Cheung, S. Tai, J. 2007. Anti-proliferative and antioxidant properties of rosemary

Rosmarinus officinalis. Oncology reports, 17, 1525-1531.

Chiorazzi, N., Rai, K. R. Ferrarini, M. 2005. Chronic lymphocytic leukemia. New England

Journal of Medicine, 352, 804-815.

Clark Jr, W. H., Elder, D. E., Guerry IV, D., Braitman, L. E., Trock, B. J., Schultz, D.,

Synnestvedt, M. Halpern, A. C. 1989. Model predicting survival in stage I melanoma based

on tumor progression. JNCI: Journal of the National Cancer Institute, 81, 1893-1904.

Cooper, G. M. Hausman, R. E. 2000. The cell, Sinauer Associates Sunderland.

Cuenda, A. Rousseau, S. 2007. p38 MAP-kinases pathway regulation, function and role in

human diseases. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1773, 1358-

1375.

Dacie, J. Lewis, S. 1991. In Practical Haematology 7th edition ELBS with Churchill

Livingstone. Longman group UK.

Dalla-Favera, R. Gaidano, G. 2001. Molecular biology of lymphomas. Cancer. Principles

and Practice of Oncology, 6, 2215-2235.

Dameshek, W. 1967. Chronic Lymphocytic Leukemia—An Accumulative Disorder of

Immunologically Incompetent Lymphocytes. Annals of Internal Medicine, 66, 1040-1041.

Dasgupta, T., Rao, A. Yadava, P. 2004. Chemomodulatory efficacy of basil leaf (Ocimum

basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin

and forestomach papillomagenesis. Phytomedicine, 11, 139-151.

References


De Flora, S. Ferguson, L. R. 2005. Overview of mechanisms of cancer chemopreventive

agents. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 591, 8-

15.

Dhanasekaran, D. N. Reddy, E. P. 2008. JNK signaling in apoptosis. Oncogene, 27, 6245.

Dimberg, J., Ström, K., Löfgren, S., Zar, N., Lindh, M. Matussek, A. 2012. DNA promoter

methylation status and protein expression of interleukin-8 in human colorectal

adenocarcinomas. International journal of colorectal disease, 27, 709-714.

Din, F. V., Valanciute, A., Houde, V. P., Zibrova, D., Green, K. A., Sakamoto, K., Alessi, D.

R. Dunlop, M. G. 2012. Aspirin inhibits mTOR signaling, activates AMP-activated protein

kinase, and induces autophagy in colorectal cancer cells. Gastroenterology, 142, 1504-1515.

e3.

Döhner, H., Estey, E. H., Amadori, S., Appelbaum, F. R., Büchner, T., Burnett, A. K.,

Dombret, H., Fenaux, P., Grimwade, D. Larson, R. A. 2010. Diagnosis and management of

acute myeloid leukemia in adults: recommendations from an international expert panel, on

behalf of the European LeukemiaNet. Blood, 115, 453-474.

Dorman, H. Deans, S. 2000. Antimicrobial agents from plants: antibacterial activity of plant

volatile oils. Journal of applied microbiology, 88, 308-316.

Dranoff, G. 2004. Cytokines in cancer pathogenesis and cancer therapy. Nature Reviews

Cancer, 4, 11.

Eastmond, D., Schuler, M., Frantz, C., Chen, H., Parks, R., Wang, L. Hasegawa, L. 2001.

Characterization and mechanisms of chromosomal alterations induced by benzene in mice

and humans. Research Report (Health Effects Institute), 1-68; discussion 69-80.

Edris, A. E. 2007. Pharmaceutical and therapeutic potentials of essential oils and their

individual volatile constituents: a review. Phytotherapy research, 21, 308-323.

Eissner, G., Kolch, W. Scheurich, P. 2004. Ligands working as receptors: reverse signaling

by members of the TNF superfamily enhance the plasticity of the immune system. Cytokine

& growth factor reviews, 15, 353-366.

Forman, H. J., Davies, K. J., Ursini, F. 2014. How do nutritional antioxidants really work:

nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free radical

biology & medicine, 66, 24-35.

Frankel, S. K., Cosgrove, G. P., Cha, S.-I., Cool, C. D., Wynes, M. W., Edelman, B. L.,

Brown, K. K. Riches, D. W. 2006. TNF-α sensitizes normal and fibrotic human lung

fibroblasts to Fas-induced apoptosis. American journal of respiratory cell and molecular

biology, 34, 293-304.

References


Gallagher, A. P., Burnett, A. K., Bowen, D. T. Darley, R. L. 1998. Mutant RAS selectively

promotes sensitivity of myeloid leukemia cells to apoptosis by a protein kinase C-dependent

process. Cancer research, 58, 2029-2035.

Gebrehiwot, H., Bachetti, R. Dekebo, A. 2015. Chemical composition and antimicrobial

activities of leaves of sweet basil (Ocimum basilicum L.) herb.

González-Trujano, M., Peña, E., Martínez, A., Moreno, J., Guevara-Fefer, P., Deciga-

Campos, M. López-Muñoz, F. 2007. Evaluation of the antinociceptive effect of Rosmarinus

officinalis L. using three different experimental models in rodents. Journal of

ethnopharmacology, 111, 476-482.

González-Vallinas, M., Molina, S., Vicente, G., Zarza, V., Martín-Hernández, R., García-

Risco, M. R., Fornari, T., Reglero, G. De Molina, A. R. 2014. Expression of microRNA-15b

and the glycosyltransferase GCNT3 correlates with antitumor efficacy of Rosemary

diterpenes in colon and pancreatic cancer. PloS one, 9, e98556.

Grayer, R. J., Kite, G. C., Veitch, N. C., Eckert, M. R., Marin, P. D., Senanayake, P. Paton,

A. J. 2002. Leaf flavonoid glycosides as chemosystematic characters in Ocimum.

Biochemical systematics and ecology, 30, 327-342.

Greaves, M. 1997. Aetiology of acute leukaemia. The Lancet, 349, 344-349.

Greenwald, P. 1996. Chemoprevention of cancer. Scientific American, 275, 96-99.

Grigore, A., Paraschiv, I., Colceru-Mihul, S., Bubueanu, C., Draghici, E. Ichim, M. 2010.

Chemical composition and antioxidant activity of Thymus vulgaris L. volatile oil obtained by

two different methods. Romanian Biotechnological Letters, 15, 5436-5443.

Güner, A., Özhatay, N., Ekim, T. Başer, K. 2000. Flora of Turkey and the East Aegean

Islands. Vol. 11. Second Supplement, Edinburgh.

Gutierrez, J., Barry-Ryan, C. Bourke, P. 2008. The antimicrobial efficacy of plant essential

oil combinations and interactions with food ingredients. International journal of food

microbiology, 124, 91-97.

Habtemariam, S. 2016. The therapeutic potential of rosemary (Rosmarinus officinalis)

diterpenes for Alzheimer’s disease. Evidence-Based Complementary and Alternative

Medicine, 2016.

Hanahan, D., Weinberg, R. A. 2011. Hallmarks of cancer: the next generation. Cell, 144,

646-674.

Hauser, A.-T. Jung, M. 2008. Targeting epigenetic mechanisms: potential of natural products

in cancer chemoprevention. Planta medica, 74, 1593-1601.

References


He, L., Mo, H., Hadisusilo, S., Qureshi, A. A. Elson, C. E. 1997. Isoprenoids suppress the

growth of murine B16 melanomas in vitro and in vivo. The Journal of nutrition, 127, 668-

674.

Höferl, M., Buchbauer, G., Jirovetz, L., Schmidt, E., Stoyanova, A., Denkova, Z., Slavchev,

A. Geissler, M. 2009. Correlation of antimicrobial activities of various essential oils and their

main aromatic volatile constituents. Journal of Essential Oil Research, 21, 459-463.

Holley, R. A. Patel, D. 2005. Improvement in shelf-life and safety of perishable foods by

plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273-292.

Hong, H. Y., Varvayanis, S. Yen, A. 2001. Retinoic acid causes MEK‐dependent RAF

phosphorylation through RARα plus RXR activation in HL‐60 cells. Differentiation, 68, 55-

66.

Horbinski, C. Chu, C. T. 2005. Kinase signaling cascades in the mitochondrion: a matter of

life or death. Free Radical Biology and Medicine, 38, 2-11.

Horváthová, E., Slameňová, D. Navarová, J. 2010. Administration of rosemary essential oil

enhances resistance of rat hepatocytes against DNA-damaging oxidative agents. Food

Chemistry, 123, 151-156.

Horvathova, E., Turcaniova, V. Slamenova, D. 2007. Comparative study of DNA-damaging

and DNA-protective effects of selected components of essential plant oils in human leukemic

cells K562. Neoplasma, 54, 478-483.

Hudaib, M. Aburjai, T. 2007. Volatile components of Thymus vulgaris L. from wild‐growing

and cultivated plants in Jordan. Flavour and fragrance journal, 22, 322-327.

Imada, K. Leonard, W. J. 2000. The jak-STAT pathway. Molecular immunology, 37, 1-11.

Inoue, O., Seiji, K., Nakatsuka, H., Watanabe, T., Yin, S., Li, G., Cai, S., Jin, C. Ikeda, M.

1989. Excretion of 1, 2, 4-benzenetriol in the urine of workers exposed to benzene.

Occupational and Environmental Medicine, 46, 559-565.

Irigaray, P., Newby, J., Clapp, R., Hardell, L., Howard, V., Montagnier, L., Epstein, S.

Belpomme, D. 2007. Lifestyle-related factors and environmental agents causing cancer: an

overview. Biomedicine & Pharmacotherapy, 61, 640-658.

Irons, R. D. 1985. Quinones as toxic metabolites of benzene. Journal of Toxicology and

Environmental Health, Part A Current Issues, 16, 673-678.

Irons, R. D. Stillman, W. S. 1993. Cell proliferation and differentiation in chemical

leukemogenesis. Stem Cells, 11, 235-242.

References


Iskander, K. Jaiswal, A. K. 2005. Quinone oxidoreductases in protection against

myelogenous hyperplasia and benzene toxicity. Chemico-biological interactions, 153, 147-

157.

Ito, Y., Mishra, N., Yoshida, K., Kharbanda, S., Saxena, S. Kufe, D. 2001. Mitochondrial

targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells. Cell death

and differentiation, 8, 794.

Jacob, S. W., Herschler, R. 1986. Pharmacology of DMSO. Cryobiology, 23(1), 14-27.

Jacob, S. W., Bischel, M., Herschler, R. J. 1964. Dimethyl sulfoxide (DMSO): A new

concept in pharmacotherapy. Current therapeutic research, clinical and experimental, 6, 134-

135.

Javanmardi, J., Khalighi, A., Kashi, A., Bais, H. Vivanco, J. 2002. Chemical characterization

of basil (Ocimum basilicum L.) found in local accessions and used in traditional medicines in

Iran. Journal of agricultural and food chemistry, 50, 5878-5883.

Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J. Thun, M. J. 2007. Cancer statistics, 2007.

CA: a cancer journal for clinicians, 57, 43-66.

Jing, L. Anning, L. 2005. Role of JNK activation in apoptosis: a double-edged sword. Cell

research, 15, 36.

Joshi, R. K. 2014. Chemical composition and antimicrobial activity of the essential oil of

Ocimum basilicum L.(sweet basil) from Western Ghats of North West Karnataka, India.

Ancient science of life, 33, 151.

Kargi, A., Yalcin, A. D., Erin, N., Savas, B., Polat, H. H. Gorczynski, R. M. 2012. IL8 and

serum soluble TRAIL levels following anti-VEGF monoclonal antibody treatment in patients

with metastatic colon cancer. Clinical laboratory, 58, 501-505.

Karimi, N., Rashedi, J., Poor, B. M., Arabi, S., Ghorbani, M., Tahmasebpour, N.

Asgharzadeh, M. 2017. Cytotoxic effect of rosemary extract on gastric adenocarcinoma

(AGS) and esophageal squamous cell carcinoma (KYSE30) cell lines. Gastroenterology and

Hepatology from bed to bench, 10, 102.

Karin, M. 2006. Nuclear factor-κB in cancer development and progression. Nature, 441, 431-

436.

Kaviarasan, S., Vijayalakshmi, K. Anuradha, C. 2004. Polyphenol-rich extract of fenugreek

seeds protect erythrocytes from oxidative damage. Plant Foods for Human Nutrition, 59,

143-147.

Kaya, I., Yigit, N. Benli, M. 2008. Antimicrobial activity of various extracts of Ocimum

basilicum L. and observation of the inhibition effect on bacterial cells by use of scanning

References


electron microscopy. African Journal of Traditional, Complementary and Alternative

Medicines, 5, 363-369.

Khan, M. K., Ansari, I. A., Khan, M. S. Arif, J. M. 2013. Dietary phytochemicals as potent

chemotherapeutic agents against breast cancer: Inhibition of NF-κB pathway via molecular

interactions in rel homology domain of its precursor protein p105. Pharmacognosy magazine,

9, 51.

Kokate, C., Purohit, A. Gokhale, S. 2006. Pharmacognosy. Nirali Prakashan, Pune, 35, 133-

525.

Kolomeichuk, S. N., Terrano, D. T., Lyle, C. S., Sabapathy, K. Chambers, T. C. 2008.

Distinct signaling pathways of microtubule inhibitors–vinblastine and Taxol induce

JNK‐dependent cell death but through AP‐1‐dependent and AP‐1‐independent mechanisms,

respectively. The FEBS journal, 275, 1889-1899.

Kontogianni, V. G., Tomic, G., Nikolic, I., Nerantzaki, A. A., Sayyad, N., Stosic-Grujicic, S.,

Stojanovic, I., Gerothanassis, I. P. Tzakos, A. G. 2013. Phytochemical profile of Rosmarinus

officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-

proliferative activity. Food Chemistry, 136, 120-129.

Koparal, A. T. Zeytinoğlu, M. 2003. Effects of carvacrol on a human non-small cell lung

cancer (NSCLC) cell line, A549. Animal Cell Technology: Basic & Applied Aspects.

Springer.

Kotla, V., Goel, S., Nischal, S., Heuck, C., Vivek, K., Das, B. Verma, A. 2009. Mechanism

of action of lenalidomide in hematological malignancies. Journal of hematology & oncology,

2, 36.

Kulisic, T., Radonic, A., Katalinic, V. Milos, M. 2004. Use of different methods for testing

antioxidative activity of oregano essential oil. Food Chemistry, 85, 633-640.

Kulkarni, A., Jan, N. Nimbarte, S. 2013. Elucidation of Secondary Metabolites from Thyme

Oil and Comparing its Antimicrobial Potential with Conventional Antibiotics against

Uropathogens. genus, 2, 3.

Lamm, S. H., Walters, A. S., Wilson, R., Byrd, D. M. Grunwald, H. 1989. Consistencies and

inconsistencies underlying the quantitative assessment of leukemia risk from benzene

exposure. Environmental health perspectives, 82, 289.

Li, Y. Ding, Y. 1990. Chromosome changes by G-banding in patients with chronic benzene

poisoning. Chin J Ind Med, 3, 29-31.

References


Lindsey, R. H., Bender, R. P. Osheroff, N. 2005. Effects of benzene metabolites on DNA

cleavage mediated by human topoisomerase IIα: 1, 4-hydroquinone is a topoisomerase II

poison. Chemical research in toxicology, 18, 761-770.

Liu, Z.-g. 2005. Molecular mechanism of TNF signaling and beyond. Cell research, 15, 24.

Looi, C. Y., Arya, A., Cheah, F. K., Muharram, B., Leong, K. H., Mohamad, K., Wong, W.

F., Rai, N. Mustafa, M. R. 2013. Induction of apoptosis in human breast cancer cells via

caspase pathway by vernodalin isolated from Centratherum anthelminticum (L.) seeds. PloS

one, 8, e56643.

Magee, T. Marshall, C. 1999. New insights into the interaction of Ras with the plasma

membrane. Cell, 98, 9-12.

Malcolm, A. 2002. The cancer handbook, Nature publishing group.

Mangan, J. K., Rane, S. G., Kang, A. D., Amanullah, A., Wong, B. C. Reddy, E. P. 2004.

Mechanisms associated with IL-6–induced up-regulation of Jak3 and its role in monocytic

differentiation. Blood, 103, 4093-4101.

Manosroi, J., Dhumtanom, P. Manosroi, A. 2006. Anti-proliferative activity of essential oil

extracted from Thai medicinal plants on KB and P388 cell lines. Cancer letters, 235, 114-

120.

Manusama, E., Nooijen, P., Stavast, J., Durante, N., Marquet, R. Eggermont, A. 1996.

Synergistic antitumour effect of recombinant human tumour necrosis factor a with melphalan

in isolated limb perfusion in the rat. British journal of surgery, 83, 551-555.

Marino, M., Bersani, C. Comi, G. 1999. Antimicrobial activity of the essential oils of

Thymus vulgaris L. measured using a bioimpedometric method. Journal of food protection,

62, 1017-1023.

Maronpot, R. R., Yoshizawa, K., Nyska, A., Harada, T., Flake, G., Mueller, G., Singh, B.

Ward, J. M. 2010. Hepatic enzyme induction: histopathology. Toxicologic pathology, 38,

776-795.

Matsumoto, K. 1984. Changes of bone marrow cell and peripheral blood cell counts in new-

born mice. Experimental Animals, 33, 339-344.

McClatchey, W. 1996. The ethnopharmacopoeia of Rotuma. Journal of ethnopharmacology,

50, 147-156.

McCubrey, J. A., Steelman, L. S., Chappell, W. H., Abrams, S. L., Wong, E. W., Chang, F.,

Lehmann, B., Terrian, D. M., Milella, M. Tafuri, A. 2007. Roles of the Raf/MEK/ERK

pathway in cell growth, malignant transformation and drug resistance. Biochimica et

Biophysica Acta (BBA)-Molecular Cell Research, 1773, 1263-1284.

References


McHale, C. M., Zhang, L. Smith, M. T. 2011. Current understanding of the mechanism of

benzene-induced leukemia in humans: implications for risk assessment. Carcinogenesis, 33,

240-252.

Medina-Holguín, A. L., Holguín, F. O., Micheletto, S., Goehle, S., Simon, J. A. O’Connell,

M. A. 2008. Chemotypic variation of essential oils in the medicinal plant, Anemopsis

californica. Phytochemistry, 69, 919-927.

Meister, A., Bernhardt, G., Christoffel, V. Buschauer, A. 1999. Antispasmodic activity of

Thymus vulgaris extract on the isolated guinea-pig trachea: discrimination between drug and

ethanol effects. Planta medica, 65, 512-516.

Mijatovic, S. A., Timotijevic, G. S., Miljkovic, D. M., Radovic, J. M., Maksimovic‐Ivanic, D.

D., Dekanski, D. P. Stosic‐Grujicic, S. D. 2011. Multiple antimelanoma potential of dry olive

leaf extract. International journal of cancer, 128, 1955-1965.

Milhau, G., Valentin, A., Benoit, F., Mallié, M., Bastide, J.-M., Pélissier, Y. Bessière, J.-M.

1997. In vitro antimalarial activity of eight essential oils. Journal of Essential Oil Research,

9, 329-333.

Miranda, M. Johnson, D. 2007. Signal transduction pathways that contribute to myeloid

differentiation. Leukemia, 21, 1363-1377.

Mitoma, H., Horiuchi, T. Tsukamoto, H. 2004. Binding activities of infliximab and

etanercept to transmembrane tumor necrosis factor-α. Gastroenterology, 126, 934-935.

Miura, K., Kikuzaki, H. Nakatani, N. 2002. Antioxidant activity of chemical components

from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) measured by the oil stability

index method. Journal of agricultural and food chemistry, 50, 1845-1851.

Mothana, R. A., Kriegisch, S., Harms, M., Wende, K. Lindequist, U. 2011. Assessment of

selected Yemeni medicinal plants for their in vitro antimicrobial, anticancer, and antioxidant

activities. Pharmaceutical Biology, 49, 200-210.

Mukherjee, D. 2002. Selective cyclooxygenase-2 (COX-2) inhibitors and potential risk of

cardiovascular events. Biochemical pharmacology, 63, 817-821.

Mukhtar, E., Mustafa Adhami, V., Khan, N. Mukhtar, H. 2012. Apoptosis and autophagy

induction as mechanism of cancer prevention by naturally occurring dietary agents. Current

drug targets, 13, 1831-1841.

Mun, S.-H., Joung, D.-K., Kim, Y.-S., Kang, O.-H., Kim, S.-B., Seo, Y.-S., Kim, Y.-C., Lee,

D.-S., Shin, D.-W. Kweon, K.-T. 2013. Synergistic antibacterial effect of curcumin against

methicillin-resistant Staphylococcus aureus. Phytomedicine, 20, 714-718.

References


Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R. De Feo, V. 2013. Effect of essential

oils on pathogenic bacteria. Pharmaceuticals, 6, 1451-1474.

Nickavar, B., Mojab, F. Dolat-Abadi, R. 2005. Analysis of the essential oils of two Thymus

species from Iran. Food Chemistry, 90, 609-611.

Nishikawa, T., Izumo, K., Miyahara, E., Horiuchi, M., Okamoto, Y., Kawano, Y. Takeuchi,

T. 2011. Benzene induces cytotoxicity without metabolic activation. Journal of occupational

health, 53, 84-92.

Noonan, D. M., Benelli, R. Albini, A. 2007. Angiogenesis and cancer prevention: a vision.

Cancer Prevention, 219-224.

Ocaña, A. Reglero, G. 2012. Effects of thyme extract oils (from Thymus vulgaris, Thymus

zygis, and Thymus hyemalis) on cytokine production and gene expression of oxLDL-

stimulated THP-1-macrophages. Journal of obesity, 2012.

Ofem, O., Ani, E. Eno, A. 2012. Effect of aqueous leaves extract of Ocimum gratissimum on

hematological parameters in rats. International Journal of Applied and Basic Medical

Research, 2, 38.

Oladipo, O., Conlon, S., O'grady, A., Purcell, C., Wilson, C., Maxwell, P., Johnston, P.,

Stevenson, M., Kay, E. Wilson, R. 2011. The expression and prognostic impact of CXC-

chemokines in stage II and III colorectal cancer epithelial and stromal tissue. British journal

of cancer, 104, 480.

Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders,

J., Sipes, G. Bracken, W. 2000. Concordance of the toxicity of pharmaceuticals in humans

and in animals. Regulatory Toxicology and Pharmacology, 32, 56-67.

Pan, L., Chai, H.-B. Kinghorn, A. D. 2012. Discovery of new anticancer agents from higher

plants. Frontiers in bioscience (Scholar edition), 4, 142.

Pandey, M. C., Sharma, J. Dikshit, A. 1996. Antifungal evaluation of the essential oil of

Cymbopogon pendulus (Nees ex Steud.) Wats. Cv. Praman. Flavour and fragrance journal,

11, 257-260.

Parmar, J., Sharma, P., Verma, P., Sharma, P. Goyal, P. 2011. Anti-tumor and anti-oxidative

activity of Rosmarinus officinalis in 7, 12 dimethyl benz (a) anthracene induced skin

carcinogenesis in mice. Am J Biomed Sci, 3, 199-209.

Parsa, N. 2012. Environmental factors inducing human cancers. Iranian journal of public

health, 41, 1.

Paul, W. E. 1989. Pleiotropy and redundancy: T cell-derived lymphokines in the immune

response. Cell, 57, 521-524.

References


Pecorino, L. 2012. Molecular biology of cancer: mechanisms, targets, and therapeutics,

Oxford university press.

Pedersen-Bjergaard, J., Christiansen, D., Desta, F. Andersen, M. 2006. Alternative genetic

pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related

myelodysplasia and acute myeloid leukemia. Leukemia, 20, 1943-1949.

Peng, C.-H., Su, J.-D., Chyau, C.-C., Sung, T.-Y., Ho, S.-S., Peng, C.-C. Peng, R. Y. 2007.

Supercritical fluid extracts of rosemary leaves exhibit potent anti-inflammation and anti-

tumor effects. Bioscience, biotechnology, and biochemistry, 71, 2223-2232.

Petiwala, S. M., Berhe, S., Li, G., Puthenveetil, A. G., Rahman, O., Nonn, L. Johnson, J. J.

2014. Rosemary (Rosmarinus officinalis) extract modulates CHOP/GADD153 to promote

androgen receptor degradation and decreases xenograft tumor growth. PloS one, 9, e89772.

Picaud, S., Bardot, B., De Maeyer, E. Seif, I. 2002. Enhanced tumor development in mice

lacking a functional type I interferon receptor. Journal of Interferon & Cytokine Research,

22, 457-462.

Pina‐Vaz, C., Gonçalves Rodrigues, A., Pinto, E., Costa‐de‐Oliveira, S., Tavares, C.,

Salgueiro, L., Cavaleiro, C., Goncalves, M. Martinez‐de‐Oliveira, J. 2004. Antifungal activity

of Thymus oils and their major compounds. Journal of the European Academy of

Dermatology and Venereology, 18, 73-78.

Pintore, G., Usai, M., Bradesi, P., Juliano, C., Boatto, G., Tomi, F., Chessa, M., Cerri, R.

Casanova, J. 2002. Chemical composition and antimicrobial activity of Rosmarinus

officinalis L. oils from Sardinia and Corsica. Flavour and fragrance journal, 17, 15-19.

Pokharel, M. 2012. Leukemia: a review article. Int J Adv Res Pharmaceut & Biosci, 1, 397-

407.

Powley, M. W. Carlson, G. P. 2000. Cytochromes P450 involved with benzene metabolism in

hepatic and pulmonary microsomes. Journal of biochemical and molecular toxicology, 14,

303-309.

Powley, M. W. Carlson, G. P. 2002. Benzene metabolism by the isolated perfused lung.

Inhalation toxicology, 14, 569-584.

Prasanth Reddy, V., Ravi Vital, K., Varsha, P. Satyam, S. 2014. Review on Thymus vulgaris

traditional uses and pharmacological properties. Med. Aromat Plants, 3, 164.

Pui, C.-H., Relling, M. V. Downing, J. R. 2004. Acute lymphoblastic leukemia. New England


Qamar, K. A., Dar, A., Siddiqui, B. S., Kabir, N., Aslam, H., Ahmed, S., Erum, S., Habib, S.

Begum, S. 2010. Anticancer activity of Ocimum basilicum and the effect of ursolic acid on

References


the cytoskeleton of MCF-7 human breast cancer cells. Letters in Drug Design and Discovery,

7, 726-736.

Rai, V., Pai, V. R., Kedilaya, H. Hegde, S. 2013. Preliminary phytochemical screening of

members of Lamiaceae family: Leucas linifolia, Coleus aromaticus and Pogestemon

patchouli. International Journal of Pharmaceutical Sciences Review and Research, 21, 131-

137.

Rane, S. G. Reddy, E. P. 1994. JAK3: a novel JAK kinase associated with terminal

differentiation of hematopoietic cells. Oncogene, 9, 2415-2423.

Rao, K., Schwartz, S. A., Nair, H. K., Aalinkeel, R., Mahajan, S., Chawda, R. Nair, M. P.

2004. Plant derived products as a source of cellular growth inhibitory phytochemicals on PC-

3M, DU-145 and LNCaP prostate cancer cell lines. Current science, 1585-1588.

Rappaport, S. M., Kim, S., Lan, Q., Vermeulen, R., Waidyanatha, S., Zhang, L., Li, G., Yin,

S., Hayes, R. B. Rothman, N. 2009. Evidence that humans metabolize benzene via two

pathways. Environmental health perspectives, 117, 946.

Rasekh, H. R., Hosseinzadeh, L., Mehri, S., Kamli-Nejad, M., Aslani, M. Tanbakoosazan, F.

2012. Safety assessment of ocimum basilicum hydroalcoholic extract in wistar rats: acute and

subchronic toxicity studies. Iranian journal of basic medical sciences, 15, 645.

Rates, S. M. K. 2001. Plants as source of drugs. Toxicon, 39, 603-613.

Ross, D. 2000. The role of metabolism and specific metabolites in benzene-induced toxicity:

evidence and issues. Journal of toxicology and environmental health Part A, 61, 357-372.

Rota, M. C., Herrera, A., Martínez, R. M., Sotomayor, J. A. Jordán, M. J. 2008.

Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis and

Thymus hyemalis essential oils. Food control, 19, 681-687.

Rothman, N., Smith, M. T., Hayes, R. B., Li, G.-L., Irons, R. D., Dosemeci, M., Haas, R.,

Stillman, W. S., Linet, M. Xi, L.-q. 1996. An epidemiologic study of early biologic effects of

benzene in Chinese workers. Environmental health perspectives, 104, 1365.

Rowley, J. D. 1973. A new consistent chromosomal abnormality in chronic myelogenous

leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature, 243, 290-293.

Rozman, C. Montserrat, E. 1995. Chronic lymphocytic leukemia. New England Journal of

Medicine, 333, 1052-1057.

Ruddon, R. W. 2007. Cancer biology, Oxford University Press.

Rushworth, S. A., Zaitseva, L., Murray, M. Y., Shah, N. M., Bowles, K. M. MacEwan, D. J.

2012. The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and

underlies its chemo-resistance. Blood, 120, 5188-5198.

References


Saad, B., Azaizeh, H., Abu-Hijleh, G. Said, O. 2006. Safety of traditional Arab herbal

medicine. Evidence-Based Complementary and Alternative Medicine, 3, 433-439.

Sáez, F. Stahl-Biskup, E. 2002. Essential oil polymorphism in the genus Thymus. Thyme, the

genus Thymus, 125-143.

Saha, S., Mukhopadhyay, M., Ghosh, P. Nath, D. 2012. Effect of methanolic leaf extract of

Ocimum basilicum L. on benzene-induced hematotoxicity in mice. Evidence-Based

Complementary and Alternative Medicine, 2012.

Sahaya, S. S., Janakiraman, N. Johnson, M. 2012. Phytochemical analysis of Vitex altissima

L. using UV-Vis, FTIR and GC-MS. Int J Pharm Sci Drug Res, 4, 56-62.

Sammett, D., Lee, E., Kocsis, J. Snyder, R. 1979. Partial hepatectomy reduces both

metabolism and toxicity of benzene. Journal of Toxicology and Environmental Health, Part

A Current Issues, 5, 785-792.

Sasiadek, M. 1992. Nonrandom distribution of breakpoints in the karyotypes of workers

occupationally exposed to benzene. Environmental health perspectives, 97, 255.

Schafer, Z. T. Kornbluth, S. 2006. The apoptosome: physiological, developmental, and

pathological modes of regulation. Developmental cell, 10, 549-561.

Schiffer, C. Schimpff, S. 1991. Acute leukemia. Comprehensive Textbook of Oncology, 2,

1203-121.

Sell, S. Max, E. E. 2001. Immunology, immunopathology and immunity.

Sertel, S., Eichhorn, T., Plinkert, P. K. Efferth, T. 2011. Cytotoxicity of Thymus vulgaris

essential oil towards human oral cavity squamous cell carcinoma. Anticancer research, 31,

81-87.

Shankaran, V., Ikeda, H., Bruce, A. T., White, J. M., Swanson, P. E., Old, L. J. Schreiber, R.

D. 2001. IFNγ and lymphocytes prevent primary tumour development and shape tumour

immunogenicity. Nature, 410, 1107-1111.

Sharma, U., Bala, M., Kumar, N., Singh, B., Munshi, R. K. Bhalerao, S. 2012.

Immunomodulatory active compounds from Tinospora cordifolia. Journal of

ethnopharmacology, 141, 918-926.

Shearwin-Whyatt, L., Harvey, N. Kumar, S. 2000. Subcellular localization and CARD-

dependent oligomerization of the death adaptor RAIDD. Cell death and differentiation, 7,

155.

Siddiqui, I. A., Sanna, V., Ahmad, N., Sechi, M. Mukhtar, H. 2015. Resveratrol

nanoformulation for cancer prevention and therapy. Annals of the New York Academy of

Sciences, 1348, 20-31.

References


Sikkema, J., De Bont, J. Poolman, B. 1995. Mechanisms of membrane toxicity of

hydrocarbons. Microbiological reviews, 59, 201-222.

Simandi, B., Hajdu, V., Peredi, K., Czukor, B., Nobik‐Kovacs, A. Kery, A. 2001. Antioxidant

activity of pilot‐plant alcoholic and supercritical carbon dioxide extracts of thyme. European

journal of lipid science and technology, 103, 355-358.

Simon, L. S. 2001. COX-2 inhibitors. Gastroenterology Clinics, 30, 1011-1026.

Smith, M. T. 1996. The mechanism of benzene-induced leukemia: a hypothesis and

speculations on the causes of leukemia. Environmental health perspectives, 104, 1219.

Smith, M. T., Zhang, L., Jeng, M., Wang, Y., Guo, W., Duramad, P., Hubbard, A. E.,

Hofstadler, G. Holland, N. T. 2000. Hydroquinone, a benzene metabolite, increases the level

of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells.

Carcinogenesis, 21, 1485-1490.

Snyder, R. Hedli, C. C. 1996. An overview of benzene metabolism. Environmental health

perspectives, 104, 1165.

Soković, M., Glamočlija, J., Ćirić, A., Kataranovski, D., Marin, P., Vukojević, J. Brkić, D.

2008. Antifungal activity of the essential oil of Thymus vulgaris L. and thymol on

experimentally induced dermatomycoses. Drug development and industrial pharmacy, 34,

1388-1393.

Sporn, M. B. 1976. Approaches to prevention of epithelial cancer during the preneoplastic

period. Cancer research, 36, 2699-2702.

Stam, K., Heisterkamp, N., Grosveld, G., de Klein, A., Verma, R. S., Coleman, M., Dosik, H.

Groffen, J. 1985. Evidence of a new chimeric bcr/c-abl mRNA in patients with chronic

myelocytic leukemia and the Philadelphia chromosome. New England Journal of Medicine,

313, 1429-1433.

Steelman, L., Pohnert, S., Shelton, J., Franklin, R., Bertrand, F. McCubrey, J. 2004.

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and

leukemogenesis. Leukemia, 18, 189-218.

Steward, W. Brown, K. 2013. Cancer chemoprevention: a rapidly evolving field. British

journal of cancer, 109, 1-7.

Subrahmanyam, V. V., Kolachana, P. Smith, M. T. 1991. Hydroxylation of phenol to

hydroquinone catalyzed by a human myeloperoxidase-superoxide complex: possible

implications in benzene-induced myelotoxicity. Free radical research communications, 15,

285-296.

References


Szlosarek, P., Charles, K. A. Balkwill, F. R. 2006. Tumour necrosis factor-α as a tumour

promoter. European journal of cancer, 42, 745-750.

Tada, H., Murakami, Y., Omoto, T., Shimomura, K. Ishimaru, K. 1996. Rosmarinic acid and

related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry, 42, 431-434.

Thomas, X., Michiels, J., Berneman, Z., Schroyens, W., Lam, K., Raeve, H., Imashuku, S.,

Shimazaki, C., Tojo, A. Imamura, T. 2013. First contributors in the history of leukemia.

World, 3, 001.

Thompson, J. D., Chalchat, J.-C., Michet, A., Linhart, Y. B. Ehlers, B. 2003. Qualitative and

quantitative variation in monoterpene co-occurrence and composition in the essential oil of

Thymus vulgaris chemotypes. Journal of Chemical Ecology, 29, 859-880.

Thuille, N., Fille, M. Nagl, M. 2003. Bactericidal activity of herbal extracts. International

journal of hygiene and environmental health, 206, 217-221.

Tian, T., Wang, M. Ma, D. 2014. TNF-α, a good or bad factor in hematological diseases?

Stem cell investigation, 1.

Trachootham, D., Lu, W., Ogasawara, M. A., Nilsa, R. D., Huang, P. 2008. Redox regulation

of cell survival. Antioxidants & redox signaling, 10, 1343-1374.

Tsai, P.-J., Tsai, T.-H., Yu, C.-H. Ho, S.-C. 2007. Evaluation of NO-suppressing activity of

several Mediterranean culinary spices. Food and chemical toxicology, 45, 440-447.

Tse, B. W., Scott, K. F. Russell, P. J. 2012. Paradoxical roles of tumour necrosis factor-alpha

in prostate cancer biology. Prostate cancer, 2012.

Tsujimoto, Y. Shimizu, S. 2005. Another way to die: autophagic programmed cell death. Cell

death and differentiation, 12, 1528.

Umerie, S., Anaso, H. Anyasoro, L. 1998. Insecticidal potentials of Ocimum basilicum leaf-

extract. Bioresource Technology, 64, 237-239.

Vainio, H. Weiderpass, E. 2006. Fruit and vegetables in cancer prevention. Nutrition and

cancer, 54, 111-142.

Valentine, J. L., Lee, S. S.-T., Seaton, M. J., Asgharian, B., Farris, G., Corton, J. C.,

Gonzalez, F. J. Medinsky, M. A. 1996. Reduction of benzene metabolism and toxicity in

mice that lack CYP2E1 expression. Toxicology and applied pharmacology, 141, 205-213.

Varney, M. L., Singh, S., Li, A., Mayer-Ezell, R., Bond, R. Singh, R. K. 2011. Small

molecule antagonists for CXCR2 and CXCR1 inhibit human colon cancer liver metastases.

Cancer letters, 300, 180-188.

References


Vaughan, A. T., Betti, C. J., Villalobos, M. J., Premkumar, K., Cline, E., Jiang, Q. Diaz, M.

O. 2005. Surviving apoptosis: a possible mechanism of benzene-induced leukemia. Chemico-

biological interactions, 153, 179-185.

Vila, R. 2002. Flavonoids and further polyphenols in the genus Thymus, Taylor & Francis:

New York.

Von Ardenne, M. Reitnauer, P. 1981. The elevation of the leucocyte and thrombocyte counts

produced by a thyme extract in the peripheral blood as compared to that caused by 2-

cyanoethylurea (author's transl). Die Pharmazie, 36, 703-705.

Wang, Q., Harrison, J., Uskokovic, M., Kutner, A. Studzinski, G. 2005a. Translational study

of vitamin D differentiation therapy of myeloid leukemia: effects of the combination with a

p38 MAPK inhibitor and an antioxidant. Leukemia, 19, 1812-1817.

Wang, Q., Salman, H., Danilenko, M. Studzinski, G. P. 2005b. Cooperation between

antioxidants and 1, 25‐dihydroxyvitamin D3 in induction of leukemia HL60 cell

differentiation through the JNK/AP‐1/Egr‐1 pathway. Journal of cellular physiology, 204,

964-974.

Wang, X.; Studzinski, G. P. 2001. Activation of extracellular signal‐regulated kinases

(ERKs) defines the first phase of 1, 25‐dihydroxyvitamin D3‐induced differentiation of HL60

cells. Journal of cellular biochemistry, 80, 471-482.

Wang, X.; Studzinski, G. P. 2006. Raf‐1 signaling is required for the later stages of 1,

25‐dihydroxyvitamin D3‐induced differentiation of HL60 cells but is not mediated by the

MEK/ERK module. Journal of cellular physiology, 209, 253-260.

Wang, Z., Richter, S. M., Gates, B. D., & Grieme, T. A. 2012. Safety concerns in a

pharmaceutical manufacturing process using dimethyl sulfoxide (DMSO) as a solvent.

Organic Process Research & Development, 16(12), 1994-2000.

Ward, A. F., Braun, B. S. Shannon, K. M. 2012. Targeting oncogenic Ras signaling in

hematologic malignancies. Blood, 120, 3397-3406.

Warner, T. D., Giuliano, F., Vojnovic, I., Bukasa, A., Mitchell, J. A. Vane, J. R. 1999.

Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are

associated with human gastrointestinal toxicity: a full in vitro analysis. Proceedings of the

National Academy of Sciences, 96, 7563-7568.

Waters, E. A., Cronin, K. A., Graubard, B. I., Han, P. K. Freedman, A. N. 2010. Prevalence

of tamoxifen use for breast cancer chemoprevention among US women. Cancer

Epidemiology and Prevention Biomarkers, 19, 443-446.

References


Wattenberg, L. W. 1990. Inhibition of carcinogenesis by minor anutrient constituents of the

diet. Proceedings of the Nutrition Society, 49, 173-183.

Wetmore, B. A., Struve, M. F., Gao, P., Sharma, S., Allison, N., Roberts, K. C., Letinski, D.

J., Nicolich, M. J., Bird, M. G. Dorman, D. C. 2008. Genotoxicity of intermittent co-exposure

to benzene and toluene in male CD-1 mice. Chemico-biological interactions, 173, 166-178.

Wikler, M. A. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that

grow aerobically: approved standard, Clinical and Laboratory Standards Institute.

Williams, J. G. 2000. STAT signalling in cell proliferation and in development. Current

opinion in genetics & development, 10, 503-507.

Witz, G., Zhang, Z. Goldstein, B. D. 1996. Reactive ring-opened aldehyde metabolites in

benzene hematotoxicity. Environmental health perspectives, 104, 1195.

Wong, F. C., Woo, C. C., Hsu, A. Tan, B. K. H. 2013. The anti-cancer activities of Vernonia

amygdalina extract in human breast cancer cell lines are mediated through caspase-dependent

and p53-independent pathways. PloS one, 8, e78021.

Woo, H. J., Lee, J. Y., Woo, M. H., Yang, C. H. Kim, Y. H. 2011. Apoptogenic activity of

2α, 3α-dihydroxyurs-12-ene-28-oic acid from Prunella vulgaris var. lilacina is mediated via

mitochondria-dependent activation of caspase cascade regulated by Bcl-2 in human acute

leukemia Jurkat T cells. Journal of ethnopharmacology, 135, 626-635.

Wu, X., Patterson, S. Hawk, E. 2011. Chemoprevention–history and general principles. Best

practice & research Clinical gastroenterology, 25, 445-459.

Yager, J. D. Davidson, N. E. 2006. Estrogen carcinogenesis in breast cancer. New England


Yamamura, K., Katoh, T., Kikuchi, M., Yoshikawa, M. Arashidani, K. 1999. Effect of

benzene exposure on hematology and hepatic drug metabolic enzymes in ethanol

administrated rats. Journal of UOEH, 21, 29-35.

Yee, C., Thompson, J., Byrd, D., Riddell, S., Roche, P., Celis, E. Greenberg, P. 2002.

Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of

patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of

transferred T cells. Proceedings of the National Academy of Sciences, 99, 16168-16173.

Yesil-Celiktas, O., Sevimli, C., Bedir, E. Vardar-Sukan, F. 2010. Inhibitory effects of

rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer

cell lines. Plant Foods for Human Nutrition, 65, 158-163.

References


Yi, W. Wetzstein, H. Y. 2011. Anti‐tumorigenic activity of five culinary and medicinal herbs

grown under greenhouse conditions and their combination effects. Journal of the Science of

Food and Agriculture, 91, 1849-1854.

Yu, S. Kong, A.-N. 2007. Targeting carcinogen metabolism by dietary cancer preventive

compounds. Current cancer drug targets, 7, 416-424.

Zaidi, M. A. Crow, S. A. 2005. Biologically active traditional medicinal herbs from

Balochistan, Pakistan. Journal of ethnopharmacology, 96, 331-334.

Zaker, F., Darley, R. L., Al Sabah, A. Burnett, A. K. 1997. Oncogenic RAS genes impair

erythroid differentiation of erythroleukaemia cells. Leukemia research, 21, 635-640.

Zhang, L., Eastmond, D. A. Smith, M. T. 2002. The nature of chromosomal aberrations

detected in humans exposed to benzene. Critical reviews in toxicology, 32, 1-42.

Zhang, L., Rothman, N., Wang, Y., Hayes, R. B., Yin, S., Titenko-Holland, N., Dosemeci,

M., Wang, Y.-Z., Kolachana, P. Lu, W. 1999. Benzene increases aneuploidy in the

lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ

hybridization in interphase and metaphase cells. Environmental and molecular mutagenesis,

34, 260-268.

Zhang, L., Venkatesh, P., Creek, M. L. R. Smith, M. T. 1994. Detection of 1, 2, 4-

benzenetriol induced aneuploidy and microtubule disruption by fluorescence in situ

hybridization and immunocytochemistry. Mutation Research/Genetic Toxicology, 320, 315-

327.

Zhao, X., Mohaupt, M., Jiang, J., Liu, S., Li, B. Qin, Z. 2007. Tumor Necrosis Factor

Receptor 2–Mediated Tumor Suppression Is Nitric Oxide Dependent and Involves

Angiostasis. Cancer research, 67, 4443-4450.

Zhong, F., Tong, Z.-T., Fan, L.-L., Zha, L.-X., Wang, F., Yao, M.-Q., Gu, K.-S. Cao, Y.-X.

2016. Guggulsterone-induced apoptosis in cholangiocarcinoma cells through ROS/JNK

signaling pathway. American journal of cancer research, 6, 226.

Zhou, G.-B., Zhang, J., Wang, Z.-Y., Chen, S.-J. Chen, Z. 2007. Treatment of acute

promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide: a paradigm of

synergistic molecular targeting therapy. Philosophical Transactions of the Royal Society B:

Biological Sciences, 362, 959-971.

Zouari, N., Ayadi, I., Fakhfakh, N., Rebai, A. Zouari, S. 2012. Variation of chemical

composition of essential oils in wild populations of Thymus algeriensis Boiss. et Reut., a

North African endemic Species. Lipids in health and disease, 11, 28.

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