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INVESTIGATIONS INTO THE EFFECTS OF TRADITIONAL CHINESE MEDICINAL HERBS USED IN
THE TREATMENT OF HUMAN BREAST CANCER
AGNES SLATER
B.A. (ELTE University, Budapest)
M.B.B.S. (Beijing Medical University, Beijing)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHARMACOLOGY
FACULTY OF MEDICINE
NATIONAL UNIVERSITY OF SINGAPORE
2005
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ACKNOWLEDGEMENTS
I would like to express my sincere appreciation and gratitude to the following:
- my supervisor, Assoc. Prof. Benny Kwong Huat Tan, for his invaluable
guidance, patience and constant encouragement
- our Senior Laboratory Officer, Ms. Annie Hsu, for her assistance, expertise
and kindness
- Head and Staff of Department of Pharmacology for making the laboratory
facilities available for my use and for their cooperation
- Dr. C.S.L. Fones, Dr. Teo Eng Kiat, Dr. Ruth Kiew, Mr. Jonathan Ward, Prof.
Chong Siew Meng for the willingness to share their expertise
- National University of Singapore for providing me with the scholarship
- my family and friends for their moral support
Without the above it would not have been possible to complete this project.
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TABLE OF CONTENTS
Page ACKNOWLEDGEMENT............................................................................... ii
TABLE OF CONTENTS ................................................................................ iii
LIST OF TABLE ............................................................................................... viii
LIST OF FIGURES ......................................................................................... ix
LIST OF ILLUSTRATIONS .......................................................................... xi
LIST OF ABBREVIATIONS ......................................................................... xii
LIST OF PUBLICATIONS AND CONFERENCE PAPERS ..................... xv
SUMMARY ...................................................................................................... xvi
1. CHAPTER ONE: GENERAL INTRODUCTION 1
1.1. Traditional Chinese Medicine (TCM)................................. 2
1.2. Herbs used in TCM for cancer treatment .......................... 5
1.3. Breast cancer: significance in modern day society ............ 20
1.4. Rationale and purpose of this study.................................... 22
2. CHAPTER TWO: MATERIALS AND METHODS 24
2.1. Materials ................................................................................ 25
2.1.1. Cell lines ........................................................................ 25
2.1.2. Animals .......................................................................... 27
2.1.3. Chemicals and reagents.................................................. 27
2.1.4. Kits................................................................................. 28
2.1.5. Facilities......................................................................... 28
2.2. Methods.................................................................................. 29
2.2.1. Quality-of-life (QOL) study ………………………….. 29
2.2.1.1.QOL study design………………..……………………………. 29
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2.2.1.2.Scoring procedures for the EORTC QLQ-30 and BR-23
questionnaires……….…………………………………………... 41
2.2.2. Extraction of herbs.........................................………… 41
2.2.2.1. Preparation of water and ethanolic extract of Astragalus
membranaceus, Curcuma aromatica, Scutellaria barbata and
Solanum nigrum .................................................................... 42
2.2.2.2. Fractionation of the water boiled extract of Astragalus
membranaceus ……… ......................................................... 42
2.2.2.3. Characterization of the ethanolic and water extract of
Solanum nigrum……… ............................................................................. 43
2.2.3. Breast cancer cell culture ............................................... 43
2.2.4. Cell viability assays: MTT test and trypan blue exclusion
examination .................................................................................. 44
2.2.5. Lymphocyte activation assay......................................... 44
2.2.6. Assay of cytokines: IL-2, IL-6 and TNF-α .................... 45
2.2.7. In-vitro cytotoxicity assay.............................................. 46
2.2.8. In-vitro analysis to determine the type of cell death...... 46
2.2.8.1. DNA fragmentation ELISA assay (Fig.1) ............... 46
2.2.8.2. Cellular DNA fragmentation ELISA assay (Fig.2)... 48
2.2.9. Animal handling............................................................. 49
2.2.9.1. Housing and maintenance ........................................ 49
2.2.9.2. Mice gavage ............................................................. 50
2.2.9.3. Blood collection by intracardiac puncture ............... 50
2.2.9.4. Intraperitoneal (i.p.) injection .................................. 50
2.2.9.5. Acute toxicity testing ............................................... 50
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2.2.10. Breast tumor model in BALB/c nude mice.................... 51
2.2.10.1.Tumor xenografts……………………………………………. 51
2.2.10.2.In-vivo treatment protocols……………………………….. 51
2.2.11. Histopathological analysis of tumor tissue sections ...... 52
2.2.12. Statistical analysis.......................................................... 52
3. CHAPTER THREE: RESULTS AND DISCUSSION 53
3.1. Quality-of-life (QOL) study in breast cancer patients .... 54
3.1.1. Aims and experimental approach................................... 54
3.1.2. Results............................................................................ 55
3.1.3. Discussion...................................................................... 68
3.2. Extraction and characterization of Solanum nigrum ..... 71
3.2.1. Aims and experimental approach................................... 71
3.2.2. Results............................................................................ 72
3.2.3. Discussion...................................................................... 74
3.3. Results of in-vitro immunomodulatory studies .............. 74
3.3.1. Aims and experimental approach.................................… 74
3.3.2. Results.............................................................................. 75
3.3.2.1.Effect of ethanolic extract of Astragalus
membranaceus and Curcuma aromatica…………………… 75
3.3.2.2.Effect of the fractions of Astragalus
membranaceus water extract…………………………. 76
3.3.3. Discussion...................................................................... 80
3.4. Cytokine studies from mice serum ...................................... 80
3.4.1. Aims and experimental approach................................... 80
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3.4.2. Results............................................................................ 81
3.4.3. Discussion...................................................................... 81
3.5. In-vitro cytotoxicity studies .................................................. 82
3.5.1. Aims and experimental approach …………………….. 82
3.5.2. Results............................................................................ 82
3.5.2.1. Results of cytotoxicity assays of Solanum nigrum and
Scutellaria barbata in MCF-7 human breast cancer cell
lines.. ............................................................................................................ ….. 82
3.5.2.2. Results of cytotoxicity assays of Solanum nigrum and
in MCF-7, MDA-MB-231, T-47D and ZR-75-1 human breast
cancer cell lines...................................................................... 85
3.5.2.3. Results of DNA fragmentation in MCF-7 cells treated
with Solanum nigrum……………………………………………………… 86
3.5.2.4. Results of cellular DNA fragmentation in MDA-MB-231
cells treated with Solanum nigrum……………………………………. 88
3.5.3. Discussion…………………………………………………………… 92
3.6. In-vivo cytotoxicity studies……………………………………. 92
3.6.1. Aims and experimental approach……………………………….. 92
3.6.2. Results…………………………………………………... 93
3.6.2.1. Acute toxicity test of Solanum nigrum in normal and
nude BALB/c mice................................................................ 93
3.6.2.2. Anti-tumor activity of Solanum nigrum on MCF-7
tumor-bearing oophorectomized BALB/c nude mice………. 94
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3.6.2.3. Estrogen-induced uterine cancer in oophorectomized
BALB/c nude mice………………………………………….. 97
3.6.2.4. Anti-tumor activity of Solanum nigrum on MDA-MB-231
tumor-bearing oophorectomized BALB/c nude mice……… 99
3.6.2.4.1. Results of therapeutic treatment with Solanum
nigrum.............................................................................. 99
3.6.2.4.2. Results of prophylactic treatment with
Solanum nigrum ............................................................... 101
3.6.2.5. Histopathological examination of Solanum nigrum
induced apoptosis in MDA-MB-231 cells........................................... 102
3.6.3. Discussion................................................................... 102
4. CHAPTER FOUR: SUMMARY AND DISCUSSION 104
4.1. Summary of results ............................................................... 105
4.2. Overall discussion ................................................................. 108
5. CHAPTER FIVE: CONCLUSION AND FUTURE STUDIES. 109
5.1. Conclusion ............................................................................. 110
5.2. Future studies ........................................................................ 110
6. BIBLIOGRAPHY.......................................................................... 111
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LIST OF TABLES
Page
Table 1. Conventional treatment of selected patients………………….......... 30
Table 2. Composition of EORTC QLQ-C30 (version 3.0) .................................. 36
Table 3. Composition of EORTC breast cancer module QLQ-BR23………….. 38
Table 4. Effect of different concentrations of 80% ethanol extract of Astragalus membranaceus and Curcuma aromatica on BALB/c mice lymphocyte proliferation……………………………………………………………… 75
Table 5. Cytokine concentration after AM treatment in BALB/c mice……………. 81
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LIST OF FIGURES
Page
Figure 1. Cell death detection ELISAPLUS. .......................................................... 47
Figure 2. Cellular DNA fragmentaion ELISA…………………………………. 49
Figure 3. QLQ-30 scores for 2 breast cancer patients receiving radio-, chemo-, hormonal and herbal TCM therapy…………………………………. 55
Figure 4. QLQ-30 scores for 1 breast cancer patient receiving radio-, hormonal and herbal TCM therapy ...................................................................... 57
Figure 5. QLQ-30 scores for 2 breast cancer patients receiving hormonal and herbal TCM therapy…………………………………………………. 59
Figure 6. QLQ-30 scores for breast cancer patient receiving only herbal TCM Therapy……………………………………………………………… 61
Figure 7. QLQ-30 reference scores compared to the baseline scores of 6 breast
cancer patients in Singapore receiving herbal TCM therapy…………. 63
Figure 8. BR-23 scores of breast cancer patients at baseline and after 11-13 weeks of TCM treatment……………………………………………… 65
Figure 9. Single item score distribution of 6 breast cancer patients at baseline and
after 11-13 weeks of TCM………………………………………….. 67
Figure 10. SN water and ethanolic extract UV spectophotometer scan……… 72
Figure 11. HPLC chromatogram of SN water extract…………………………… 73
Figure 12. Immunomodulatory effect of the ethanolic extract of AM on mouse lymphocytes…………………………………………………………. 77
Figure 13. Immunomodulatory effect of the aqueous extract of AM on mouse splenocytes…………………………………………………………. 77
Figure 14. Immunomodulatory effect of butanolic fraction of aqueous extract of AM on mouse splenocytes………………………………………. 78
Figure 15. Immunomodulatory effect of ethyl acetate fraction of aqueous extract of AM on mouse splenocytes………………………………… 78
Figure 16. Immunomodulatory effect of remaining water fraction of aqueous
Extract of AM on mouse splenocytes………………………………… 79
Figure 17. Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells using MTT-tetrazolium assay …………………………… 83
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Figure 18. Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells (log scale)…..………………………………. 84 Figure 19. Cytotoxic effect of the ethanol extract of SN on MCF-7, MDA-MB-231, T-47D and ZR-75-1 cell lines………………………………………….. 85
Figure 20. DNA fragmentation in MCF-7 cells (SN 4h)……………………….. 86
Figure 21. DNA fragmentation in MCF-7 cells (SN 8h)…………………...……… 86 Figure 22. DNA fragmentation in MCF-7 cells (SN 12h)…………………...…… 87 Figure 23.. DNA fragmentation in MCF-7 cells (SN 24h)…………………....…. … 87 Figure 24. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of
SN on MDA-MB-231 after 2h incubation …………………………… 88 Figure 25. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of
SN on MDA-MB-231 after 4h incubation …………………………… 89 Figure 26. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of
SN on MDA-MB-231 after 6h incubation …………………………… 89 Figure 27. Acute toxicity test of Solanum nigrum (water extract) in Swiss albino mice……………………………………………………………………………… 93 Figure 28. Mortality of 50 beta-estradiol implanted nude mice over time……… 97 Figure 29. Prophylactic treatment of Solanum nigrum (ethanolic extract) on MDA-MB-231 implanted nude mice…………………………………………… 101
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LIST OF ILLUSTRATIONS
Page
Picture 1. Dried roots of Astragalus membranaceus………………………………….. 11
Picture 2. Dried roots of Curcuma aromatica…………………………………… 13
Picture 3. Reconstruction of Solanum nigrum…………………………………… 15
Picture 4. Number of death from breast cancer in Singapore among women aged 0-85 between years 1963-2002 (WHO IARC, 2005) …… ……………. 20 Picture 5. MCF-7 cells................................................................................................... 25 Picture 6. MDA-MB-231 cells……………………………………………………………………….. 26 Picture 7a, 7b. EORTC QOL-C30 (version 3) questionnaire…………………………………. 32 Picture 8a, 8b. EORTC QOL-BR23 questionnaire…………………...……………………………34 Picture 9. MDA-MB-231 cells incubated with SN extract (100X) ……………………….. 90 Picture 10. Large solid MCF-7 tumor in estrogen-primed mouse #1…………………… 95 Picture 11. Two solid breast tumors grown from mouse #1……………………….. 95 Picture 12. Multiple solid tumors grown from MCF-7 implants from mouse #2……………………………………………………………………………. …… 96 Picture 13. Cachexic mouse on the left compared to SN-treated mouse
on the right……………………………………………………………………………….… 96 Picture 14. Mouse uterine tissue H&E staining………………………………………............... 98 Picture 15. Enlarged mouse uteri on post-mortem examination…………………………. 98 Picture 16 (a, b, c). MDA-MB-231 metastasis to the spine ………..……….……… 99 Picture 17. (a, b, c). MDA-MB-231 metastasis to the orbit………………..……. …….. 100 Picture 18. SN extract versa saline treated MDA-MB-231 tumors………………….... 102
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LIST OF ABBREVIATIONS
ABTS: 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
AM: Astragalus membranaceus
BALB: Bagg albino
BRCA1: breast cancer gene 2
BRCA2: breast cancer gene 1
BrdU: 5-bromo-2-deoxyuridine
BW: body weight
CA: Curcuma aromatica
ConA: Concanavalin A
DMSO: Dimethyl sulphoside
DOX: Doxorubicin
EDTA: Ethylenediaminetetraacetic acid
EORTC: European Organization of Research and Treatment of Cancer
EGF: epidermal growth factor
FBS: fetal bovine serum
5-FU: 5-fluorouracil
HPLC: high performance liquid chromatography
IC50: 50% inhibition concentration
IL-2: interleukin-2
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IL-6: interleukin-6
i.p.: intraperitoneal
LPS: Lipopolysaccharide
6-MP: 6-mercaptopurine
MTP: micro titer plate
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thiazol blue)
NSAID: non-steroidal anti-inflammatory drug
O.D.: optical density
PBS: Phosphate-buffered saline
QOL: quality-of-life
QLQ: quality-of-life questionnaire
QLQ-C30: quality-of-life core questionnaire
QLQ-BR23: quality-of-life breast cancer supplementary questionnaire
ROS: reactive oxygen species
RS: Raw Score
S: score
s.c.: subcutaneous
SB: Scutellaria barbata
SDS: Dodecyl-sulphate sodium salt
SN: Solanum nigrum
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TCM: traditional Chinese medicine
TNF-α: tumor necrosis alpha
WHO IARC: World Health Organization, International Agency for Research on Cancer
xv
LIST OF PUBLICATIONS
1. Slater A, Tan BKH (2004): Immunomodulating property of Astragalus membranaceus and Curcuma aromatica. In: Novel Compounds from Natural Products in the New Millennium. Potential and Challenges, Eds; TanBKH, Bay BH and Zhu YZ, pp.217-221. World Scientific Press, Singapore. 2. Tan BKH, Slater A (2003): Traditional Chinese Medicines in breast cancer: clinical and experimental data. Int. Journal of Molecular Medicine. 12(1): S68.
3. Slater A, Fones CSL and Tan BKH (2001): The use of herbal Traditional Chinese Medicine as supportive treatment in breast cancer patients. In: Traditional Healing Systems: Negotiating Science & Technology Challenges. Indigenous Knowledge Systems Research & Development Studies (INDAKS) Series, Ed: Quah S, pp.81-93. Leiden Ethnosystems and Development Programme (LEAD), Institute of Cultural and Social Studies, Leiden University, Holland.
LIST OF CONFERENCE PRESENTATIONS
1. Tan BKH, Hsu A, Slater A: Solanum nigrum – a traditional Chinese medicinal herb for use in human breast cancer. 4th International Conference on Natural Products, Leysin, Switzerland, 28-31 May, 2006 2. Tan BKH, Slater A: Effects of Solanum nigrum in breast cancer: Clinical and experimental data. Ist International Conference on Complementary and Alternative Medicine, Singapore, 25-27 Feb 2005 3. Slater A, Tan BKH: In-vivo studies to investigate effects of Solanum nigrum extract on human breast cancer growth in nude mice. Leura V International Breast Cancer Conference Sydney, Australia 10 - 14 November 2004
4. Slater A, Tan BKH: Some observations on tumor growth in estradiol-primed oophorectomized nude mice. Berries in Cancer Prevention: from experimental findings to humans, Lahti, Finland 10-11 July 2004 5. Slater A, Tan BKH: Cytotoxic property of Solanum nigrum on MCF-7 human breast cancer cell line. Annual Oncology Symposium, Singapore, 25-27 October 2002 6. Slater A, Tan BKH: Immunomodulating property of Astragalus membranaceus and Curcuma aromatica in breast cancer patients. 2nd International Conference on Natural Products, Singapore 1-4 July 2002
xvi
SUMMARY
The incidence of breast cancer is the highest among Singaporean women, with more
then 1000 new cases diagnosed each year. There is an urgent need for new forms of
treatment i.e. complementary/ alternative medicine (CAM). Traditional Chinese
Medicine (TCM) is the most popular type of CAM for the treatment of cancer among
the Chinese population in Singapore. In most cases it is used to complement
conventional treatment (surgery, radio-, chemo- and/or hormonal therapy).
We sought a scientific evaluation of the medicinal herbs that might have
immunomodulatory and/or anti-tumor effects and could palliate the side-effects
(nausea, insomnia, pain and fatigue) of chemo- and radiotherapy.
The investigation was carried out in two phases. In the clinical phase of our study, a
quality-of- life (QOL) analysis of completed questionnaires from 6 patients was
performed using the European Organization for Research and Treatment of Cancer
(EORTC) QOL questionnaire (QLQ-C30 version 3.0) and the supplementary breast
cancer module (QLQ-BR23). We compared the herbal prescription of the patients
who had better scores on the symptoms scale (i.e. less fatigue, pain, dyspnoea,
insomnia and appetite loss).
In the laboratory phase, we selected the 2 herbs, Astragalus membranaceus (AM) and
Curcuma aromatica (CA) that we found to be the most frequently prescribed for their
possible immunomodulatory effect. We tested these herbs for their effects on
lymphocyte activation and cytokine (IL-2, IL-6, TNF-α) production.
We also selected Solanum nigrum (SN) which was used for its cytotoxic property
against breast cancer. We evaluated the tumor cell growth inhibitory effect of the
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crude ethanolic extract of SN on MCF-7, T-47D, ZR-75 and MDA-MB -231 human
breast cancer cell lines. The lowest ED50=7.75 µg/ml was obtained with the estrogen-
dependent MCF-7 cell line. We also demonstrated apoptosis as the cause of cell
death, both by ELISA and histopathology.
Experiments were also conducted in oophorectomized nude mice models with breast
tumor implants. The anti-tumor activity of SN was assumed in these animals by
measuring tumor growth and life span prolongation as parameters. We found that SN
treatment caused irreversible tumor regression where the tumor size was relatively
small (MCF-7 and MDA-MB-231) or when it was given simultaneously with the
tumor implant. SN 300 mg/kg BW was effective against small volume tumors but not
efficient against larger, multiple tumors. SN 500 mg/kg BW could be used as
prevention (producing a prophylactic rate of 75%) but was not an efficient dose of
invasive tumors. Mice bearing human breast tumors treated with SN maintained their
original body weight without any change in behavior.
This study suggests there is potential usefulness in using herbal TCM to treat breast
cancer patients.
1
CHAPTER ONE: GENERAL INTRODUCTION
2
1.1 Traditional Chinese Medicine (TCM)
Complementary and alternative medicine (CAM) is a booming healthcare industry in
Europe and U.S.A. and is used by 25%-69% of the general population of developed
countries (Richardson et al., 2000).
CAM has been defined as “diagnosis, treatment and/or prevention which complements
mainstream medicine by contributing to a common whole, by satisfying a demand not
met by orthodoxy or by diversifying the conceptual frameworks of medicine” (Ernst et
al., 1995). It has also been described as “medical and healthcare practices outside the
realm of conventional medicine, which are yet to be validated using scientific methods”
(National Center for Complementary and Alternative Medicine, Strategic Plan, 2000).
In South-East Asia, the most frequently used CAM interventions are Traditional
Chinese Medicine (TCM) and Ayurvedic Medicine systems (Dobs, 2001). TCM
includes different treatment modalities such as herbal medicine, acupuncture,
moxibustion, oriental massage, qi-gong and diet. The practice of herbal TCM
(“phytotherapy”) has been defined as “the use of products derived from plants to treat
diseases or eliminate pain” (The Cochrane Collaboration, 1993).
In Singapore, acupuncture and herbal TCM are the most popular forms of alternative
medicine due to the majority of the population being ethnic Chinese (personal
communication with Dr. Teo Eng Kiat; 2001), principal of the Singapore College of
TCM and Chairman, TCM Practitioners Board Examination Committee, Singapore.
Singaporeans of Chinese descent, who comprise 76.8% of the total population
(Singapore Department of Statistics, 2000), have used TCM for many decades, and tend
to turn to this form of treatment for a variety of diseases, from simple colds to advanced
cancer (Ho et al., 1980).
3
According to Dr. Teo, as many as 40% of the Singapore population seek TCM
treatment as the primary form of treatment. Statistics compiled in 1994 indicate that an
estimated 12% of the daily outpatient attendance of about 10,000 people was seen by
TCM practitioners (Hsu et al.,1995).
With the recent legislation requiring all TCM practitioners in Singapore to upgrade their
skills and knowledge to obtain certification to practice by the local health regulatory
authority, the percentage of TCM users is likely to increase in the coming years (Health
Science Authorities, Singapore, 2002).
One important reason for the choice of TCM is economic - it is considered a more
affordable form of health care, compared to the more sophisticated form of Western
medicine. Some of the clinics in Singapore, like the Chung Hwa Free Clinic, offer free
consultation and treatment to cancer sufferers. Some of the patients who seek TCM
treatment do so because they believe it has less side effects and toxicity, and can
improve their immune system. Other patients have incurable end-stage disease (e.g.
chronic renal failure, liver cirrhosis, cancer), where conventional treatment has failed to
cure, and TCM is tried as a last choice treatment to relieve their symptoms and prolong
life (Ernst et al.,, 1998; Richardson et al., 2000).
Some cancer patients in Singapore do take TCM at some stage of their illness. Most of
these patients do not disclose this to their physicians as they fear losing their doctor’s
trust or being discriminated against. Thus primary care physicians usually
underestimate their patients’ use of complementary medicine. In USA, 72% of the
patients fail to report to their physicians the use of CAM. In Israel, one study showed
that only 18.7% of CAM-using patients reported having ever used CAM (Eisenberg et
al., 1993; Kitai et al., 1998; Giveon et al., 2003). A similar problem was reported in
4
Canada, where only 46.4% of breast cancer patients reported their use of CAM to their
physician (Boon et al., 2000).
The current approach of physicians to complementary medicine in primary care is
essentially “don’t ask, don’t tell” (Perlman et al., 1999).
There is a generally low acceptance of herbal TCM by oncologists and other
conventional medical practitioners here and elsewhere. Their main concerns are that
herbal medicine might be contaminated with high levels of toxic heavy metals, such as
mercury or lead (Wong et al., 1985) and could cause direct adverse effects such as
allergic reactions, nausea, vomiting and sedation (Ernst et al., 1996; Shaw et al., 1997).
There is also concern that TCM treatment could indirectly interfere with the effects of
radio- or chemotherapy, delaying the recovery of patients and putting them at
unnecessary risk (Brown , 1986; Kaegi , 1998). There is an urgent need that the health
care practitioners understand the CAM products their patients are using and are familiar
with the information about the safety and efficacy of these herbal products (Smith et al.,
1999).
Western medicine is based on an understanding of anatomical structures or organs and
the cause of disease is viewed as the pathological function of these organs – a diseased
structure causes dysfunction of the body. In contrast, TCM is based on the “system
theory” and the human body is viewed as a part of a complex structure of organs – liver,
heart, spleen, lung and kidney (these do not correspond to the exact anatomical
structures known in Western medicine). This system undergoes spatial, functional and
temporal changes and allows open exchange of substance and energy with the
environment and interaction/ interrelation between the organs/ compartments in the
body. The main substance of energy transfer is qi which can’t be seen but can be
detected by modern technology (Seto et al., 1992). The imbalance in the fluctuation of
5
internal and external energy and a weakened self-regulatory power can cause disease.
This is a chain reaction of chemical, physiological and biological changes. In TCM, the
etiology of a disease is attributed to the malfunction of the system which causes a
change in the organs – the emphasis is based on the function/ dysfunction of an organ
which triggers pathological structural changes in the organ. The main principle of TCM
treatment is to cure this deviation by eliminating the pathological factors and by
strengthening the self healing (Cheng, 2005).
Appropriate application of dosages of Chinese herbs based on differentiation of
symptoms and signs is important in order to achieve better therapeutic results with less
adverse effects. The diagnosis is not disease- but patient-specific. The TCM physicians
place great emphasis on the severity of disease, the form/ formula/ dosage of the herbs,
gender and age of the patients. Different patients may have different reactions to the
same herbs and the same formulae with different dosage/ amounts/ proportions of
Chinese herbs may have different functions and indications, thus making the treatment
individually tailored (Deng, 2002). This also makes TCM-related clinical trial designs
very challenging.
1.2 Herbs used in TCM for cancer treatment
In TCM, cancer is viewed as the result of imbalance of the whole body-mind network
by taking into account the human body as whole. The tumor is due to stagnation or
accumulation of pathological elements (qi, blood, phlegm or body fluids). This
functional disorder leads to structural changes (tumor growth) when the body loses its
capacity for recovery. The aim of herbal treatment is to strengthen and restore the yin
and yang balance, the Five Organs System, the external and internal factors in order to
cure the cancer. In conventional medicine, the therapeutic aim is to destroy the cancer
6
cells and prevent their spread by applying chemo-, radio, hormonal and surgical
treatments.
There is emerging evidence that TCM can play an important role in the supportive
treatment of cancer patients. Though the way cancer and its treatment is viewed in
Western (disease-specific) and Chinese medicine (patient-specific) is very different, the
holistic approach of TCM could possibly be integrated into conventional Western
medicine to palliate the side-effects of the latter (Wong et al., 2001).
Plants have been used as medicinal agents for centuries, and they continue to play an
important role in primary health care. More than 50% of all drugs in clinical use are
derivatives of natural products and 12 of the world’s 25 best–selling pharmaceuticals
originate from plants (Baker et al., 1995).
Natural product extracts like Aloe vera (to treat burns and skin abrasions), Echinacea
(to boost the immune system and fight common colds), Allium sativum (to lower high
cholesterol levels), Ginger (for immunostimulation and inhibition of cancer metastasis),
Ginkgo biloba (antioxidant, for improving microcirculation in the brain, reducing
tendency for blood clot formation), Glucosamine (to rebuild and maintain worn
cartilage), Artemisia annua (for treating fever and drug resistant malaria), Green tea
(antioxidant), Lingzhi (to stimulate the immune system, anticancer properties), Royal
jelly (high vitamin, amino acid and mineral content), Vitex Agnus castus (to balance
estrogen-progesterone level), Lavandula augustifolia (anti-inflammatory), Pinus
sylvestris (adrenal cortex stimulant) and Tanacetum annum (anti-histamine) are popular
health supplements (Dewick, 1999; Effert, 2005; Schnaubelt, 2005).
The most well-known anti-cancer drugs derived from natural products are the Vinca
alkaloids (Vinscristine, Vinblastin, Vindesin), Epipodophyllotoxins (Etoposide),
7
Taxanes (Paclitaxel, Docelatel), Camptothecins (Topotecan, Irinotectan), and the
antibiotics (Doxorubicin, Mitomycin, Bleomycin) [Rang et al., 2003].
According to TCM, herbal treatment can eliminate the disease indirectly by boosting
the host immune system (immunostimulation) and directly by eliminating the disease
(cytotoxicity). The herbs prescribed for tumors are usually (a) tonifying herbs which act
on multiple organ systems and stimulate the immune system and (b) tumor-dispersing
herbs which eliminate the pathological external factors (i.e. stress, unhealthy diet and
lifestyle).
The guiding principles in TCM treatment of tumors are: “supplementing qi and
nourishing blood” (in biomedical terms: to stimulate the bone marrow and increase the
white blood cell count); “dispersing the lucid and sending down the turbid” (to protect
renal function, decrease serum urea nitrogen and creatinine level); relieving abdominal
distention (due to accumulation of gas from long-term bed rest), “ regulating the flow of
qi”, (removing blood stasis to alleviate the pain and reduce fever due to
infections)[Zhou et al., 2001].
The specific aim of herbal TCM is to slow disease progression by modulating the
neuro-endocrine and immune systems, to inhibit the tumor growth and to improve the
quality of life of the patients. Different herbs with different disease fighting pattern are
used simultaneously, assuming that groups of chemicals in these herbs produce a cluster
of actions on various cells, cellular organs and multiple receptors, thus achieving a more
efficient cure. In TCM, these herbs are used for their organ system properties (the way
they can rectify the functional and structural deviation in the body) and not for their
chemical properties. In Western medicine usually a single chemical compound is used
to act on a single cell type and receptor, however (especially with chemotherapeutic
drugs) the physicians tend to use multi-drug treatments to palliate the side-effects of the
8
anticancer drugs. TCM takes into consideration the property of a whole herb thus using
multiple chemical compounds which work synergistically on multiple systems,
receptors and enzymes (Swee, 2005). For example, a mixture of TCM herbs for breast
cancer patients would contain herbs which aim not only to shrink the tumor itself but
also palliate insomnia and anxiety, enhance appetite and boost the immune system, thus
having an integrated effect on the different symptoms of a breast cancer patient.
Chinese herbs have been reported to have the effect of blocking precancerous lesions by
removing carcinogenic factors and by chemoprophylaxis, as well as slow the
development of tumors by inhibiting cancer cell infiltration into the surrounding tissue
and thus reduce metastasis (Wu et al., 2001; Niu et al., 2003).
Treatment with CAM interventions could also reduce the oxidative stress in cancer cells
(Nelson et al., 2001).
At the cellular level, the tumor-reversing effects of Chinese herbs could occur either by
inducing early stage apoptosis of tumor cells or by repressing proliferation and causing
arrest of cancer cells at the G0/G1 stage (Yano et al., 1994).
The use of TCM is very much embedded in Chinese culture both in Singapore and
Hong Kong. As many breast cancer patients believe that diet is responsible for their
cancer, food, diet and herbs should also play an important role in the management of
their illness (Simpson, 2003).
In Singapore, herbal TCM is the most popular type of alternative medicine. Most of the
patients seek TCM treatment because they believe it has less side-effects and toxicity,
and can enhance their immune system.
In the breast cancer patients we studied, herbal TCM was used to complement
conventional treatment (surgery, radio-, chemo- and hormonal therapy). We sought a
9
scientific evaluation of the immunomodulatory effects of some Chinese herbs used
widely in breast cancer patients at two TCM Clinics that we surveyed.
While the patients in this study had the same diagnosis (breast cancer), their diagnosis
according to TCM principles could be very different and variable at each visit
(stagnation of qi, blood, phlegm, body fluids; dampness type; heat type etc).
Each TCM practitioner has his/her preference for the type of herb(s) to prescribe,
whether raw herbs or processed mixtures of herbs. It is also very challenging for the
practitioner to standardize the prescriptions since it is given individually for every
patient depending on his/her medical condition.
The most commonly prescribed TCM herbs for our six breast cancer patients were:
herbs to tonify the qi - Codonopsis pilosula, Astragalus membranaceus, Atractylodes
macrocephala, Ziziphus jujuba (red date), Lycium barbarum (wolfberry fruit); herb to
invigorate the blood - Curcuma aromatica; herb to relieve food stagnation – Crataegus
pinnatifida; herbs to drain dampness - Poria cocos, Coix lacryma Jobi (Job’s tears
seed); herbs to clear heat and relieve toxicity- Scutellaria barbata, Solanum nigrum
(Reid, 1993; Keys, 1997).
Herbs that tonify qi decrease lethargy, fatigue, lack of appetite, abdominal pain,
diarrhea, dyspnoea, edema and stimulate the body and the sympathetic nervous system.
Herbs that invigorate the blood alleviate pain, while those which relieve food stagnation
stimulate digestion and increase gastrointestinal secretion. Herbs which drain dampness
are diuretics and have tranquilizing effect, while those which clear heat and relieve
toxicity have anti-inflammatory, anti-infectious and diuretic effects. The combination of
these herbs might explain their efficacy in improving the quality-of-life (QOL) in
cancer patients (Read, 1977).
10
The effects of the prescription herbs initially were evaluated through a QOL analysis
using the European Organization for Research and Treatment of Cancer (EORTC) QOL
questionnaire (QLQ-C30 version 3.0) and the supplementary breast cancer module
(QLQ-BR23). We compared the herbal prescriptions of the patients who had better
scores on the symptoms scale (i.e. less fatigue, pain, dyspnoea, insomnia and appetite
loss) and selected the 6 most frequently used herbs: Astragalus membranaceus (AM),
Curcuma aromatica (CA), Solanum nigrum (SN), Scutellaria barbata (SB) for studies
on their immunomodulatory and cytotoxic effect. Crataegus pinnatifida (CP) and
Ziziphus jujube (ZJ) were well documented in the literature for their appetite enhancer
and anxiolytic effect respectively, thus in our research project we did not conduct
biomedical experiments on these 2 herbs.
11
Picture 1. Dried root of Astragalus membranaceus
12
Astragalus membranaceus L. (AM) (Huang Qi, Mongolian Milk Vetch,) is native to
China, Korea and Japan. It contains glycosides (astragalosides I, II, III, IV, V, VI, VII)
[Hirotani et al., 1994], isoflavonoids (Wu et al., 2005; Zhang et al., 2005),
polysaccharides (astroglucan A, B and C), more than forty types of saponins (Yang et
al., 2005), vitamin A, betaine, beta-sitosterol (Kim et al.2003), hexuroic acid,
rumatakenin and sugars (He et al., 1990). In TCM, AM has been used to correct the
deficiency of qi, repair and tonify emptiness. It is used as a diuretic, a cardiotonic and to
relief fatigue hypotension (Wu et al., 2000; Fetrow et al., 2001).
In Western medicine, it has been reported that the saponins and polysaccharide
compounds found in the Astragalus root support and stimulate the cellular immune
system, strengthen the natural defence mechanism by enhancing antibody production
through increased T helper cell activity (Yoshida et al., 1997; Shan et al., 1999; Lee et
al., 2005). It was reported that Astragaloside VII is a potent IL-2 inducer thus being a
strong immunostimulator (Yesilada et al., 2005). It was shown that the extract of AM
stem and leaves could promote lymphocyte proliferation, elevate T cell count and
enhance IL-2-induced LAK activity thus having antineoplastic effect (Wang et al.,
1992; Jiao et al., 1999). It was also reported that AM is effective in treating Coxsackie
B virus induced myocardial muscle injury (Yang et al., 1990; Yuan et al., 1990). Used
in combination with other herbs, it could reduce serum virus count in some AIDS
patients (Chang et al., 1988; Yao et al., 1992). The saponins have been found to have
anti-diabetic (Li et al., 2004), neuro-protective (Luo et al., 2004), cardio-protective
(Zhang et al., 2005), anti-inflammatory (Zhang et al., 2003), diuretic and
antihypertensive properties as well (Yang et al., 2005).
13
Picture 2. Dried roots of Curcuma aromatica
14
Curcuma aromatica L. (CA) (Yu Qin, Wild Turmeric) is a member of the
Zingiberaceae family, like ginger and cardamon. It grows in tropical countries like
Samoa, Tonga, Jamaica, Peru, Brazil, Jordan, India, Indonesia and South-China
(Govindarajan, 1980). It contains, among others, curcumin, curcumenol, tumerone,
atlantone, diaryl heptanoids, zingiberone, sugars, resins, proteins, different vitamins and
minerals (Kojima et al., 1998; Navarro et al., 2002; Xia et al., 2005; Yang et al., 2005).
It is known to be an energy regulator and is used in energy stagnant illnesses. CA is a
mass-reducing herb and has been used to remove the stagnation of qi, reduce
inflammation, chest pain and colic (Bensky et al., 1986; Reid,1993; Dewick, 1997;
Dharmananda 1997; Fetrow et al., 2001; Park et al., 2004; Jayaprakasha et al., 2005).
In Western medicine, it has been reported to enhance fibrinolysis (Srivastava et al.,
1989), to treat atherosclerosis (Ashraf et al., 2005), pain (Navarro et al., 2002),
cholelithiasis (Niederau et al., 1999), irritable bowel syndrome (Brinkhaus et al., 2005),
bacterial and fungal infections ( Grosvernor et al., 1995). It has been shown to be useful
in the prevention and treatment of breast cancer (Inano et al., 2000), colorectal cancer
(Goel et al., 2001), skin (Huang et al., 1997) and liver cancer (Cheng et al., 2001).
Curcumin, a potent bioactive compound in CA, was shown to have anti-HIV activity (
Mazumder et al., 1995) to inhibit carcinogenesis by being a strong antioxidant ( Selvam
et al., 1995; Abas et al., 2006) and to induce apoptosis in cancer cells without cytotoxic
effect on healthy cells (Duvoix et al., 2005). Curcuminoids are effective by inhibiting
leukotriene biosynthesis, reducing prostaglandin formation thus having a potential anti-
inflammatory and anticoagulant effect (Selvam et al., 2005).
15
Picture 3. Reconstruction of Solanum nigrum (authenticated Dr. Ruth Kiew and stored as a voucher specimen BT4, Herbarium, Singapore Botanical gardens by)
16
Solanum nigrum L (SN) (Black nightshade) is a common herb that grows wildly and
can be found worldwide. SN has been used in many different countries (China, India,
Israel, Turkey, Madagascar, Lesotho, Tanzania, Mexico, Brazil, Hawaii, Cook Islands)
as traditional folk medicine and for its various biological activities (Dafni et al., 1994;
Ankli et al., 2002). It belongs to the Solanacae family and contains, depending on the
parts of the plant (stem, leaves, fruit), steroidal glycosides (α and β2-solamargine,
solamargine, degalactotigonin), steroidal alkaloids (solasodine, solanidine), flavonols,
sitosterol, stigmasterol (Dewick, 1997; Keys, 1997; Hu et al., 1999; Fetrow et al., 2001;
Zhao, 2004). There is a lot of debate over whether or not the leaves or fruit of SN are
poisonous and carcinogenic. However, in some countries it is consumed as a vegetable
(Purchase et al., 1975; Blankmeyer et al., 1998, Sammon, 1992). It was reported that
the unripe fruit contains the highest concentration of solasodine which is a cytotoxic
substance for cancer cells. It is active against anaphylactic and hypoglycaemic shocks,
and shocks resulting from burns (Eltayeb et al., 1997; Latoxan, 2005). It was shown
that SN and some of its compounds has antitumor (Chiang et al., 1991; Yen et al.,
2001; Son et al., 2003; Cai et al., 2004), antifungal (Locher et al., 1995), cholesterol-
lowering (Lee et al., 2005), antipyretic and diuretic (Bensky et al., 1986) effects. It is
also used to treat inflammation, edema, mastitis (Sultana et al., 1995), liver complaints
(Goodman et al., 1988), neurological conditions ( Perez et al., 1998) and for
cytoprotection in drug induced kidney damage (Kumar et al., 2001). The steroidal
glycosides from Solanacae species can be used to treat herpes simplex and skin cancer
(Cham et al., 1987; Bensky et al., 1986; Ikeda et al., 2000). It has also been reported
that solamargine together with cisplatin was effective in treating lung and liver cancer
(Kuo et al., 2001).
17
Scutellaria barbata (SB) (Skullcap) contains flavonoids (scutallarein, scutellarin),
sesquiterpenes, wogonin, lignin, resin etc. It is known to have anti-inflammatory,
anticonvulsant, sedative and cardioprotective action (Fetrow et al., 2001). It was
reported that SB induces apoptosis in human leukemia cells (Cha et al., 2004) and has
anti-cancer/ cytotoxic activity in lung and ovarian cancer (Yin et al., 2004; Powell et
al., 2003).
Ziziphus jujuba (ZJ) (Spina Date Seed) is rich in phytoestrogens, which are known to
have anxiolytic activity and increase appetite (Lund et al., 2001). It is used in TCM
treatment of insomnia, neurasthenia and palpitation (Zhao, 2004).
Crataegus pinnatifida (CP) (Hawtorn) has high content of bioflavonoids and was
reported to have antioxidant and cardioprotective properties (Fetrow et al., 2001). It
was published that the corosolic acid isolated from the fruit of CP is a protein kinase C
inhibitor and has cytotoxic effect on leukemia cells (Ahn et al., 1998). In TCM it is
used for dyspepsia, diarrhea, abdominal pain, hypertension and hyperlipidemia (Zhao,
2004).
Herbal TCM (due to the many different compounds used together) usually exhibit
synergistic antitumor activity. One of the mechanisms of TCM in cancer treatment is
immune system modulation by stimulatory, suppressive or regulatory activity or a
combination of these effects (Patwardhan et al., 2005). Some of the most common
TCM- stimulated cytokines are Tumor Necrosis Factor- α (TNF-α), Interleukin-2 (IL-
2), and Interleukin-6 (IL-6). TNF-α (cachectin) is a potent mediator of immune and
inflammatory responses. It is produced by different activated cells including
macrophages, granulocytes, T and B lymphocytes, natural killer (NK) cells, fibroblasts,
and certain tumor cells. TNF can co-stimulate the proliferation of activated T (Thelper)
and B lymphocytes. TNF-α is selectively cytotoxic for some transformed cell lines and
18
can exert cytotoxic effects against certain solid tumors in lower dose. In vivo, TNF-α
serves as a primary mediator in protective immune responses against microbial and
viral pathogens. However, TNF-α in higher dose has also been implicated as an
inflammatory mediator in a number of pathologic responses including septic shock,
cachexia and autoimmune diseases (Levinson et al., 1996; Parslow et al., 2003). IL-6 is
secreted by Thelper cells and macrophages. It is a multifunctional cytokine which
regulates immune responses, acute-phase reactions and hematopoiesis. It potentiates the
effect of IL-1 and TNF-α to further promote T- cell activation. IL-6 also enhances B
lymphocyte proliferation and differentiation and promotes multi-potent hematopoietic
cells. It is also an endogenous pyrogen.
IL-2 (T-cell growth factor, TCGF) is a lymphokine which is produced by antigen-
activated Thelper cells. It is involved in activating all types of acquired immune response:
Tcytotoxic, Thelper, B-cell growth and differentiation; lymphokine-activated killer (LAK)
cells and NK cell generation and proliferation. It plays an important immunoregulatory
role and it is frequently used in cancer therapy. IL-2 also induces other lymphokines
such as interferon and B-cell growth factor (Gupta et al., 1996; Weir et al., 1996a; Weir
et al., 1996b; Kresina, 1998).
Another effect of TCM in cancer treatment is cytotoxicity, inducing cell death in solid
tumors. It was shown that apoptosis (programmed cell death) is the most common type
of cell death, though it has been reported that cancer cells can undergo autoschizis, a
novel type of necrosis (Jamison et al., 2002). It has been reported that most cytotoxic
drugs induce necrosis (of cancer cells in-vitro) at high doses and apoptosis at low doses
(Schwartzman et al., 1993).
19
Apoptosis is a biochemical and physiological response characterized by distinct
morphological changes, including pre-lytic DNA fragmentation, cell shrinkage and
appearance of apoptotic bodies. The underlying mechanism of apoptosis induction
involves different signal transduction pathways controlled by pro- and anti-apoptotic
factors (death signals, genetic regulation by transcription factor manipulation, activation
of effector enzymes) [Renehan et al., 2005].
The most common apoptosis initiators are: (a) stress (ultraviolet and γ-irradiation,
hypoxia, growth factor deprivation), cytotoxic T-cells releasing granzyme-B – these
trigger changes in the nucleus (p53, cyclin D, cyclin E regulation) and/or mitochondria
(cytochrome c release, (b) cytotoxic drugs and cytokines which bind to the “death
receptors” (Fas, Apo1, TNFR1, TNRF2). These activate either the effector caspases-3, -
6,-7 or through caspase-9 / poly (ADP)-ribose polymerase (PARP) pathway to cause
DNA fragmentation in the nucleus (Hengartner, 2000; Desagher et al., 2000; Kaufmann
et al., 2001; Reed, 2001; Renehan et al., 2005).
The protein bcl-2 has been shown to play an important role in the apoptotic pathway
both as pro- and anti-apoptotic regulator (Song et al., 1999). Overproduction of bcl-2
can result in blockage of apoptosis. The anti-apoptotic property of bcl-2 is possibly
modulated by the protein Bax, and the Bcl-2: Bax ratio could determine whether a cell
would undergo apoptosis. Among other chemicals, steroid sex hormones (estrogen) and
certain natural compounds (flavonoids, terpenoids, alkaloids, polysaccharides) may
regulate Bax and Bcl-2 levels. Breast epithelium undergoes cyclic apoptosis and
fluctuation in Bcl-2 protein level during the menstrual cycle and pregnancy. Changes in
Bcl-2 levels have also been associated with breast cancer (Oltvai et al., 1993; Gee et al.,
1994; Reed, 1994; Sato et al., 1994; Sabourin et al., 1994; Wang et al., 1995).
20
1.3 Breast cancer: significance in modern day society
Breast cancer is the most common cancer in Singaporean women and the 3 major ethnic
groups (Chinese, Malay, Indian) are equally affected. The incidence rate is about 5%
and more than 1000 new cases are diagnosed yearly (Singapore Cancer Society, 2002),
while 300 deaths from this cancer occur per year (WHO IARC, 2005). According to the
World Health Organization (WHO), more than 1.2 million people will be diagnosed
with breast cancer in 2005 worldwide. While breast cancer is less common at a young
age (in their thirties), younger women tend to have more aggressive breast cancers than
older women. The survival rates are lower among younger women (Imaginis, 2005).
Picture 4. Number of deaths from breast cancer in Singapore among women age of 0-85 between years 1963-2002 (WHO IARC, 2005)
The risk factors associated with breast cancer are: female, age above 40, family history
(having a first degree relative with breast cancer), late first pregnancy, early onset of
21
menses, late menopause, diet (high intake of saturated animal fats, decreased fruit and
vegetable intake), alcohol consumption, weight gain, lack of physical exercise and
intake of hormones (oral contraceptive pills, hormone replacement therapy) [Ng et al.,
1997].
In Singapore, the median age at diagnosis of breast cancer is 50 years, approximately 10
years younger than the Caucasian population (Seow et al., 1996). This could be due to
the different type of BRCA1 mutations observed in patients of Asian ethnicity,
especially in those younger than age 36 years (Ho et al., 2000). Breast carcinoma
presenting at a younger age is usually the invasive type thus requiring very complex
treatment.
The conventional breast cancer treatment involves multimodal approaches, such as
surgery, radio-, chemo- and/or hormonal therapy. In most of the breast cancer cases we
observed in Singapore, herbal TCM was used to complement conventional treatment.
It has been established earlier that most of the breast cancer cases are caused by
hormonal (estrogen, progesterone) imbalance, estrogen being a risk factor in the
development of this disease. Preventive and therapeutic treatments often target
estrogen. The most common drugs used are (a) Selective Estrogen Receptor Modulators
(SERMs): Tamoxifen, Toremifene, Raloxifene; Soy, Flaxseed in CAM (b) Aromatase
Inhibitors: Anastrozole, Exemestane, Letrozole; Genistein in CAM (c) Hormonal
agents: Fulvestrant, Goserelin acetate, Megestrol.
A wide range of conventional chemotherapy is also commonly used: Capecitabe,
Cyclophosphamide, Docetaxel, Doxorubicin, Epirubicin, Fluorouracil (5-FU),
Paclitaxel, Vinorelbine (Jellin, 2005).
Against HER2+ breast cancer the current treatment is Transtuzumab, which is a
recombinant antibody against the HER2 gene (Piccart-Gebhart et al., 2005).
22
There are also promising results with the angiogenesis inhibiting anti-Vascular
Endothelial Growth Factor (anti-VEGF) drug , Bevacizumab.
It has been reported that certain Chinese medicinal plants have estrogenic activities thus
might have estrogen agonist/antagonist effect. Some of these herbs (Epimedium
brevicornum, Antrodia camphorat) were tested in-vitro for MCF-7 human cancer cell
growth inhibition, others are already used for the management of menopausal
symptoms (Yap et al., 2005; Yang et al., 2005; Zhang et al., 2005).
There is very little available scientific evidence to validate the efficacy of TCM.
Singapore, with its dual health-care system, is in a very unique position for clinical and
laboratory studies to be done to evaluate the effectiveness of the combined use of
Western and traditional medicine in the treatment of disease states like breast cancer.
1.4 Rationale and purpose of this study Breast carcinoma is a common cancer in women and is a leading cause of cancer-
related deaths in Singapore and worldwide (Polyak, 2001). Early detection and
intervention have shown promising results. However, the incidence of this disease is
constantly growing, causing a greater impact on health care services to demand new
cures for this disease.
It is known that CAM therapies provide psychological benefits (optimism, hope for
disease control, cure and longer survival) for cancer patients (Di Gianni et al., 2002).
The objective of our study was to obtain data that may show whether TCM use could
have beneficial effect on breast cancer patients through improving their quality of life
(by palliating the side effects of conventional therapy), stimulating their immune system
and inhibiting tumor growth.
23
The conventional treatment for breast cancer includes surgery, radio-, chemo- and/or
hormonal therapy. The side-effects of these medical interventions and the stress of
living with breast cancer can negatively affect the patients’ quality of life. QOL
research has become an important part of the assessment of cancer treatment in Western
society. However only two studies (one in Shanghai and the other in Hong Kong) have
been conducted on Chinese breast cancer patients (Yu et al., 2000; Fung et al., 2001;
Cui et al., 2004).
The purpose of our study was to gain scientific evidence that TCM, an ancient type of
medicine based on empirics and widely used for centuries, could be potentially useful in
the modern biomedical setting.
The results obtained from our study may contribute towards improved treatment of
patients with breast cancer.
24
CHAPTER TWO: MATERIALS AND METHODS
25
2.1 Materials
2.1.1. Cell lines and cell cultures
All cell lines are adherent type and were obtained from American Type Culture
Collection (ATCC) (Rockville, MD, USA).
MCF-7
Picture 5.MCF-7 cells
MCF-7 was derived from pleural effusion of a metastasised adenocarcinoma
(epithelial) of the human breast. It is estrogen receptor positive and its growth can be
inhibited by TNF-α. The MCF-7 line retains several characteristics of differentiated
mammary epithelium including the ability to process estradiol via cytoplasmic
26
estrogen receptors and the capability of forming domes. Doubling time is 29 hours. If
exposed to estrogen, it can develop into tumors in nude mice (ATCC, 2005).
MDA-MB-231
Picture 6.MDA-MB-231 cells
MDA-MB-231 was derived from metastasised adenocarcinoma (epithelial) of the
human breast. It expresses epidermal growth factor (EGF) and transforming growth
factor alpha (TGF- α). Doubling time is around 19 hours. In nude mice, MDA-MB-
231forms poorly differentiated (grade III) adenocarcinoma (ATCC, 2005).
T-47-D
T-47-D was derived from a patient with infiltrating ductal carcinoma of the breast. It
expresses androgen, progesterone, glucocorticoid, prolactin, calcitonin and estrogen
receptors. Doubling time is 32 hours (ATCC, 2005).
27
ZR-75-1
T-47-D was derived from a patient with infiltrating ductal carcinoma of the breast. It
is estrogen receptor positive. Doubling time is 80 hours. It forms tumors in nude mice
(ATCC, 2005).
2.1.2. Animals
Inbred male and female BALB/c mice, 5 to 6 weeks old, were obtained from the
Animal Holding Unit, National University of Singapore.
Inbred female oophorectomized BALB/c nude mice, 8 to 12 weeks old, were obtained
from the Animal Research Centre (Canning Vale, Australia). The autosomal recessive
nude gene in homozygous (nu/nu) mice causes the lack of fur and an abnormal
thymus. The deficiency in T cell function allows athymic mice to accept and grow
xenografts as well as allografts of normal and malignant tissues.
All animal experiments were conducted in accordance with the Animal Welfare Act
and the Animal Research Extension Act, in facilities approved by the Institutional
Animal Care and Use Committee.
2.1.3. Chemicals and reagents
BDH Laboratory Supplies (UK)
Dimethyl Sulphoxide (DMSO)
Hyclone (UK)
Inactivated fetal bovine serum (FBS)
Merck KgaA (Darmstadt, Germany)
28
Methanol, Ethanol, Hexane, HCl, H2SO4, Dodecyl-sulphate sodium salt (SDS), Ethyl
acetate, n-butanol
Innovative Research of America (Sarasota, Florida, USA).
60 days release 17-β-estradiol (0.72 mg) pellets for nude mice
Sigma-Aldrich (St Louis, MO, USA)
All other chemicals and reagents used in the present study
2.1.4. Kits
BD Biosciences Pharmingen (San Diego, CA, USA)
Mouse IL-2 BD OptEIATM ELISA Set
Mouse IL-6 BD OptEIATM ELISA Set
Mouse TNF-α (Mono/Mono) BD OptEIATM ELISA Set
Roche Diagnostics GmbH, Roche Applied Sciences (Penzberg, Germany)
Cell Death Detection ELISAPLUS
Cellular DNA Fragmentation ELISA
2.1.5. Facilities/ Equipment
-86°C Freezer (Forma Scientific)
-20°C & 4°C ACMA Refrigerator
Balance (Precisa 40SM-200A, Switzerland)
Biological Safety Cabinet (NUAIRE, Plymouth, USA)
CO2 Incubator (SANYO Electronic Co. Ltd, Japan)
ELX 800 Microplate Reader (Bio-Tek Instruments Inc., USA)
Hemocytometer (Fortuna, Germany)
29
HPLC with Phenomenex Synergi 4µ Hydro- RP80A column (Japan)
Kubota Centrifuge KR400 (Kubota Seisakusho Co. Ltd., Japan)
Nikon Light Microscope (Nikon Co. Ltd, Japan)
Beckman pH Meter (Fullerton, CA, USA)
Tecan Sunrise Microplate Reader (Grödig, Austria)
UV-1601 Spectrophotometer (Shimadzu, Japan)
2.2. Methods
2.2.1. Quality-of-life (QOL) study
2.2.1.1. QOL study design
Our study started in February 2001 at two TCM clinics in Singapore. One clinic is the
Chung Hwa Free Clinic at Toa Payoh, which is financed by individual donors and
Buddhist charity organizations. Most of the patients here are from low-income
households. The other clinic is Teo Acupuncture Hall, a private clinic at Rochor
Centre, which provides TCM treatment for mainly higher income patients.
We chose breast cancer patients for the study since the incidence of this disease is
increasing worldwide, creating a need for a more satisfactory treatment. Breast cancer
patients are also ideal candidates for observing the side-effects of chemo- and
radiotherapy since they have little complications from the tumor itself (compared to
gastric or pancreatic cancer), thus any change in quality-of-life is most likely to be
due to the applied treatment itself.
Thirteen female Chinese patients, age 31 to 53 years, who previously had been
diagnosed with breast cancer by Western-trained physicians, were selected as the first
subjects for our study between February 2001 and July 2001. After a month, seven
30
patients dropped out (mainly because of not returning the questionnaires) and we
continued to follow up the remaining six patients. Two had been treated with
radiotherapy, chemotherapy and the hormone receptor modulator, Tamoxifen; one
with radiotherapy and Tamoxifen; two with Tamoxifen only; one with herbal TCM
only. It is noted that five of the six patients recruited had some form of surgery as part
of their prior conventional medical treatment (Table 1).
Table 1.Conventional treatment of selected patients
R: radio-therapy C: chemotherapy T: Tamoxifen TCM: Traditional Chinese Medicine
Our inclusion criteria were that the patients (a) must have primary, not metastatic,
breast cancer diagnosed by a biomedical practitioner, (b) should not have any other
treated or untreated underlying medical condition, (c) should have had previous or
current conventional treatment for breast cancer, (d) should comply with TCM and (e)
should be able to come back for follow-up. After initial evaluation of the patients’
medical history, with regard to the modality of breast cancer treatment including
previous surgery and the type of herbs used (if any), we explained the nature of our
non-invasive, purely observational study and obtained the patients’ consent to
Modality of treatment at the time of
study initiation
Number with
prior surgery Number of patients
R, C, T 1 2
1 R, T 1
T 2 2
1 TCM only 1
31
participate in the study by showing them the questionnaire in their preferred language
(English or Chinese).
These patients were followed up for their herbal TCM treatment by the same Chinese
physician, Dr. Teo. They responded weekly, bi-weekly, monthly or bi-monthly to the
European Organization for Research and Treatment of Cancer (EORTC) QOL
questionnaire (QOL-C30 version 3.0) and to the supplementary breast cancer module
(QOL-BR23) questionnaire (Aaronson et al., 1993) [see Picture 7a, 7b, 8a, 8b and
Table 2 and Table 3 for their composition].
32
Picture 7a.EORTC QOL-C30 (version 3) questionnaire
33
Picture 7b.EORTC QOL-C30 (version 3) questionnaire
34
Picture 8a.EORTC QOL-BR23questionnaire
35
Picture 8b.EORTC QOL-BR23 questionnaire
36
Table 2.Composition of EORTC QLQ-C30 (version 3.0)
Scale QLQ-C30 item numbers
GLOBAL HEALTH STATUS
Global health status/QoL QL2 29, 30
FUNCTIONAL SCALES
Physical functioning PF2 1 to 5
Role functioning RF2 6, 7
Emotional functioning EF 21 to 24
Cognitive functioning CF 20, 25
Social functioning SF 26, 27
SYMPTOM SCALES
Fatigue FA 10, 12, 18
Nausea and vomiting NV 14, 15
Pain PA 9, 19
Dyspnoea DY 8
Insomnia SL 11
Appetite loss AP 13
Constipation CO 16
Diarrhea DI 17
Financial difficulties FI 28
37
The QLQ-C30 questionnaire is designed to measure the physical, psychological and
social functions of the cancer patients (Kaasa et al., 1995). It is composed of both
multi-item and single-item scales. These include five functional scales (physical, role,
emotional, social and cognitive function), three symptom scales (fatigue, pain and
nausea), a global health status scale and six single items (dyspnoea, insomnia, appetite
loss, diarrhea, constipation and financial difficulties). The physical and role function
scales have a choice of “yes/no” for response; the two global quality-of-life questions
have a 7-point linear analogue scale for answers (1 being “very poor”, 7 being
“excellent”). All other items have a 1 (”not at all”) to 4 (“very much”) point score on
the answer scale.
38
Table 3.Composition of EORTC breast cancer module QLQ-BR23 Scale QLQ-BR23 item numbers FUNCTIONAL SCALES
Body image BRBI 9 – 12
Sexual functioning BRSEF 14,15
Sexual enjoyment BRSEE 16
Future perspective BRFU 13
SYMPTOM SCALES
Systemic therapy side effects
BRST 1 – 4,6,7,8
Breast symptoms BRBS 20 – 23
Arm symptoms BRAS 17,18,19
Upset by hair loss BRHL 5
39
The breast cancer BR-23 module is meant for use among patients varying in disease
stage and treatment modality (i.e. surgery, chemotherapy, radiotherapy and hormonal
treatment) (Sprangers et al., 1996). The module comprises 23 questions assessing
disease symptoms, side effects of treatment (surgery, chemotherapy, radiotherapy and
hormonal treatment), body image, sexual functioning and future perspective.
Validation studies on the module have been completed in The Netherlands, Spain and
the United States. It had been field-tested in a large cross-cultural study involving 12
countries (EORTC Protocol 15931) (Fayers et al., 1998). All of the scales and single-
item measures range in scores from 0 to 100. A high score for an item on the
functional scale represents a high / healthy level of functioning. Similarly, a high
score for the global health status represents a high quality-of-life, but a high score for
an item on the symptom scale represents a high level of problems.
Patients on chemo- and radiotherapy answered the questionnaire more frequently
because it was assumed that their state of well-being would be more likely to fluctuate
during follow-up. The questionnaire was self-administered in English or in validated
Chinese (Mandarin and “Taiwanese Chinese”) translation, depending on the patients’
language preference.
After completing the first QLQ-30 and BR-23 questionnaires, five patients had their
first herbal TCM treatment, while one patient (who previously had mastectomy) was
already using TCM at the time of induction into the study. They subsequently made
several visits to the clinic, the frequency varying between 3 times per week to once in
2 months. Their herbal prescriptions were altered according to their condition at each
visit, and comprised different air-dried plants which were soaked in water for half-an-
hour and then boiled for two hours. The supernatant decoction was then consumed
40
twice a day, in the morning and in the evening, before or after meals. The patients
were requested by the TCM practitioner to avoid taking any other type of medicine
within 1 hour of taking the decoction to minimize the possibility of direct drug
interactions with TCM.
Scores were obtained from the completed questionnaires and transformed for
interpretation and comparison in two ways: (a) baseline (pre-treatment) scores were
compared with post-TCM treatment scores and (b) baseline scores were compared
with scores of breast cancer patients published in the manual of EORTC QLQ-30
Reference Values (Fayers et al., 1999). Our patients were matched by age, gender and
initial conditions with those from the reference population (Canada, Norway, Sweden,
Denmark). The Reference Values (general population data based on large random
samples from the general population in the above-mentioned countries) are based on
pre-treatment data and give baseline quality-of-life scores for patients according to
specific cancer sites (breast, prostate, lung, brain etc.) and stage of disease (local/
loco-regional/advanced).
All of our patients were diagnosed with early or advanced but not yet metastasised
breast cancer and were treated by Western-trained physicians. Comparing our
patients’ baseline mean scores to the published EORTC reference values, we observed
a moderate difference in physical and emotional functioning, pain and appetite loss,
and a little change on the role functioning and dyspnoea scale. This might indicate
that our six patients in Singapore were different from the reference population in their
psychological and physiological responses to their cancer, and might have a higher
threshold of pain perception.
The patients used herbal TCM as an alternative, complementing the conventional
therapy. The prescription was adjusted at each visit according to the actual condition
41
at the time of the medical consultation by adding or removing herbs and increasing or
decreasing the dosage and frequency of the herbal medication. The better quality of
life could be attributed partially to the patients’ favorable prognosis and the use of
moderately toxic conventional therapies (Burstein et al., 1999). The higher scores on
the functional scale and lower scores on the symptoms scale could also indicate that
herbal TCM can ease some of the symptoms in cancer patients.
SN 300 mg/kg BWsmall volume tumorsnot efficient larger, multiple tumorsSN 500
mg/kg BW could be use as prevention (prophylactic rate75%)not efficient dose of
tumors
2.2.1.2. Scoring procedures for the EORTC QLQ-30 and BR-23
questionnaires
As a first step, we estimated the average of the items on the functional or symptom
scale on the QLQ-30 and BR-23 questionnaires. This average is the Raw Score (RS) =
(I1 + I2 +…+ In) / n (n: number of items). As a next step we applied a linear
transformation to 0-100 to obtain the Score (S) which was calculated for the
respective scales as it is shown below (range is the difference between the maximum
and the minimum possible value of RS):
Functional scales: S = {1-(RS-1)/range} x 100
Symptom scales / items and Global health status/ QOL: S = {(RS-1)/ range} x 100
2.2.2. Extraction of herbs
The dried forms of Astragalus membranaceus (AM) root, Curcuma aromatica (CA)
tuber, Scutellaria barbata (SB) leaves and Solanum nigrum (SN) leaves were
purchased from Teo Acupuncture & Medical Hall, where the breast cancer patients
42
obtained their prescription. The herbs were harvested in Southern China (Personal
communications with Teo Eng Kiat, 2001).
2.2.2.1. Preparation of water and ethanolic extract of Astragalus
membranaceus, Curcuma aromatica, Scutellaria barbata and Solanum
nigrum
The dried herbs were crushed into small pieces with an electronic micronizer. As a
first step in the fractionation process, we chose the ethanolic extract of these herbs.
170 g of AM, 180 g of CA, 120g of SB and 130 g of SN were extracted with 80%
ethanol at room temperature until exhaustion. The preparation was filtered with
Whatman filter paper GF/A, concentrated under reduced pressure and freeze dried.
The yield was 19%, 13.3%, 9.2% and 6.5% respectively.
We also prepared boiled water extracts of 200 g each of AM, CA, SB and SN by
covering the herbs with sufficient water, bringing to boiling point. After cooling, we
filtered the extract and proceeded to freeze dry. The yield was less compared to the
ethanolic extracts, 12.2%, 7.8%, 4.7% and 2.9% respectively.
2.2.2.2. Fractionation of the water boiled extract of Astragalus membranaceus
As a next step we also fractionated the water-boiled extract of AM. The water extract
of AM was partitioned to obtain ethyl acetate (0.2% yield), butanol (3.3% yield) and
the remaining water fraction (15.9% yield).
43
2.2.2.3. Characterization of the ethanolic and water extract of Solanum
nigrum
Following the in-vitro cytotoxic experiments with the ethanolic extract of Solanum
nigrum, we were interested to further investigate and compare the ethanolic and water
extract of this herb. The reason being was to see whether there is any major difference
between the ethanolic extract used in our laboratory testing and the water extract the
patients were taking.
We ran both extracts on reversed-phase high performance liquid chromatography
(HPLC). It was a linear gradient elution at a flow rate of 0.8ml/min on Phenomenex
Synergi 4µ Hydro- RP80A column (250 x 4.6mm, 4 micron) with acetonitrile and de-
ionized water as mobile phase. Detection was done with a Shimadzu Diode Array
detector. During the linear gradient elution program, the percentage of acetonitrile in
mobile phase changed as follows: 0.1 to 5 mins: 0%; 5 to 30 mins from 0% to 100%;
45 to 50 mins: from 100% to 0%; 50 to 60mins: 0%.
The peaks obtained were detected at four different wavelengths: UV 240, 254, 260
and 280nm.
2.2.3. Breast cancer cell culture
The human breast cancer cells were cultured for 4-8 passages: MCF-7, T-47D and
ZR-75 cells were cultured in RPMI-1640, while MDA-MB-231 cells were cultured in
DMEM medium at 37°C in 5% CO2 humidified incubator. The medium contained
10% fetal bovine serum, penicillin (100 IU/ml) and streptomycin (100μg/ml). Before
the cellular DNA fragmentation experiment, the cells were labeled with BrdU
overnight.
44
2.2.4. Cell viability assays by MTT test and trypan blue exclusion Evaluation of cell activation in the immunomodulatory and cytotoxicity studies was
carried out using the MTT tetrazolium assay. Briefly, after 48-72 h incubation of
tissue culture plates at 37°C in 5% humidified CO2 incubator, 10 μl MTT (stock
solution 5 mg/ml PBS) was added to each well. The plates were again incubated for 4
h after which 100 μl/well 10% SDS in 0.01N HCl was added to dissolve the formazan
crystals. After overnight incubation, the plates were read in a microplate reader (ELX
800, Bio-Tek Instruments, USA) at 570 nm. Wells with medium, MTT and SDS, but
without cells, were used as blanks (Gerlier et al., 1986; Swamy et al., 2000; Liu,
2002).
2.2.5. Lymphocyte-activation assay
Fresh splenocytes were prepared aseptically into single cell suspension (Nakamura et
al., 1986; Wu et al., 1990). These were suspended at 1.6 x 106 cells/ml in medium
containing RPMI 1640 supplemented with 10% heat-inactivated FBS. A total of 90 μl
of the cells were added to each well of a flat-bottom 96-well plate using a multi-
channel pipette. Studies were carried out to evaluate the effects of 10.0, 1.0 and
0.1μg/ml AM and CA extracts on the proliferation of mouse lymphocytes under
various conditions, in the absence or presence of 10 μl of (a) suboptimal doses of
ConA (0.3 μg/ml) or LPS (1.0 μg/ml) and (b) optimal doses of ConA (3.0 μg/ml) or
LPS (6.0 μg/ml).
The spleen-cell suspensions were incubated for 48-72 h at 37°C in 5% humidified
CO2 incubator with 10 μl serially diluted extracts, starting with highest concentration
of 10.0 μg/ml for each extract in quadruplicate wells. Experiments were repeated in
four replicates for extracts showing stimulation of the splenocytes. Evaluation of cell
activation was carried out using the MTT tetrazolium assay.
45
2.2.6. Assay of cytokines IL-2, IL-6 and TNF-α
6-8 weeks old BALB/c female mice were fed orally for 14 days with AM (50,100,
200 mg/BW kg). Blood samples were collected by cardiac puncture and the serum
was analyzed for IL-2, IL-6 and TNF-α.
To measure cytokine production we used an assay that employs the quantitative
'sandwich' enzyme immunoassay technique. The sample was blood serum, obtained
by cardiac puncture from BALB/c 8 weeks old female mice.
Nunc Maxisorb 96 MTP was coated with 100 μl/well diluted capture antibody and
incubated overnight at 4°C. After washing 3 times, the wells were blocked with 200
μl/well assay diluent. Following an hour incubation at room temperature (RT), the
plates were washed 3 times. 100 μl/well standard solution, serial dilution and serum
sample was added to the plates. After 2 hours of incubation at RT, the plates were
washed 5 times.
For the TNF-α and IL-6 assays 100 μl/well detection antibody plus Avidin-HRP was
added. After 1 hour of incubation at RT, the wells were washed 7 times and 100 μl
substrate solution was added to each well. Following 30 minute incubation at RT in
the dark, 50 μl/well stop solution was added.
For the IL-2 assay 100 μl/well detection antibody was added first, then after 5 washes
100 μl/well Avidin-HRP was added.
As a last step for all 3 assays, the MTP was read @ 450 nm within 30 minutes. Color
development was in proportion to the amount of cytokine bound in the initial step. By
comparing the optical density of the samples to the standard/ serial dilution curve, the
concentration of the cytokine in the unknown samples was then determined.
46
2.2.7. In-vitro cytotoxicity assay
Sub-cultured, trypsinized human breast cancer cells MCF-7, T-47D, ZR-75 and
MDA-MB-231 were centrifuged @ 900 rpm for 5 minutes. After discarding the
supernatant, the cells were re-suspended in medium. The cell suspension was diluted
to 5x105 cell/ml concentration using trypan blue to determine total cell counts and
viable cell number (non-viable cells dye blue). 95 μl of cell suspension was added to
each well. After 2 hour incubation, 5 μl of serially diluted AM, CA, SB and SN
extracts were added to the respective wells. 5 μl 6-MP was the positive control and
10% DMSO in sterile water was the negative control. After a 48 hour incubation at
37°C in 5% humidified CO2 incubator, MTT-tetrazolium assay (see 2.2.4.) was
applied and the absorbance was read @ 570 nm using microplate reader. The
inhibition rate and ED50 were calculated as follows:
inhibition rate%= (T cells with extract - cells with medium) X 100%
(T cells with solvent - T cells with medium)
T: optical density of cells under different treatments (with or without extract)
ED50 is the calculated effective dose which inhibits growth by 50%
2.2.8. In-vitro analysis to determine the type of cell death
2.2.8.1. DNA fragmentation ELISA assay (Fig.1)
After the promising in-vitro cytotoxic experiments we were focusing on SN as a
potential cytotoxic agent which could be used in breast cancer treatment.
Exponentially growing human breast cancer cells MCF-7 were diluted with culture
medium to obtain a cell concentration of 1x105 cells/ml. 100 µl of diluted cells (104
cells) were added to each microplate well. The cells were incubated with SN extract
47
for 4, 8, 12 and 24 hours at 37° C and 5% CO2. Negative control was 10% DMSO in
sterile water, positive control was DNA-histone-complex, background control was the
incubation buffer. After the respective incubation periods, the MTPs were centrifuged
(200 x g for 10 minutes). The supernatant (S1: contained necrotic DNA that leaked
through the membrane during the incubation) was used to analyze necrosis, the pellet
was lysed and centrifuged again. The supernatant (S2: the cytoplasmic fraction) was
used to detect apoptosis. 20 µl of S1 and S2 were transferred into the streptavidin-
coated MTP and continued with ELISA: 80 µl of the Immunoreagent was added to
each well and incubated on a microplate shaker (300 rpm for 2 hours at 15–25°C).
After rinsing MTP 3 times, 100 µl ABTS solution/well was added. After incubation on
a plate shaker (250 rpm 10-20 min) the samples were measured @ 405 nm against
ABTS solution as a blank (reference wavelength @ 490 nm).
Figure 1. Cell death detection ELISAPLUS (Roche, 2005a) A: sample preparation B: ELISA
48
The values were averaged and the background value (Incubation buffer + ABTS
solution) was substracted. The specific enrichment of mono- and oligonucleosomes
released into the cytoplasm was calculated from these values using the following
formula:
mU of the sample (dying/ dead cells)
enrichment factor = -----------------------------------------------------------
mU of the corresponding negative control (cells without SN treatment)
mU = absorbance [10-3]
2.2.8.2. Cellular DNA fragmentation assay (Fig.2)
Sub-cultured MDA-MB-231 breast cancer cells (2-4 x 105) in tissue culture flask were
pre-labeled with the non-radioactive thymidine analogue BrdU overnight at 37°C in
5% humidified CO2 incubator. The labeled cells were further incubated in the
presence of SN extract at different concentrations (10-400 μg/ml) for 2, 4 and 6 hours.
The negative control was 10% DMSO in sterile water, the positive control was
camptothecin (0.1 μg/ml).
At the end of each incubation period, the cells were centrifuged @ 900 rpm for 5
minutes. The supernatant (S1) was analyzed for necrosis and the cellular lysate was
further centrifuged. The supernatant of this cellular lysate (S2) was used for apoptosis
detection.
49
Figure 2. Cellular DNA fragmentaion ELISA (Roche, 2005b)
The wells of a MTP were coated with anti-DNA antibody, S1 and S2 samples were
added into duplicate wells and incubated for 90 minutes. After washing the MTP, the
samples (some of them containing immunocomplexed BrdU-labeled DNA-fragments)
were denaturized by microwave irradiation (500W for 5 minutes). Anti-BrdU
antibody peroxidase conjugate (Anti-BrdU-POD) was added to form an
immunocomplex with the BrdU-labeled DNA. Following 90 minutes of incubation,
peroxidase substrate (TMB) was added to the wells and after 10-30 minutes of
incubation the absorbance was measured @ 405nm wavelength using an ELISA plate
reader.
2.2.9. Animal handling
2.2.9.1. Housing and maintenance
The animals (Swiss albino, BALB/c, BALB/c nude mice) were kept in filter-covered
plastic cages, housed in a temperature-controlled (28°C) room with a diurnal 12 hours
light cycle, and provided with tap water and standard rodent diet ad libitum. The nude
mice were kept in a pathogen-free environment under similar maintenance
50
2.2.9.2. Mice gavage
BALB/c mice were restrained by grasping the nape, and a gavage tube was then
inserted into the oral cavity of the mouse, moved downwards into the esophagus and 1
ml herbal extract was administered.
2.2.9.3. Blood collection by intracardiac puncture
BALB/c mice were euthanized by cervical dislocation and a hypodermic needle was
inserted on the left lateral side of the sternum. Blood was aspirated and centrifuged to
separate plasma and serum.
2.2.9.4. Intraperitoneal (i.p.) injection
After restraining the mouse, the abdominal area was wiped with alcohol swab and a
hypodermic needle was inserted into the lower left/ right quadrant of abdomen. To
confirm proper placement of the needle, the syringe was aspirated and the herbal
extract (average 0.2 ml) was then administered in a steady, fluid motion.
2.2.9.5. Acute toxicity test
For the investigation of the acute toxicity of SN and to estimate the LD50, we used
both normal (SN water extract) and nude (SN ethanolic extract) BALB/c mice. We
tested ethanolic extract in nude mice since the in-vitro experiments demonstrated
stronger cytotoxic effect than the water extract and our future in-vivo experiments
involved nude mice.
The doses were 10, 100, 1000, 2000, 5000 mg/BW intraperitonially (i.p.) in normal
and 1500, 1750, 1875, 2000, 2250, 2500 mg/BW i.p. in nude mice. Both experiments
included control groups, each treatment group had 3 animals. Acute poisonous effect,
51
body weight and behavior change was observed daily for a period of 2 weeks. The
tests were carried out according to the protocols established by Lorke, 1983.
2.2.10. Breast tumor model in BALB/c nude mice
2.2.10.1. Tumor xenografts
In the first phase, 50 mice received subcutaneous intrascapular sustained release 17-β-
estradiol (0.72 mg, released over 60 days) pellet implant (Hawkins et al., 2000). 24
mice were inoculated with 107 MCF-7 cancer cells into the left and/or right flank
within 4 weeks of estradiol pellet implant.
To determinate the size of the subcutaneous tumors, we used the ellipsoid volume
formula (Osborne et al., 1985; Ware et al., 1985; Riondel et al., 1986) which was
reported to be the most accurate estimation of actual tumor volume (Tomayko et al.,
1989) and was applied by other laboratories in several breast cancer xenograft models
(Gottardis et al., 1988; Mimnaugh et al., 1991; Cameron et al., 1997). The tumors
were measured every four days with calipers in three dimensions (length (A), width
(B), depth (C) ) in order to calculate tumor volume, V (V=A x B x C x π / 6 mm3).
2.2.10.2. In-vivo treatment protocols
Animals with tumors larger than 8 x π / 6 mm3 (length, width and dimension
measuring at least 2mm respectively), were selected for treatment with SN extract
administered via i.p. injection: 300 mg/kg BW in 0.2 ml vehicle for three consecutive
days per week . The negative control was vehicle (10% DMSO in saline) alone, also
for three consecutive days each week. The positive control was 5 mg/BW kg
Doxorubicin in sterile saline, given once weekly.
In the second phase of the study, 25 mice were implanted with 107 MDA-MB-231
cancer cell implant into the left and/or right flank. 10 mice started to be treated at the
52
same time of the tumor implant (prophylactic study), 15 mice were selected for
treatment only after the tumor volume, V, was larger than V=A x B x C x π /6 mm3
(therapeutic study). The SN extract was administered via i.p. injection: 500 mg/kg
BW in 0.2 ml vehicle for three consecutive days per week. The negative control was
vehicle alone, also injected i.p. for three consecutive days each week. The positive
control was Doxorubicin 5 mg/kg BW in sterile saline, given i.p. once weekly.
Despite that the patients were consuming the TCM drugs per os, in our cytotoxic
laboratory experiments all nude mice were given i.p. injection as a more accurate and
reliable administration route and easily adjustable dose of the SN extract. This is in
line with the common cytotoxic/ chemotherapeutic drug administration in cancer
treatment.
2.2.11. Histopathological analysis of tumor tissue section
The uterine specimens and solid breast tumors were fixed in 10% formalin and
embedded in paraffin. The sections/ slides were stained with haematoxylin and eosin
and viewed under light microscope at 40x, 100x or 200x magnification.
2.2.12. Statistical analysis
The significance of the differences between control and treated values was analyzed
using Students’ t-test. Differences with p values < 0.05 were considered to be
statistically significant.
53
CHAPTER THREE: RESULTS AND DISCUSSION
54
3.1 Quality-of-life (QOL) study in breast cancer patients
3.1.1. Aims and experimental approach
The objective of the first part of our study was to conduct a QOL analysis among
breast cancer patients who are using conventional therapy and herbal Traditional
Chinese Medicine (TCM), in order to identify the medicinal herbs that might have
immunomodulator and/or anti-tumor effects and could palliate the side effects of
chemo- and radio-therapy.
A total of 13 female Chinese patients, age 31 to 53 years, were recruited to respond to
the EORTC questionnaires QLQ-C30 (version 3.0) and to the supplementary breast
cancer module (QLQ-BR23).
The questionnaire was administered in English or in validated Chinese translation.
During the study, 7 patients dropped out, thus we had only 6 cases to follow up at the
start (baseline) and 11-13 weeks after the herbal TCM treatment. The scores from the
questionnaires were transformed, interpreted and compared against breast cancer
patients published in the EORTC QLQ-30 Reference Values manual.
55
3.1.2 Results
Figure 3. QLQ-30 scores for 2 breast cancer patients receiving radio-, chemo-, hormonal and herbal TCM therapy
0
10
20
30
40
50
60
70
80
90
100
quali
ty of
life
phys
ical fu
nctio
ning
role f
uncti
oning
emoti
onal
functi
oning
cogn
itive f
uncti
oning
socia
l func
tionin
gfat
igue
naus
ea an
d vom
iting
pain
dysp
noea
insom
nia
appe
tite lo
ss
cons
tipati
on
diarrh
ea
finan
cial d
ifficu
lties
scor
e %
baselineafter 11 weeks of TCM treatment**
**
**
***
**
*
**
**
56
For two patients receiving chemo-, radio-, hormonal and herbal TCM therapy (Fig. 3),
the scores for global health status were 70.3% pre-treatment (70.3% post-treatment).
The mean scores for each of the functional scales pre- and (post) treatment were:
physical 85.0% (90.0%), role 75.0% (66.7%), emotional 79.2% (83.3%), cognitive
83.3% (83.3%), and social 66.7% (83.3%).
The mean scores for each of the symptom scales were: fatigue 33.3% (27.8%), nausea
and vomiting 25.0% (16.7%) and pain 8.3% (8.3%), dyspnoea 33.3% (16.7%),
insomnia 66.7% (16.7%), appetite loss 0% (0%), constipation 0% (16.7%), diarrhea
16.7% (0%), and financial difficulties 16.7% (33.3%).
On comparing the differences (Δ) between pre- and post-TCM treatment scores, we
found moderate improvements in social functioning (Δ=16.7%), dyspnoea
(Δ=16.7%), diarrhea (Δ=16.7%) and clinically very significant decrease of insomnia
(Δ=50.0%), and little improvement in nausea and vomiting (Δ=8.3) and fatigue
(Δ=5.5%). The scores were a little worse for role functioning (Δ=8.3%) and
moderately worse for constipation (Δ=16.7%) and financial difficulties (Δ=16.7%).
57
Figure 4. QLQ-30 scores for 1 breast cancer patient receiving radio-, hormonal and herbal TCM therapy
0
10
20
30
40
50
60
70
quality
of life
physica
l functi
oning
role
functioning
emotio
nal functi
oning
cognitiv
e functi
oning
socia
l functi
oning
fatigue
nause
a and vo
miting
pain
dyspnoea
insomnia
appeti
te loss
consti
pation
diarrh
ea
finan
cial d
ifficu
lties
scor
e %
baselineafter 11 weeks of TCM treatment
****
**
**
***
58
For one patient receiving radio-, hormonal and herbal TCM therapy (Fig. 4), the mean
scores for global health status were 33.3% (33.3%). The mean scores for the functional
scales were: physical 60.0% (66.7%), role 66.7% (50.0%), emotional 50.0% (50.0%),
cognitive 50.0% (50.0%) and social 33.3% (66.7%). The mean scores for the symptom
scales were: fatigue 55.7% (44.3%), nausea and vomiting 0% (0%) and pain 33.3%
(33.3%), dyspnoea 0% (0%), insomnia 0% (33.3%), appetite loss 33.3% (33.3%),
constipation 0% (0%), diarrhea 0% (0%), and financial difficulties 33.3% (33.3%).
On comparing the differences between the pre- and post-TCM treatment scores, we
found little improvement in physical functioning (Δ=6.7%), moderately less fatigue
(Δ=11.3%) and very significant improvement in social functioning (Δ=33.3%). The
scores were moderately worse for role functioning (Δ=16.7%) and there was a very
significant (Δ=33.3%) increase in insomnia.
59
Figure 5. QLQ-30 scores for 2 breast cancer patients receiving hormonal and herbal TCM therapy
0
20
40
60
80
100
120
quali
ty of
life
phys
ical fu
nctio
ning
role
func
tioning
emotio
nal fun
ction
ing
cogn
itive f
uncti
oning
socia
l func
tioning
fatigu
e
naus
ea an
d vom
iting
pain
dysp
noea
insomnia
appe
tite lo
ss
cons
tipati
on
diarrh
ea
finan
cial d
ifficu
lties
scor
e %
baselineafter 11 weeks of TCM treatment
*
*
* *
**
**
***
60
For two patients receiving hormonal and herbal TCM therapy (Fig. 5), the mean scores
for global health status were 54.2% (62.5%). The mean scores for the functional scales
were: physical 90.0% (93.3%), role 100.0% (91.7%), emotional 91.7% (83.3%),
cognitive 91.7% (83.3%), and social 75.0% (75.0%).
The mean scores for the symptom scales were: fatigue 22.2% (11.2%), nausea and
vomiting 0% (0%) and pain 16.7% (16.7%), dyspnoea 0% (16.7%), insomnia 33.3%
(33.3%), appetite loss 0% (0%), constipation 50.0% (33.3%), diarrhea 0% (0%), and
financial difficulties 0% (0%).
On comparing pre- and post-TCM treatment scores, we found insignificant change for
global health status (Δ=8.3%), moderate improvement for fatigue (Δ=11.0%) and
constipation (Δ=16.7). The scores were little worse on role, emotional and cognitive
function scales and the patients had much more dyspnoea.
61
Figure 6. QLQ-30 scores for breast cancer patient receiving only herbal TCM therapy
0
20
40
60
80
100
120
quality
of life
physica
l functi
oning
role
functioning
emotio
nal functi
oning
cognitiv
e functi
oning
socia
l functi
oning
fatigue
nause
a and vo
miting
pain
dyspnoea
insomnia
appeti
te loss
consti
pation
diarrh
ea
finan
cial d
ifficu
lties
scor
e %
baselineafter 11 weeks of TCM treatment
*
***
**
***
62
For 1 patient receiving only herbal TCM therapy (Fig. 6), we used one survey point
instead of a mean of data. The scores for global health status were 100.0% (post-TCM
treatment 100.0%). The scores for the functional scales were: physical 93.3%
(100.0%), role 100.0% (100.0%), emotional 100.0% (100.0%), cognitive 100.0%
(100.0%), and social 100.0% (100.0%).
The scores for the symptom scales were: fatigue 33.3% (11.0%), nausea and vomiting
0% (0%) and pain 16.7% (0%), dyspnoea 0% (0%), insomnia 33.3% (0%), appetite loss
0% (0%), constipation 0% (0%), diarrhea 0% (0%), and financial difficulties 0% (0%).
We found little difference between pre- and post-TCM treatment scores on the physical
functioning scale (Δ=6.7%), moderately less pain (Δ=16.7%) and clinically very
significant improvement for fatigue (Δ=22.3%) and insomnia (Δ=33.3%).
63
Figure 7. QLQ-30 reference scores compared to the baseline scores of 6 breast cancer patient in Singapore receiving herbal TCM therapy
0
10
20
30
40
50
60
70
80
90
100
quality
of life
physica
l functi
oning
role
functioning
emotio
nal functi
oning
cognitiv
e functi
oning
socia
l functi
oning
fatigue
nause
a and vo
miting
pain
dyspnoea
insomnia
appeti
te loss
consti
pation
diarrh
ea
finan
cial d
ifficu
lties
scor
e %
EORTC reference values
baseline scores for 6 Singaporeanbreast cancer patients
**
** **
*
*
**
*
*
64
We also calculated each patient’s baseline score on each scale of the QLQ-30
questionnaire, then took the means of the scores of the six patients as our referral
values, and compared these values with the reference values given in the QLQ-C30
Reference Value Manual (Fayers et al., 1999).
The mean score of our six patients for global health status was 63.7% (reference value
66.3%).
The mean scores for the functional scales were: physical 83.9% (73.6%), role 86.1%
(76.6%), emotional 81.9% (67.3%), cognitive 83.3% (83.1%), and social 69.4%
(77.3%).
The mean scores for the symptom scales were: fatigue 33.3% (31.4%), nausea and
vomiting 8.3% (8.8%), pain 16.7% (29.1%), dyspnoea 11.1% (20.1%), insomnia 38.9%
(31.1%), appetite loss 5.6% (19.9%), constipation 16.7% (13.9%), diarrhea 5.6%
(7.0%), and financial difficulties 11.1% (13.4%).
On comparing the six breast cancer patients’ baseline scores from Singapore with the
scores from the QLQ-C30 Reference Value Manual of breast cancer patients (Fig. 7)
[Fayers et al., 1999], we found moderately lower scoring on the symptom scales of pain
and appetite loss and little less dyspnoea among our patients. On the functional scales,
the scores for our patients were little better for role function and moderately higher for
physical and emotional functions; there was no difference between patient populations
on the other scales.
65
Figure 8. BR-23 scores of breast cancer patients at baseline and after 11-13 weeks of TCM treatment
0
10
20
30
40
50
60
70
80
90
body imag
e
sexu
al functi
on
sexu
al en
joymen
t
future pers
pectiv
e
syste
mic thera
py side e
ffects
breast
symptoms
arm sy
mptoms
upset b
y hair
loss
scor
e %
baseline
after TCM treatment
***
***
***
* *
*
66
To interpret the scores from the QLQ-BR23 questionnaire, we compared the mean
QLQ-BR23 scores of our patients before and after herbal TCM treatment (Fig. 8). The
scoring for the following items was as follows: body image pre-TCM treatment 84.7%
(post-TCM treatment 83.3%), sexual functioning 30.6% (43.3%), sexual enjoyment
66.7% (44.4%), future perspective 27.8% (55.6%). The scores for the following items
on the symptom scales were systemic therapy side-effects 23.9% (18.0%), breast
symptoms 12.5% (18.5%), arm symptoms 12.9% (11.1%) and “upset by hair loss”
33.3% (16.7%).
As reference values for the BR-23 module are not available, we compared our
findings as changes in scores over the 11-13 week period of follow up, using the
baseline measurement as reference value. We concluded from the findings that after
herbal TCM treatment, our patients had very much more positive “future
perspective”. They also had a little better sexual functioning and the systemic therapy
side effects decreased a little. On the other hand, their sexual enjoyment decreased
markedly and they had a little increase in breast symptoms.
67
Figure 9.Single item score distribution of 6 breast cancer patients at baseline and after 11-13 weeks of TCM
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Dyspnoea
at bas
eline
Dyspnoea
post-TCM
Insomnia
at bas
eline
Insomnia
post-TCM
Appetite
loss at
baseli
ne
Appetite
loss post-
TCM
Constipati
on at bas
eline
Constipati
on post-TCM
Diarrh
ea at
baseli
ne
Diarrh
ea postT
CM
Financia
l diffi
cultie
s at b
aseli
ne
Financia
l diffi
cultie
s post-
TCM
SCALES
patie
nts
not at alla littlequite a bitvery much
68
On the single item score distribution (Fig. 9), we can see that the proportion of
patients complaining of having “quite a bit” or “very much” symptoms decreased
after herbal TCM treatment: at baseline, 50% of the patients were complaining of
“quite a bit” of insomnia, which was reduced to 0% after treatment; 20% of the
patients reported “very much” to having constipation, this changed to 50% of patients
complaining of “a little bit” of constipation. But the proportion of patients who had
“a little bit” of dyspnoea increased from 33% to 50%. After the treatment, no
patients complained of having insomnia, appetite loss or diarrhea.
As reference values for the BR-23 module are not available, we compared our findings
as changes in scores over the 11-13 week period of follow up, using the baseline
measurement as reference value. We concluded from the findings that after herbal TCM
treatment, our patients had very much more positive “future perspective”. They also had
a little better sexual functioning and a little less systemic therapy side effects. On the
other hand, their sexual enjoyment decreased markedly and they had a little increase in
breast symptoms.
3.1.3. Discussion
The responses of the 6 breast cancer patients to herbal TCM were generally favorable.
The two patients who were using radio, chemo, hormonal (Tamoxifen) and herbal
TCM therapy had much less dyspnoea, insomnia and diarrhea. The patient who was
receiving radio-, hormonal therapy and herbal TCM had moderately less fatigue,
while the two patients who were on herbal TCM treatment at the same time with
Tamoxifen had much less constipation. When herbal TCM was the only treatment, the
69
patient had a very significant decrease in fatigue, pain and insomnia without
decreasing other functions or increasing disease symptoms. These might indicate that
herbal TCM had reduced the side effects of radio-, chemo- and hormonal therapy and
could be especially useful in palliative cancer treatment.
Our study has several limitations. Interpreting quality of life data can be approached
in different ways. According to Fayers (2001), one method is to use the normative
approach and compare the scores against published data. In our study, we used the
QLQ-C30 Reference Value Manual scores (derived from population in Canada,
Denmark, Norway and Sweden) as our population-based reference values because
there are no reference values for Asian populations as yet.
One of the points we need to discuss is how we can compare our findings with
reference values from a different population having national and cultural differences
i.e. Singapore’s Chinese population vs Scandinavian population. A Singapore
population-based set of national reference values is long overdue. Such values will be
of great use to future Asian QOL studies investigating the efficacy of TCM.
The second issue is how to decide whether a finding is significant. Clinical
significance is subjective and statistical significance is not an appropriate method to
define it, because “for any given change or treatment effect, the P values are affected
by the sample size” (Fayers, 2001). Osoba et al., (1998) found that when the scale
scores changed by 5 to 10 points (on the 0-100 scale), patients described their
condition as a “little” better or worse. A change of 10 to 20 points was described as a
“moderate” change, while one greater than 20 was “very much” better or worse. In
our study, we found that the 1 patient who was receiving only herbal TCM therapy
had a “little” (6.7 points) better post-TCM treatment score on physical functioning,
70
moderately less pain (16.7 points) and “very much” less fatigue (22.3 points) and
insomnia (33.3 points). Other score differences of less than 5 points were thus
considered to be clinically insignificant.
The third issue is the possible influence of repeated measurements from the learning
effect through using the same questionnaire in the same patient over relatively short
time intervals (1-2 weeks). This influence could perhaps be minimized by
administering only 2 questionnaires per patient (baseline and post-treatment).
The fourth issue is the sample size and selection of the patients. We recruited 13
patients for this study, but over time 7 dropped out – questionnaires were not returned
perhaps due to the lack of incentives.
Last but not least is the issue of the extent of missing values, which were the highest
(51%) on the sexual functioning scale, thus affecting the validity of the scores for that
scale. Our impression was that in line with Asian standard pattern of behavior - the
patients were not willing to answer questions related to their sexual life.
It needs to be pointed out that the findings from these case studies are subjective;
evidence from laboratory tests and other investigations (i.e. computer tomography
scans, X-ray, ultrasound) would be helpful to document the clinical improvement of
the patients and so provide further support for the role of herbal TCM in the care of
cancer patients.
71
3.2. Extraction and characteristics of Solanum nigrum
3.2.1. Aims and experimental approach
It has been shown that SN crude extract and some of its individual compounds have
growth inhibiting effects on different cancer cell lines: colon, nasopharynx, uterine
and cervix, glioma, melanoma (Chiang et al., 1991), human hepatoma (Kuo et al.,
2001) breast cancer (Son et al., 2003), basal cell and squamous cell carcinoma (Cham
et al., 1987). Cai et al., in 2004 reported that phenolic compounds found in SN and
other traditional Chinese medicinal herbs were the dominant antioxidant components
to which the anti-cancer property of these herbs are attributed to.
It has also been reported that the bioactive compounds are most likely to be the
steroidal glycosides β2-solamargine, solamargine and galactotigonin (Hu et al., 1999).
As the breast cancer patients used the boiled water extract of SN but (as a standard,
first line laboratory procedure) we used ethanolic extract of SN in our experiments,
we were interested to know whether there was a major difference between the water
and ethanolic extracts of SN.
Our aim was to characterize the SN.
We ran both SN ethanolic and SN water extract on UV spectrophotometer and HPLC.
72
3.2.2. Results
Figure 10.SN water and ethanolic extract UV spectophotometer scan
A
A
B
The UV spectrophotometer scan suggested that SN water extract has a high peak at a
wavelength of 200-400nm (A). The SN ethanolic extract showed 2 peaks, one is
similar to the water extract, the second one at 650-700nm (B).
73
Figure 11.HPLC chromatogram of SN water extract
A B C
Figure 12.HPLC chromatogram of SN ethanolic extract
A B C
The HPLC chromatogram showed three similar peaks (A,B,C ) in both extracts, most
of the compounds being highly polar.
74
3.2.3. Discussion
From the above experiments we can conclude that most of the SN compounds are
highly polar. Both the water and ethanolic extract had a high peak at 200-500 nm
where most lipids, steroids (solamargine, solanidine), vitamins and flavoproteins
show absorbance. The peak at 650-700 nm in the SN ethanolic extract was perhaps
due to the porphyrins, chlorophylls and derivates ( Teledyne, 2003).
These experiments revealed that there was no major difference between the boiled
water and ethanolic extracts of SN. The components present in both extracts perhaps
were heat resistant steroidal glycosides.
3.3. Results of in-vitro immunomodulatory studies
3.3.1. Aims and experimental approach
Following the EORTC QOL-C30 and QLQ-BR23 analysis, we evaluated the herbal
prescription of the patients and selected the 2 most frequently used medicinal herbs,
Astragalus membranaceus (AM) and Curcuma aromatica (CA) for their possible
immunomodulatory property.
The immunopotentiating effect of the roots of AM has been associated with its
polysaccharide fractions (Shao et al., 2004)
Lymphocyte activation assay was carried out to evaluate the effects of the ethanolic
extracts of AM and CA on the proliferation of mouse lymphocytes under various
conditions: in the presence of optimal /suboptimal doses or absence of the mitogens,
concanavalin A (ConA) and lipopolysaccharide (LPS). The MTT tetrazolium assay
was carried out to evaluate lymphocyte activation by measuring lymphocyte activation.
75
We also compared whether the AM or CA ethanolic extract has stronger
immunomodulatory property. After obtaining the results, we carried out lymphocyte
proliferation assays comparing ethanolic, aqueous and fractionated aqueous AM
extracts. This was
Furthermore, it has been reported in the literature that AM has cytokine production
modulatory effect (Mao et al., 2004), thus we were interested in the levels of IL-2, IL-6
and TNF-α cytokines in the mouse serum following AM treatment.
3.3.2. Results
3.3.2.1. Effect of ethanolic extract of Astragalus membranaceus and Curcuma
aromatica
Table 4.Effect of different concentrations of 80% ethanol extract of Astragalus membranaceus and Curcuma aromatica on BALB/c mice lymphocyte proliferation. AM CA
0.1 1.0 10.0 0.1 1.0 10.0 μ g / m l
No mitogen + + + ConA suboptimal + + ConA optimal - - - + + + LPS suboptimal
LPS optimal
(+): stimulation, (-): inhibition, empty square: no statistically significant change (p value < 0.05).
Results are from six individual experiments.
76
As shown in Table 4, in the absence of mitogens, both 0.1 μg/ml and 1.0 μg/ml of
AM with 10.0 μg/ml of CA stimulated splenocyte proliferation. Neither extract
caused significant splenocyte proliferation in the presence of B cell mitogen, LPS.
In the presence of suboptimal dose of ConA, AM at 0.1 μg/ml and 10.0 μg/ml
significantly potentiated splenocyte activity. AM depressed splenocyte activity in the
presence of optimal dose of ConA.
In the presence of optimal dose of ConA, CA at 0.1 μg/ml, 1.0 μg/ml and 10.0 μg/ml
significantly potentiated splenocyte activity.
3.3.2.2. Effect of the fractions of Astragalus membranaceus water extract
Lymphocyte activation assay was carried out according to (Nakamura et al., 1986;
Wu et al., 1990) to evaluate the effects of 10 μg/ml, 1 μg/ml and 0.1μg/ml ethanolic,
aqueous and fractionated aqueous AM extract on the proliferation of mouse
lymphocytes under various conditions:
[a] in the presence of suboptimal doses of ConA (0.3 μg/ml), LPS (1 μg/ml)
[b] In the presence of optimal doses of ConA (3 μg/ml), LPS (6 μg/ml)
[c] without mitogens.
C0.3: conA suboptimal, C3: optimal, LPS1: LPS suboptimal, LPS6 optimal dose AM0.1, AM 1, AM10: Astragalus membranaceus 0.1, 1, 10 mg/ml concentration
The results are from 2 different experiments, each concentration combination in 6 wells per
experiment.
77
Figure 12.Immunomodulatory effect of the ethanolic extract of AM on mouse lymphocytes
* statistically significant (p value < 0.05) ** statistically significant (p value < 0.005)
Figure 13.Immunomodulatory effect of the aqueous extract of AM on mouse splenocytes
78
Figure 14.Immunomodulatory effect of butanolic fraction of aqueous extract of AM on mouse splenocytes
Figure 15. Immunomodulatory effect of ethyl acetate fraction of aqueous extract of AM on mouse splenocytes
79
Figure 16. Immunomodulatory effect of remaining water fraction of aqueous extract of AM on mouse splenocytes
The ethanolic AM extract has shown to be a stronger immunomodulator than the
boiled aqueous AM extract or any of its fractions (butanol, ethyl acetate or remaining
water fraction). All boiled water fractions showed a similar trend in the lymphocyte
activation assay: in the presence of suboptimal dose of ConA the AM extracts
stimulated the lymphocyte activity, while in the presence of optimal dose of mitogen
ConA the AM extracts in higher concentration suppressed lymphocyte proliferation.
However, these changes were not statistically significant. None of the AM extracts
stimulated the LPS induced splenocytes proliferation.
80
3.3.3. Discussion
The results indicate that both AM and CA have immuno-stimulatory effect.
AM acts best in the presence of suboptimal dose of ConA, while CA caused
significant splenocyte proliferation in the presence of optimal dose of ConA.
The responder cells are probably T-cells, as there was increased splenocyte
proliferation with the T-cell mitogen, ConA. The suppression of splenocytes in the
presence of optimal doses of ConA with 10.0 and 1.0 μg/ml dose of AM might be due
to the toxic effect of this extract. It appears that AM has a stronger
immunomodulating property as it potentiated splenocyte activity at a lower dose than
CA.
The ethanolic extract of AM displayed stronger immunomodulatory property then the
butanolic, ethyl acetate or the remaining water fraction of same.
The results of these experiments indicate that AM and CA may have a role not only in
palliative care, but also in enhancing the effects of conventional oncology therapy.
The stimulating activity on human lymphocytes deserves future investigation.
3.4. Cytokine studies from mice serum
3.4.1. Aim and experimental approach
It was reported earlier that AM is able to produce pro- and anti-inflammatory
cytokines (Chu et al., 1988a; Chu et al., 1988b): IL-2, TNF-α (Hong et al., 2005),
6-8 weeks old BALB/c female mice were fed orally daily for 14 days with ethanolic
extract of AM (50,100, 200 mg/BW kg). Blood samples were collected by cardiac
puncture and the serum was analyzed for levels of IL-2, IL-6 and TNF-α.
81
3.4.2. Results
Table 5. Cytokine concentration after AM treatment in BALB/c mice
CYTOKINE CONCENTRATION (pg/ml)
TREATMENT DOSE (mg/Bw kg) IL-2 IL-6 TNF-α
Control 28.4 (+SD0.36) Not enough sample < 3.8
50 18.8 (SD+0.35) 13.9 (SD+0.02) < 3.8
100 14.6 (SD+1.1) 24.2 (SD+0.14) < 3.8
200 19.2 (SD+0.1.1) Not enough sample < 3.8 three readings for each group, duplicate samples (mean + SD)
3.4.3. Discussion
IL-2 level fluctuated between the different AM treatment groups. There was no
consistent IL-2 inducing activity observed.
We were able to detect a more then 70% increase in IL-6 serum level in animals
which where treated with a double dose (50 versa 100 mg/BW kg). The limitation of
our study was that we had insufficient blood sample from the control and 200 mg/BW
kg treatment group.
TNF-α levels were undetectable < 3.8 pg/ml. The lack of detection might be due to
the sensitivity of the test
Our collaborator in USA (Gangemi, 2003) conducted tests with the ethanolic AM
extract provided by us. Dr. Gangemi tested the inductive effect of AM on human
lymphocytes in-vitro, measuring the levels of IL-6 and TNF-α cytokines. His findings
were in line with our results: the ethanolic extract of AM augmented IL-6 production,
82
however seemed to reduce LPS-induced TNF-α levels in a dose dependent manner.
The underlying mechanism remains to be determined.
3.5. In-vitro cytotoxicity studies
3.5.1. Aims and experimental approach
We sought a scientific evaluation of the cytotoxic effects of some Chinese herbs
widely used in breast cancer patients at 2 TCM Clinics we surveyed. The herbs were
identified through a quality-of- life (QOL) analysis using the European Organization
for Research and Treatment of Cancer (EORTC) QOL questionnaire (QLQ-C30
version 3.0) and the supplementary breast cancer module (QLQ-BR23). We compared
the herbal prescription of the patients who had better scores on the symptoms scale
(i.e. less fatigue, pain, dyspnoea, insomnia and appetite loss) and selected two of the
most frequently used herbs, Solanum nigrum (SN) and Scutellaria barbata (SB) for
their possible cytotoxic effects. First we tested them on MCF-7 to confirm the herb
with the stronger cytotoxic property, then tested its effects on other human breast
cancer cell lines: MDA-MB-231, T-47D and ZR-75-1.
3.5.2. Results
3.5.2.1. Results of cytotoxicity assays of Scutellaria barbata and Solanum
nigrum on MCF-7, MDA-MB-231, T-47D, ZR-75-1 human breast
cancer cell lines
83
Figure 17.Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells using MTT-tetrazolium assay
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
5 10 20 30 40
Concentration (μg/ml)
Abs
orba
nce
ratio
@ 5
70nm
from
aliv
e ce
lls
Solanum nigrum Scutellaria barbata
The results from both SN and SB treatment groups were statistically significant
(except SB 10 μl/ml and SN 40 μl/ml) in cell growth inhibition (p value < 0.005). SN
has shown stronger cytotoxic activity.
84
Figure 18.Comparative cytotoxic effect of Solanum nigrum and Scutellaria barbata on MCF-7 cells (log scale)
0
50
100
1 10
Concentration (μg/ml)
Inhi
bitio
n (%
)
100
Solanum nigrum Scutellaria barbata
A
B
SN appeared to be a stronger cytotoxic herb then SB.
The dose-response curve showed that the effect of SN declined after reaching a
plateau (maximal effective dose) at approximately 30 μg/ml, while the SB curve did
not plateau. ED50 for SN was 7.76 µg/ml (A) and for SB > 40 μg/ml (B).
We continued our cytotoxic experiments with SN on other human breast cancer cell
lines.
85
3.5.2.2. Results of cytotoxicity assays of the ethanol extract of Solanum
nigrum on MCF-7, MDA-MB-231, T-47D and ZR-75-1 cell lines
Figure 19.Cytotoxic effect of the ethanol extract of SN on MCF-7, MDA-MB-231, T-47D and ZR-75-1 cell lines
ED50=106.26µg/ml
ED50=37.08 µg/ml
ED50=24.32 µg/ml
ED50=7.76 µg/ml
0
50
100
1 10 100Concentration (µg/ml)
Inhi
bitio
n (%
)
Solanum nigrum on MCF-7 Solanum nigrum on ZR-75-1Solanum nigrum on MDA-MB-231 Solanum nigrum on T-47D
The ED50 values for SN treatment were: MCF-7 ED50=7.76 µg/ml < ZR-75-1
ED50=24.32 µg/ml < MDA-MB-231 ED50=37.08 µg/ml < T-47D ED50=106.26 µg/ml.
MCF-7 and ZR-75-1 (ERα+, bcl-2+)cell lines were the most sensitive to SN, while
MDA-MB-231 (ER α-, bcl-2+) was less sensitive, T-47D (ERα+, bcl-2-, prolactin +,
androgen +) was the least inhibited by SN. Comparing the biochemical markers and
genes expressed in the different cell lines, bcl-2 and p53 might have a role in the
mechanism of cytotoxicity.
86
3.5.2.3. Results of DNA fragmentation in MCF-7 cells treated with Solanum
nigrum
Figure 20.DNA fragmentation in MCF-7 cells (SN 4h)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10 100 1000Concentration (µg/ml)
Abso
rban
ce (4
90-4
05 n
m)
supernatant lysate
Figure 21.DNA fragmentation in MCF-7 cells (SN 8h)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10 100 1000
Concentration (µg/ml)
Abs
orba
nce
(490
-405
nm
)
supernatant lysate
87
Figure 22.DNA fragmentation in MCF-7 cells (SN 12h)
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
1 10
Concentration (µg/ml)
Abs
orba
nce
(490
-405
nm
)
100
supernatant lysate
Figure 23.DNA fragmentation in MCF-7 cells (SN 24h)
0
0.5
1
1.5
2
2.5
3
1 10
Concentration (µg/ml)
Abso
rban
ce (4
90-4
05 n
m)
100
supernatant lysate
88
Preliminary ELISA DNA fragmentation study suggested that cell death in MCF-7 cell
line occurred via apoptosis. Higher dosage (50-400) µg/ml with shorter incubation
time (4, 8 h) and lower dosage (5-50 µg/ml ) with longer incubation time (12, 24 h)
were also tested. SN stimulated nucleosome release in a concentration- and time-
dependent manner: (a) after 4 hours of incubation, the necrosis was more dominant,
(b) after 8 hours of incubation, nucleosome release was present, (c) after 12 hours, the
intensity of the nucleosome release reached a plateau.
3.5.2.4. Results of DNA fragmentation in MDA-MB-231 cells treated with Solanum nigrum
The supernatant (S1, blue) was analyzed for necrosis and the cellular lysate was
further centrifuged. The supernatant of this cellular lysate (S2, pink) was used for
apoptosis detection. The logarithmic curve was generated by measuring the
nucleosome content of lysate S1 and S2 treated with SN after different incubation
time.
Figure 24. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 2h incubation
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
1 10 100
Concentration (µg/ml)
Abso
rban
ce (4
50nm
)
2 hours supernatant 2 hours lysate
89
Figure 25. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 4h incubation
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
1 10 100
Concentration (µg/ml)
Abso
rban
ce (4
50 n
m)
4 hours supernatant 4 hours lysate
Figure 26. Cytotoxic effect and cellular DNA fragmentation of the ethanol extract of SN on MDA-MB-231 after 6h incubation
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
1 10 100
Concentration (µg/ml)
Abso
rban
ce
6 hours supernatant 6 hours lysate
After 2 hours of incubation of MDA-MB-231 cells with SN, the DNA fragment
release was more prominent then the high molecular weight DNA release. After 4
hours, the nucleosome release reached a plateau and decreased after 6 hours.
90
Picture 9. MDA-MB-231 cells incubated with SN extract (100X)
2 hours 4 hours Negative control
20 µg/ml SN
40 µg/ml SN
80 µg/ml SN
91
2 hours 4 hours SN 100µg/ml SN
SN 200µg/ml
SN 400µg/ml
Positive control
92
Examination by light microscopy showed increased cellular damage in a time- and
dose-dependent manner. The characteristics of apoptosis (cell shrinkage, nuclear
condensation, apoptotic bodies, cell debris) were visible after 2 hours of incubation
with higher then 80 μg/ml SN dose and after 4 hours of incubation with higher then
40 μg/ml SN dose.
3.5.3. Discussion
DNA fragmentation studies and microscopic examination revealed that incubation
with SN extract caused apoptotic cell death in both caspase-3 negative MCF-7 and
caspase-3 positive MDA-MB-231 human breast cancer cells in-vitro. The apoptosis
occurred within 4 hours, after which secondary necrosis was more prominent. In the
MCF-7 cells the apoptosis probably occurred in a caspase-3 independent manner.
The distinctive morphological markers of apoptosis (membrane blebbing, cell
shrinkage, condensation of chromatin, presence of apoptotic bodies) and the DNA
fragmentation results were suggesting that the type of cell death could be apoptosis.
3.6. In-vivo cytotoxicity studies
3.6.1. Aims and experimental approach
Our previous in-vitro experiences showed that certain Chinese herbs, including SN,
can cause cytotoxicity via apoptosis in human breast cancer cells. We found that the
ethanolic extract of SN appears to have a dose-dependent cytotoxic effect on cancer
cells in-vitro, with an average ED50 of 7.75 µg/ml for MCF-7 and ED50 of 37.08
µg/ml for MDA-MB-231.
Thus we investigated further whether SN crude extract could cause tumor regression
in athymic mouse model xenografted with hormone-dependent MCF-7 (after priming
93
with estrogen) and hormone independent MDA-MB-231 human breast cancer cells.
This model is ideal to investigate the actions of antitumor drugs ( Gottardis et al.
1988; Tomayko et al., 1989; Mimnaugh et al., 1991; Hawkins et al., 2000).
3.6.2. Results
3.6.2.1. Acute toxicity test of Solanum nigrum in normal and nude BALB/c
mice
Figure 27.Acute toxicity test of Solanum nigrum (water extract) in Swiss albino mice
30.00
32.00
34.00
36.00
38.00
40.00
42.00
44.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
days after single SN dose administration
body
wei
ght a
fter t
reat
men
t (m
g)
0 mg/kg BW 10 mg/kg BW 100 mg/kg BW 1000 mg/kg BW 2000 mg/kg BW 5000 mg/kg BW
The oral acute toxicity test showed that in Swiss albino mice LD50 was higher then
5000 mg. There was no significant change in the weight, appearance, grooming and
activity of the animals after a 2 week period of observation (Fig.29).
The i.p. administration of SN in nude mice appeared to be more toxic, LD50 being
around 2000 mg. The animals injected with SN doses higher then 2000 mg showed
symptoms of tachycardia, psychomotor agitation, paralysis, circulatory and
94
respiratory depression and eventually death within 20 minutes of SN administration.
Yen et al., 2001, reported that the toxicity could be due to the SN compound
solasonine. This glycoalkaloid can be transformed into a secondary metabolite in the
body, entering the blood-brain barrier causing disruption in essential life functions
(Schwartz et al., 2005).
Nude mice treated with non-toxic dose had no change in their weight, appearance or
behavior.
3.6.2.2. Anti-tumor activity of Solanum nigrum treated MCF-7 tumor
bearing oophorectomized nude mice
Prior to SN-treatment, estradiol stimulated the growth of MCF-7 breast tumor
implants (Pic.10,11,12 ) in BALB/c oophorectomized nude mice. Most of the tumors
were continuously growing even after estrogen treatment had completed.
In this study we implanted (estradiol-primed ) 15 mice with MCF-7 cells, chose 20
weeks as the cut off period to shorten the duration of suffering. 9/15 did not grow
tumors after 20 weeks and but maintained their body weight (20-26 grams). 6/15 grew
tumors larger then 8 x π / 6 mm3. 5 of these tumor bearing mice were treated with SN
from the time when the tumor volume reached 8 x π / 6 mm3. 3 out of these 5 died and
2 survived. In one of these mice the tumor disappeared, while the other mouse
maintained its body weight, though the tumor continued to grow. One negative
control (no SN treatment), died within a week after the tumor volume reached 8 x π /
6 mm3.
The total mortality among the MCF-7 implanted mice was 6/15: 2/15 died without
visible tumor but were cachexic. 3/15 died despite SN 300mg/kg BW treatment, 1/15
died without SN treatment. The mortality rate for tumor bearing mice was 60%.
95
Picture 10. Large solid MCF-7 tumor in estrogen-primed mouse #1
Picture 11.Two solid tumors grown from mouse #1
96
Picture 12 Multiple solid tumors grown from MCF-7 implants from mouse #2
Mice treated with SN did maintain their original weight of 20-26 grams without any
change in behavior. The tumor-bearing mice without SN treatment became cachexic,
with bodyweights below 20 grams (Pic.13).
Picture13. Cachexic mouse on the left compared to SN-treated mouse on the right
97
3.6.2.3. Estrogen-induced uterine cancer in oophorectomized BALB/c nude
mice
We investigated tumor regression in athymic mouse model xenografted with
hormone-dependent MCF-7 human breast cancer cells after priming with oestrogen.
As mentioned previously, this model is ideal to investigate the actions of antitumor
drugs.
Fifty inbred female oophorectomized BALB/c nude mice (Animal Research Centre,
Canning Vale, Australia), 8 to 12 weeks old, received implants of 60 days release 17-
β-oestradiol (0.72 mg) pellets (Innovative Research of America, Sarasota, Florida,
USA).
Figure 28.Mortality of 50 beta-estradiol implanted nude mice over time
0
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20weeks from beta-estradiol implant
num
ber o
f mic
e
However, incidental findings revealed that 0.012 mg daily (0.72 mg over 60 days)
estrogen dose induced endometrial carcinoma (Pic.14) with an 11/50 total mortality
rate (Fig.30). On post mortem examination (Pic.13), the mice had enlarged uteri with
poorly differentiated cell infiltration. The average enlarged uterine diameter was 10
mm, compared to a normal of 2-3 mm.
98
Picture 14. Mouse uterine tissue H&E staining
Picture 15.Enlarged mouse uteri on post-mortem examination
99
3.6.2.3.Anti-tumor activity of Solanum nigrum treated MDA-MB-231 tumor bearing oophorectomized nude mice
3.6.2.4.1. Results of therapeutic Solanum nigrum treatment
In this study we implanted 15 mice with MDA-MB-231 cells. The cut-off for the
observation period was 8 weeks post-implant since this type of tumor is very aggressive
and spreads easily.
6/15 did not grow tumors and they maintained their body weight (20-25 grams). 7/15
grew tumors, out of which 5 had multiple metastases - 1 to the spine (Pic.16a,b,c,d), 1
to the orbit (Pic.17a,b,c) These mice received SN treatment – the tumors were growing
though the body weight did not drop below 20grams. 2 animals with tumors which did
not received SN treatment became cachexic. 2 mice without measurable tumor but on
SN treatment also became cachexic. Picture 16 (a, b, c). MDA-MB-231 metastasis to the spine
(a) (c)
(b)
100
Picture 17 (a, b, c). MDA-MB-231 metastasis to the orbit
It is shown on both Picture16. and 17. the extensive damage the MDA-MB-231
implant caused – despite the SN-treatment. Picture 16. shows (a) spinal metastasis, (b)
a paraffin section of this multi-lobular tumor, (c) H&E staining of a section of the
above mentioned mass. On Picture 17. is shown (a) a large, left orbital, bony
metastatic mass, (b) the paraffin section of the tumor site, (c) H&E staining of a tumor
slide under light microscope (40x).
On both H&E slides is evident the presence of tumor cells with poorly differentiated
nuclei. Some of the apoptotic bodies are marked with arrows on Picture 16c.
(a)
(b) (c)
Poorly differentiated nuclei ( ) and apoptotic bodies ( ) are marked with arrows on Picture 16(c)and 17(c).
101
3.6.2.4.2. Results of prophylactic Solanum nigrum treatment
In this study we chose 10 mice, cut-off period at 8 weeks. The grouping was the
following: positive control Doxorubicin 5 mg/kg (n=3), negative control 10%DMSO
in saline (n=3) and SN treatment (n=4). The mortality rate in the positive control
group was 100% probably due to Doxorubicin-induced cardiac toxicity. In the
negative control group 67% grew tumors, while 33% had no tumor. In the 500 mg/kg
BW SN treatment group, 25% grew tumor, 75% had no tumor (Fig. 29). SN showed a
75% prophylactic rate against the growth of the tumor implants.
Figure 29. Prophylactic treatment of Solanum nigrum (ethanolic extract) on MDA-MB-231 cancer cell implanted BALB/c nude mice
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6 7 8 9
weeks after MDA-MB-231 implants
tum
or v
olum
e m
m3
saline (n=3) doxorubicin (n=3) SN500 (n=4)
102
3.6.2.5. Histopathological examination of Solanum nigrum induced apoptosis
in MDA-MB-231 cells
Picture 18. SN extract versa saline-treated MDA-MB-231 tumors SN-treated saline-treated
The H&E stained paraffin MDA-MB-231 tumor sections on the left were treated with
SN and shows several apoptotic bodies (black arrows) as a marker of apoptotic cell
death.
On the right panel we can see mainly necrotic tissue and less apoptotic bodies.
3.6.3. Discussion
40x40x
200x
Our experiment with the MCF-7 induced nude mice showed that the growth of the
implanted breast cancer cells was highly dependent on estrogen.
103
The results also revealed that the 0.012 mg daily (0.72 mg over 60 days) estrogen
dose was very high and induced endometrial carcinoma with a 11/50 total mortality
rate. This seems to suggest that mice models with estrogen-dependent human breast
cancer need to be hysterectomized in order to avoid the mortality rate from uterine
cancer.
We noted that cessation of estrogen at 60 days after pellet implant did not prevent
MCF-7 cells from growing tumors as occurred even at 10 days after estrogen
cessation.
SN treatment caused irreversible tumor regression where the tumor size was relatively
small.
In the cases where the mice grew multiple tumors, SN at 300 mg/bw kg was not
efficient in inhibiting the tumor growth, but the treated animals retained their original
body weight despite there being tumor growth, while untreated animals became
cachexic.
SN treatment could be used as prevention but the dose is not efficient for the
treatment of already existing aggressive tumors.
The acute toxicity test showed that the LD50 for oral SN treatment is higher then 5000
mg which is within relative safety limits.
104
CHAPTER FOUR: SUMMARY AND OVERALL
DISCUSSION OF THE RESULT
105
4.1 Summary of the results
In the clinical phase of our study we showed that most of our breast cancer patients
responded favorably to herbal TCM. The two patients who were using radio, chemo,
hormonal and herbal TCM therapy had much less dyspnoea, insomnia and diarrhea.
The patient who was receiving radio-, hormonal therapy and herbal TCM had
moderately less fatigue, while the two patients who were on herbal TCM treatment at
the same time with Tamoxifen had much less constipation. When herbal TCM was the
only treatment, the patient had a very significant decrease in fatigue, pain and
insomnia without decreasing other functions or increasing disease symptoms.
Our experimental results showed that breast cancer patients benefited from herbal
TCM use. The possible explanation for this could be that the prescribed herbal
mixture with its different compounds had different actions: This might indicate that
herbal TCM had reduced side effects of radio-, chemo- and hormonal therapy and
could be especially useful in palliative cancer treatment.
In the laboratory phase of our study we were seeking scientific evidence for the
therapeutic activity of respective herbs prescribed for breast cancer patients.
The results indicate that both AM and CA have immuno-stimulatory effect.
AM acts best in the presence of suboptimal dose of ConA, while CA caused
significant splenocyte proliferation in the presence of optimal dose of ConA.
The responder cells are probably T-cells, as there was increased splenocyte
proliferation with the T-cell mitogen, ConA. The suppression of splenocytes in the
presence of optimal doses of ConA with 10.0 and 1.0 μg/ml doses of AM might be
due to the toxic effect of this extract. It appears that AM has a stronger
immunomodulating property as it potentiated splenocyte activity at a lower dose
than CA.
106
The ethanolic extract of AM displayed stronger immunomodulatory property then
the butanolic, ethyl acetate or the remaining water fraction.
The results of the immunomodulating experiments suggest that AM and CA may
have a role not only in palliative care, but also in enhancing the effects of
conventional oncology therapy.
The stimulating activity on human lymphocytes deserve future investigation. The
herbs could have a chemopreventive action by blocking carcinogen activation and
suppressing malignant cell proliferation (Duvoix et al., 2005).
MCF-7 and ZR-75-1 cell lines (ERα+, bcl-2+) were the most sensitive to SN, MDA-
MB-231 (ER α-, bcl-2+)was less sensitive, T-47D (ERα+, bcl-2-, prolactin +,
androgen +) was the least inhibited by SN. Comparing the biochemical markers and
genes expressed in the different cell lines, bcl-2 and p53 might have a role in the
mechanism of cytotoxicity of SN.
The possible chemotherapeutic mechanisms of SN could involve blocking of tumor
cell cycle progression and/or signal transduction pathways by bonding to TNFR1 or
TNFR2 receptors, inhibiting the activities of transcription factors (NF-κB), increasing
the production of nitric oxide thus triggering apoptosis ( Lee et al., 2003; Son et al.,
2003; Heo et al., 2004).
Preliminary ELISA DNA fragmentation study suggested that cell death in MCF-7 cell
line occurred via apoptosis. Tested at higher dosages (50-400) µg/ml with shorter
incubation time (4, 8 h) and lower dosages (5-50 µg/ml ) with longer incubation time
(12, 24 h), SN stimulated nucleosome release in a concentration- and time-dependent
manner: a) after 4 hours incubation the necrosis was more dominant, b) after 8 hours
incubation the nucleosome release was present, c) after 12 hours the intensity of the
nucleosome release reached a plateau.
107
Further cellular DNA fragmentation studies and microscopic examination revealed
that incubation with SN extract caused apoptotic cell death in MDA-MB-231 human
breast cancer cells in-vitro. The apoptosis occurred within 4 hours, after which
secondary necrosis was more prominent. The distinctive morphological markers of
apoptosis were evident.
Our in-vivo experiment with the MCF-7 induced nude mice model showed that the
growth of the implanted breast cancer cells was highly dependent on estrogen.
Accidental observations also revealed that the 0.012 mg daily (0.72 mg over 60 days)
estrogen dose was very high and induced endometrial carcinoma with a 22% total
mortality rate. This seems to suggest that mice models with estrogen-dependent human
breast cancer need to be hysterectomized in order to avoid mortality from uterine
cancer.
We noted that cessation of estrogen at 60 days after pellet implant did not prevent
MCF-7 cells from growing into tumors as this occurred even 10 days after estrogen
cessation.
SN treatment caused irreversible tumor regression where the tumor size was relatively
small.
In the cases where the mice grew multiple tumors, SN at 300 mg/BW kg was not
efficient in inhibiting the tumor growth, but the treated animals retained their original
body weight despite there being tumor growth, while untreated animals became
cachexic.
SN treatment could be used in breast cancer prevention. It was however not efficient
for the treatment of pre-existing aggressive tumors at the doses studied.
The acute toxicity test showed that the LD50 for oral SN treatment (5000 mg) was
within relative safety limits.
108
4.2 Overall discussion
The present work is an attempt to evaluate the efficacy of herbal TCM treatment in
breast cancer. Scientific evaluation of TCM is extremely challenging. Several
biomedical experiments have described many possible mechanisms behind the
success of TCM herbal therapy in cancer: some plants/ components have anti-oxidant,
anti-angiogenesis, immunostimulator, cytotoxic, cytostatic effects. The molecular,
genetical mechanisms behind these therapeutic effects are not clear. There is a need
for scientific proof and clinical validation, including chemical standardization of
complex herbal formulations, in-vitro and in-vivo biological assays, animal models
and clinical studies (Yuan et al., 2000).
Future research and development in herbal TCM should adopt a clinical efficacy-
driven strategy and plan randomized, double-blind placebo trials to evaluate their
efficacy. However, the obstacle is how to generalize an individualized treatment?
In our clinical case studies we described 6 patients whose QOL improved and we
analyzed the prescribed medicinal herbs. This study has demonstrated in the
laboratory that these herbs are effective individually, but our clinical findings suggests
that the efficacy of TCM herbs is increased when they are used together, probably due
to their synergistic interaction.
As herbal TCM is already in use for over two thousand years, the efficacy driven
approach should have priority over the current mechanism centered approach (Tang,
2001).
TCM has a profound influence on the health care system in Singapore and should be
integrated into supportive cancer care, combined with biomedical practices.
109
CHAPTER FIVE: CONCLUSION AND FUTURE STUDIES
110
5.1 Conclusion
The following results have been demonstrated:
1. Herbal TCM improved quality of life of the recruited breast cancer patients.
2. Astragalus membranaceus and Curcuma aromatica exhibited
immunostimulatory effect in splenocytes.
3. Solanum nigrum had cytotoxic effects against both MCF-7 and MDA-MB-231
breast cancer cells in-vitro and in-vivo, and appeared to have a protective
effect against the growth of metastatic breast cancer.
The findings of this study indicate that:
1. Astragalus membranaceus and Curcuma aromatica enhanced T lymphocyte
proliferation and indirectly stimulated cytokine production.
2. Solanum nigrum induced cytotoxicity (in both estrogen-dependent and -
independent human breast cancer cells) was shown to be by apoptosis.
5.2 Future studies
The results of this study show that Solanum nigrum may be useful in the prevention
and/or treatment of invasive, non-hormone responsive breast cancer.
It would be worthwhile to identify the bioactive compound(s) of this herb, to further
investigate the molecular mechanisms of its cytotoxic action and to evaluate the effect
of the compound(s) on the apoptotic pathway.
111
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