Hong Kong Baptist University

DOCTORAL THESIS

A novel nucleolin aptamer-celastrol conjugate (NACC) with superantitumor activity on advanced pancreatic cancerLiu, Biao

Date of Award:2017

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HONG KONG BAPTIST UNIVERSITY

Doctor of Philosophy

THESIS ACCEPTANCE

DATE: August 8, 2017 STUDENT'S NAME: LIU Biao THESIS TITLE: A Novel Nucleolin Aptamer-celastrol Conjugate (NACC) with Super Antitumor

Activity on Advanced Pancreatic Cancer This is to certify that the above student's thesis has been examined by the following panel members and has received full approval for acceptance in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Chairman: Prof. Yu Zhiling

Professor, Chinese Medicine - Teaching and Research Division, HKBU (Designated by Dean of School of Chinese Medicine)

Internal Members: Dr. Yang Zhijun Associate Professor, Chinese Medicine - Teaching and Research Division, HKBU (Designated by Director of Chinese Medicine - Teaching and Research Division) Dr. Zhang Hongjie Associate Professor, Chinese Medicine - Teaching and Research Division, HKBU

External Members: Prof. Xu Hongxi Professor and Dean School of Pharmacy Shanghai University of Traditional Chinese Medicine Prof. Lin Zhixiu Associate Professor School of Chinese Medicine The Chinese University of Hong Kong

In-attendance: Prof. Lyu Aiping

Dean, School of Chinese Medicine, HKBU Issued by Graduate School, HKBU

A Novel Nucleolin Aptamer-Celastrol Conjugate (NACC) with Super

Antitumor Activity on Advanced Pancreatic Cancer

LIU Biao

A thesis submitted in partial fulfilment of the requirements

for the degree of

Doctor of Philosophy

Principal Supervisor:

Prof. LYU Aiping (Hong Kong Baptist University)

August 2017

i

DECLARATION

I hereby declare that this thesis represents my own work which has been done

after registration for the degree of PhD at Hong Kong Baptist University, and has

not been previously included in a thesis or dissertation submitted to this or any

other institution for a degree, diploma or other qualifications.

I have read the University’s current research ethics guidelines, and accept

responsibility for the conduct of the procedures in accordance with the

University’s Committee on the Use of Human & Animal Subjects in Teaching and

Research (HASC). I have attempted to identify all the risks related to this research

that may arise in conducting this research, obtained the relevant ethical and/or

safety approval, and acknowledged my obligations and the rights of the

participants.

Signaure:

Date: August 2017

ii

Abstract

Advanced pancreatic cancer (APC) has a poor prognosis due to the high degree

of resistance after systemic chemotherapy. Celastrol (CSL), a quinone methyl

triterpenoid monomer extracted from Tripterygium wilfordii Hook F, exhibits

superior antitumor activity on pancreatic cancer (PC) both in vitro and in vivo. In

addition, CLS counteracts multiple mechanisms involved in multi-drug resistance

(MDR) of PC cells. However, CSL induced toxicity to normal tissues (e.g. liver) is

the major impediment to its clinical application. Thus, it is desirable to seek

strategy to facilitate CSL selectively targeting PC tissues and simultaneously

reducing its exposure to healthy tissues (e.g. liver).

Aptamers are single-stranded oligonucleotides which specifically recognize and

bind to targets by distinct secondary or tertiary structures. Nucleolin, a protein

overexpressed on the plasma membrane of PC cells than that of normal cells (e.g.

liver cell), which shuttle between cell surface, cytoplasm and nucleus, work as a

cell surface receptor. Nucleolin aptamer is an anti-proliferative G-rich

oligonucleotide with high affinity and specificity to nucleolin, which has been

proved to be safe in clinical research. Then, nucleolin aptamer, as a target moiety,

provide an approach to facilitate CLS selectively targeting PC cells. Taken together,

our hypothesis is that the nucleolin aptamer modification could facilitate the

conjugated CSL selectively targeting pancreatic cancer cells to achieve higher

antitumor activity and less liver toxicity.

iii

In our study, CSL was conjugated to nucleolin aptamer to form Nucleolin

Aptamer-Celastrol Conjugate (NACC). A CRO Aptamer-Celastrol conjugate

(CACC) was also synthesized as a control for comparison. The water solubility of

NACC was significantly higher than that of CSL. Then, the molecular weight of

NACC was detected by ESI mass sepectrum (MS). The anti-proliferative efficacy

of NACC was higher than CSL in vitro. NACC could selectively bind to PANC-1

cells over normal liver cells. The cellular uptake of NACC by PANC-1 cell was

stronger than CSL. Moreover, NACC could be taken up by PANC-1 cells mainly

via macropinocytosis. Tissue distribution study revealed that NACC could

selectively accumulate in pancreatic tumor tissue and reduce the distribution in

liver in vivo. In addition, NACC demonstrated higher antitumor activity and less

liver toxicity in vivo, compared with CSL and CACC.

The above results revealed that the nucleolin aptamer modification could facilitate

the conjugated CSL selectively targeting PC cells to achieve higher antitumor

activity and less liver toxicity.

iv

Acknowledgement

I would like to express my sincere gratitude to my supervisors, Prof. Aiping Lyu,

Prof. Ge Zhang and Dr. Hailong Zhu for their guidance and support on the

research project during my Ph.D. program study. I would like to thank Prof. Aiping

Lyu for the encouragement and support in both project and daily life. I would also

like to thank Prof. Ge Zhang and Dr. Hailong Zhu for the careful guidance on the

ideas, experimental design and article written.

I would also express my gratitude to all the researchers and students of the TMBJ

for their valuable suggestions and support including: Dr. Baosheng Guo and Dr.

Defang Li for their guidance on the experimental design; Mr. Shaikh Atik Badshah

and Ms. Luyao Wang for their assistance on the animal study; Dr. Fangfei Li, Dr.

Yuanyuan Yu and Dr. Chao Liang for the support on molecular biology and cell

biology; Dr. Feng Jiang, Dr. Cheng Wang, Mr. Jun Lu and Ms. Lei Dang for their

assistance on chemosynthesis; Dr. Jin Liu and Dr. Bing He for their support on data

collation and statistical analysis.

I would also like to thank the researchers and students of Institute of Basic

Research in Clinical Medicine of China Academy of Chinese Medical Sciences

including: Dr. Miao Jiang, Dr. Cheng Lu, Dr. Xiaojuan He and Dr. Nannan Shi for

v

their support to the project; Mr. Kang Zheng and Ms.Qing qing Guo for the

assistance in the animal study.

I would also like to thank the lab technicians of CMTR including Ms. Cheung Yeuk

Siu, Hilda, Ms. Lee Sally, Ms. Chan Wai Kei, Nickie and Ms. Koo Ching, Irene for

their technical support.

I really appreciate the opportunity and support provided by Hong Kong Baptist

University.

Last but not the least, I would like to express my sincere gratitude to my family and

friends for their understanding and support.

vi

Table of Contents

DECLARATION .................................................................................................. i

Abstract ............................................................................................................... ii

Acknowledgement .............................................................................................. iv

Table of Contents ............................................................................................... vi

Lists of Figures and Tables .................................................................................. x

List of Abbreviation............................................................................................ xi

Chapter 1 Background ......................................................................................... 1

1.1 The epidemiology of pancreatic cancer and the major challenges ........... 1

1.2 The causes of chemotherapy tolerance in pancreatic cancer and the related

molecular mechanisms ................................................................................. 3

1.2.1 P-glycoprotein and MDR in pancreatic cancer ............................. 4

1.2.2 NF-kB pathway and MDR in pancreatic cancer ........................... 7

1.2.3 HSP90 and MDR in pancreatic cancer ......................................... 9

1.2.4 Tumor angiogenesis and MDR in pancreatic cancer ....................12

1.3 Celastrol as a potential drug for treatment of pancreatic cancer and the

impediment to its clinical application ..........................................................15

1.4 The application of aptamer in tumor targeted therapy ............................18

1.5 Nucleolin aptamer as a moiety to target pancreatic cancer .....................21

1.6 Research Hypothesis .............................................................................22

Chapter 2 Materials and Methods .......................................................................24

2.1 Materials for chemical synthesis ...........................................................24

2.2 Preparative high performance liquid chromatography (Pre-HPLC) analysis

for purifying compounds.............................................................................25

2.3 Nuclear magnetic resonance (NMR) analysis for indentifying compounds

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

2.4 Reversed-phase high performance liquid chromatography (RP-HPLC)

analysis for indentifying compounds ...........................................................26

vii

2.5 Cell culture ...........................................................................................26

2.6 Cell proliferation study .........................................................................27

2.7 Animal handing.....................................................................................27

2.8 Xenograft tumor model of pancreatic cancer .........................................28

2.9 Flow cytometry .....................................................................................28

2.10 Cellular uptake pathways ....................................................................29

2.11 In vivo injection of Microfil and Micro CT ..........................................30

2.12 Western blot analysis ...........................................................................30

2.13 Tissues and cellular distribution ..........................................................31

2.14 Blood biochemical analysis .................................................................32

2.15 Histological analysis ...........................................................................32

2.16 Statistical analysis ...............................................................................32

Chapter 3 The effect of modification on the carboxylic acid group on the antitumor

activity of celastrol .............................................................................................33

3.1 Aim .......................................................................................................33

3.2 Design ..................................................................................................33

3.3 Results ..................................................................................................34

Chapter 4 The synthesis and characterization of Nucleolin Aptamer-Celastrol

Conjugate (NACC) .............................................................................................36

4.1 Aim .......................................................................................................36

4.2 Synthesis of the celastrol derivative ......................................................36

4.2.1 Experimental design ...................................................................36

4.2.2 Results .......................................................................................37

4.3 Synthesis process of Nucleolin Aptamer-Celastrol Conjugate (NACC). 38

4.3.1 Experimental design ...................................................................38

4.3.2 Results .......................................................................................39

4.4 Synthesis process of the CRO Aptamer-Celastrol Conjugate (CACC) ...40

4.4.1 Experimental design ...................................................................40

4.4.2 Results .......................................................................................41

4.5 The water solubility and the stability of the NACC in vitro ...................42

viii

4.5.1 Experimental design ...................................................................42

4.5.2 Result .........................................................................................42

Chapter 5 The effect of the nucleolin aptamer modification on the cytotoxicity of

the conjugated CSL in NACC in vitro .................................................................44

5.1 Aim .......................................................................................................44

5.2 Experimental design..............................................................................44

5.3 Results ..................................................................................................44

Chapter 6 The effect of the nucleolin aptamer modification on the cellular uptake

and the cellular internalization of NACC in vitro ................................................46

6.1 Aim .......................................................................................................46

6.1.1 To investigate the selectivity of NACC to pancreatic cells and the

cellular uptake in vitro.........................................................................46

6.1.2 To investigate the mechanisms of cellular uptake of NACC by

pancreatic cancer cells in vitro.............................................................46

6.2 Experimental design..............................................................................46

6.3 Results ..................................................................................................47

Chapter 7 The effect of the nucleolin aptamer modification on the distribution of

NACC in a xenograft mouse model of human pancreatic cancer .........................52

7.1 Aim .......................................................................................................52

7.2 Experimental design..............................................................................52

7.3 Results ..................................................................................................53

Chapter 8 The effect of the nucleolin aptamer modification on the antitumor

activity of the NACC in a xenograft mouse model of human pancreatic cancer ..57

8.1 Aim .......................................................................................................57

8.1.1 To investigate the antitumor efficacy of NACC in xenograft tumor

model of pancreatic cancer. .................................................................57

8.1.2 To investigate the effect of NACC on angiogenesis of tumor

microenvironment. ..............................................................................57

8.1.3 To investigate the effect of NACC on expression level of NF-κB,

HSP 90 and P-glycoprotein in tumor tissue. ........................................57

ix

8.1.4 To investigate the effect of NACC on survival of tumor bearing

mice. ...................................................................................................57

8.2 Experimental design..............................................................................57

8.3 Results ..................................................................................................59

Chapter 9 The effect of the nucleolin aptamer modification on the liver toxicity of

NACC ................................................................................................................66

9.1 Aim .......................................................................................................66

9.2 Experimental design..............................................................................66

9.3 Result ...................................................................................................67

Chapter 10 Discussion ........................................................................................70

References ..........................................................................................................76

CURRICULUM VITAE .....................................................................................96

x

Lists of Figures and Tables

Figure 1………………………………………………………………………..16

Table 1…………………………………………………………………………18

Table 2…………………………………………………………………………19

Table 3…………………………………………………………………………26

Scheme 1………………………………………………………………………35

Figure 2………………………………………………………………………..35

Scheme 2………………………………………………………………………36

Figure 3………………………………………………………………………..37

Scheme 3………………………………………………………………………38

Figure 4………………………………………………………………………..39

Figure 5………………………………………………………………………..41

Figure 6………………………………………………………………………..43

Figure 7………………………………………………………………………..45

Figure 8-1……………………………………………………………………..49

Figure 8-2……………………………………………………………………..51

Figure 9……………………………………………………………………….56

Figure 10-1……………………………………………………………………62

Figure 10-2……………………………………………………………………63

Figure 10-3……………………………………………………………………64

Figure 10-4……………………………………………………………………65

Figure 11………………………………………………………………………68

xi

List of Abbreviation

ABC ATP-binding Cassette

ALT Alanine aminotransferase

AST Aspartate transaminase

Bcl-2 B-cell lymphoma-2

CACC CRO Aptamer-Celastrol Conjugate

CSL Celastrol

DIPEA N,N-Diisopropylethylamine

Dox Doxorubicin

DMF N,N-Dimethylformamide

EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-HCl

EMT Epithelial-mesenchymal transition

FasL FAS Ligand

FDA Food and Drug Administration

HATU o-(7-Azabenzotriazol-1-yl)-N,N,N',N'-te-tramethyluronium

hexafluoro-phosphate

H&E Haematoxylin-eosin

HSP Heat shock proteins

Hsp90 Heat shock protein 90

HUVEC Human umbilical vascular endothelial cells

xii

IκB Inhibitor of NF-κB

IKKβ Inhibitor of nuclear factor kappa-B kinase

L-Ala-OMe L-Alanine methyl

LC-MS Liquid Chromatograph-Mass Spectrometer

MAPK Mitogen-activated protein kinase

MDR Multidrug Resistance

MMP-9 Matrix metalloprotein 9

NACC Nucleolin Aptamer-Celastrol Conjugate

NF-κB Nuclear factor-κB

NMR Nuclear magnetic resonance

PBS Phosphate-buffered saline

PC Pancreatic cancer

P-gp P-glycoprotein

PKB Protein kinase B

PKC Protein kinase C

PSMA Prostate specific membrane antigen

Pre-HPLC Preparative high performance liquid chromatography

RP-HPLC Reversed-phase high performance liquid chromatography

ss DNA Single-stranded DNA

SELEX Systematic evolution of ligands by exponential enrichment

Sulfo-NHS N-Hydroxysulfosuccinimide sodium salt

TFA Trifluoroacetic acid

xiii

THF Tetrahydrofuran

TLC Thin layer chromatography

TMS Tetramethylsilane

TNF-α Tumor necrosis factor-α

VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

Wnt Wnt/β-catenin signaling

XIAP X-linked apoptotic proteins

1

Chapter 1 Background

1.1 The epidemiology of pancreatic cancer and the major challenges

Pancreatic cancer (PC) is a common digestive system tumor with high degree of

malignancy and dismal prognosis (Vera R et al. 2016, Bond-Smith G et al. 2012).

The 5-year survive rate of PC patients is less than 6% and the median survival time

of patient is less than 6 months (Kleeff J et al. 2016). In recent years, the incidence

of PC worldwide has been increasing year by year. In the United States, according

to statistical data, the number of PC patients accounted for 2%-3% of the total

number of tumor patients. But the death rate of PC ranked fourth among

cause of cancer-related deaths (Kamisawa T et al. 2016). In China, PC ranks sixth

in the death rate of malignant tumors and the incidence of PC increased by 6 times

during the past two decades (Jones OP et al. 2014, Ko AH et al. 2015). In Hong

Kong, PC is the No. 6 cancer killer with nearly 400 deaths every year (Bosetti C et

al. 2012). Therefore, as a malignant tumor, PC has become a huge challenge

to human health and life.

Different studies have demonstrated that PC is among the most complex cancers

due to the special tumor microenvironment and high metastatic property (Heestand

GM et al. 2015, Vincent A et al. 2011). Currently, surgical resection is the

only curative strategy offering the best long-term outcomes for PC patients. The

poor survival rate of PC is attributed to the late detection caused by lack of specific

2

biomarkers (Heinemann V et al. 2012). Due to the lack of specific symptoms of PC,

vascular invasion and distant metastasis were happened at the early stage of disease

and 85% of patients were diagnosed as late stage by the first diagnosis, losing the

best opportunity to surgery (Tang SC et al. 2014). Only a minority of patients with

PC were diagnosed as resectable disease at the first time. Even with early diagnosis

and surgical resection, there are still many patients with local recurrence or distant

metastasis soon after surgery (Hidalgo M et al. 2010).

Adjuvant chemotherapy, radiotherapy and interventional therapy are common

treatments for PC patients. Although the above treatments could extend the survival

time of patients to some extent, the overall efficacy is very limited. Gemcitabine (2',

2'-difluorodeoxycytidine, dFdC) was approved by the US Food and Drug

Administration (FDA) as the first-line chemotherapeutic agent for the treatment of

PC (Yang ZY et al. 2013). A large number of clinical trials have shown that

gemcitabine could effectively alleviate the symptoms of PC patients (Corbo V et al.

2012). Therefore, single gemcitabine treatment is still the standard treatment for

late stage of PC so far (Matsuoka T et al. 2016). However, during the past decade,

innumerable clinical trials were performed to investigate the curative effects of

gemcitabine. Unfortunately, gemcitabine chemotherapy could not significantly

prolong the patient's survival time and serious side effects were reported as well

(Oettle H et al. 2013). Thus, the efficacy of gemcitabine in treating PC is far from

satisfactory (De Sousa Cavalcante L et al. 2014). The major reason for the high

mortality of PC is the high chemoresistance after chemotherapy (Garrido-Laguna I

3

et al. 2015). Thus, in order to provide an effective therapy for PC patients, it is

necessary to develop novel drugs aiming at chemoresistant-cancer cells.

1.2 The causes of chemotherapy tolerance in pancreatic cancer and the

related molecular mechanisms

At present, cancer is still a major threat to human health. Chemotherapy plays an

irreplaceable role in the treatment of malignant tumors (Sato T et al. 2012, Duong

HQ et al. 2012). However, with the widespread use of chemotherapeutic drugs,

intrinsic or acquired multidrug resistance (MDR) has become an important cause

for the failure of treatment in malignant tumors (Chen Y et al. 2012, Hung SW et al.

2012).

Tumor multidrug resistance (MDR) is a complex biological process involving

multiple genes and signaling pathways (Kang M et al. 2013). An important feature

of PC is the high tolerance to traditional chemotherapy and radiation therapy,

including the intrinsic (primary) and acquired (treatment-induced) chemical

resistance behavior of cancer cells. In the past 30 years, despite the improvement in

the diagnosis method and prognosis, PC is still a great challenge (Hasegawa S et al.

2014). Drug resistance is a key factor that limits the choice of therapy and

significantly reduces the efficacy of chemotherapy drugs in PC. Thus,

understanding the mechanism of chemotherapy tolerance in PC will help develop

specific strategies to overcome drug resistance and improve the efficacy of

4

treatment (Fiorini C et al. 2015).

The mechanism of drug resistance in PC is so complex that many genes and

signaling pathways are involved in the process (Skrypek N et al. 2015, Asuthkar S

et al. 2013). In recent years, domestic and foreign studies have shown that the

resistance of PC to chemotherapy is associated with multiple factors, including

oncogenes, inflammatory signaling pathways, gemcitabine metabolic enzymes,

cytokines, drug transport proteins and multidrug resistance genes (Nath S et al.

2013, O'Shea LK et al. 2014, Song WF et al. 2013). Numerous studies

demonstrsted that the resistance of PC is related to abnormal gene expression,

critical signaling pathways (NF-κB, Akt and apoptotic pathways), stromal cells,

highly resistant cells and the presence of stem cells (Queiroz KC et al. 2014 ).

1.2.1 P-glycoprotein and MDR in pancreatic cancer

Tumor MDR is induced by multiple mechanisms, one of the most representative

mechanisms is MDR mediated by ATP-binding Cassette (ABC) transporter.

(Lopes-Rodrigues V et al. 2017, Seebacher NA et al. 2016). It has been reported

that ABC transporters are responsible for drug outflow and MDR in different

kinds of cancer cells (Zhang R et al. 2016, Sivak L et al. 2017). The ABC

transporter is thought to act as a hydrophobic vacuum cleaner by discharging

nonpolar compounds from the plasma membrane using the energy released from

ATP hydrolysis (Schwarz T et al. 2016, Kohan HG et al. 2015). At present, the

5

most widely investigated ATP binding (ABC) transporter is P-glycoprotein (P-gp)

transporter.

P-gp is the first ATP-dependent transmembrane transporter that was found to be

associated with MDR. P-gp is encoded by human MDR1 gene, synthesized in the

endoplasmic reticulum, folded, and then transported to the Golgi body for

processing, modification and ultimately located in the cell membrane (Kiuchi S, et

al. 2015). Only the P-gp located in the cell membrane is demonstrated to be

responsible for induction of MDR (Harpstrite SE et al. 2014). Numerous studies

showed that P-gp is able to transport a range of compounds. The drug binds to the

specific site of P-gp embedded in the lipid layer of the cell membrane and is

pumped out of the cell by the energy released by ATP hydrolysis (Chen X et al.

2013).

The expression of P-gp is also found in some normal tissues (intestine, bile duct),

which can prevent the body to absorb harmful substances and promote the effluent

efflux, so as to protect the brain, testis, fetus and bone marrow and other important

tissues from poison damage (Schwarz T et al. 2016). However, P-gp is mainly

located on the cytoplasmic membrane of tumor cells, which can pump a series of

different substrate (including anti-cancer drugs such as paclitaxel, doxorubicin,

vincristine and so on) out of the cells using the energy released by ATP hydrolysis

(Kohan HG et al. 2015).

P-gp has been reported to overexpressed in all type of human tumor cells and the

expression level is negatively correlated with the differentiation of tumor cells.

6

However, tumor cells that are insensitive to chemotherapy often have a higher

level of P-gp expression (Lopes-Rodrigues V et al. 2017). Numerous studies

showed that the expression level of P-gp in clinical samples is positively

correlated the extent of clinical drug resistance, particularly in hematological

malignancies and solid tumors (Seebacher NA et al. 2016).

P-gp has been shown to directly or indirectly participate in cell metabolism,

proliferation, differentiation and other aspects of regulation, revealing P-gp has a

potential multiple physiological functions (Harpstrite SE et al. 2014).

Overexpression of P-gp specifically inhibits caspase-dependent pathway and

delays the apoptotic cascade, which then protects drug-resistant tumor cells from

apoptosis induced by cytotoxic drugs, fas ligand (FasL), tumor necrosis factor-α

( TNF-α) and radiation(Schwarz T et al. 2016).

P-gp is able to transport the antitumor drugs out of cells using the energy released

by ATP hydrolyzation, resulting in tumor resistance. P-gp on the cytoplasmic

membrane also redistributes intracellular drugs and accumulates drugs into

unrelated organelles such as lysosomes to reduce the drug concentration at the

target site. In addition, recent studies have shown that P-gp expression is

associated with many signaling pathways including MAPK, Wnt/β-catenin,

Protein kinase C (PKC) and NF-κB (Kohan HG et al. 2015).

The inhibition of ABC transporters can serve as a strategy for targeted therapy of

chemoresistant PC because of the pivotal role of P-gp in MDR. Currently, the

reversal strategy targeting P-gp is mainly achieved by inhibiting the activity or

7

expression of P-gp.

1.2.2 NF-kB pathway and MDR in pancreatic cancer

Besides the abnormal gene expression, MDR is also associated with activation and

inhibition of transcription factors and critical signaling pathways. It has been

reported that many signaling pathways are related to MDR in PC, such as NF-κB,

Akt and Notch pathways (Galvani E et al. 2015). Nuclear factor-κB (NF-κB) is a

kind of multifunctional present in eukaryotic cells, which was widely involved in

the physiological processes such as inflammatory reaction, immune stress, cell

proliferation, apoptosis and growth regulation (Nadiminty N et al. 2013).

NF-κB is a heterodimer consisting of p50 and p65 subunits. Under normal

conditions, it is inhibited by IκB in the cytoplasm and remains inactive state. When

activated by oxidants, inflammatory cytokines, NF-κB activation leads to the

phosphorylation of IκB and the degradation of the protease pathway (Huang H et al.

2012, Davoudi Z et al. 2014). Then, the activated NF-κB translocates into the

nucleus and binds to the promoter region of the target gene to induce expression of

the target gene. The activated NF-κB signaling pathway can also protect the cells

from apoptosis by regulating their downstream target genes such as Bcl-2, X-linked

apoptotic proteins (XIAP) and surviving (Xin Y et al. 2013).

NF-κB can be involved in the pathogenesis of cancer through the following

pathways including inhibiting apoptosis, promoting the tumor angiogenesis,

8

promoting cell proliferation and migration (Dermawan JK et al. 2014). In

conclusion, NF-κB is an essential condition for the process of tumor occurrence

and development. The chemotherapy tolerance in pancreatic cancer is also

associated with constitutive activation of NF-κB, which could subsequently result

in cell proliferation, invasion, angiogenesis and MDR of PC. (Raninga PV et al.

2016). It has been reported that tumor cells growth and metastasis could be

significantly suppressed by antagonizing NF-κB activation (Oiso S et al. 2012).

Arlt et al. found that NF-κB pathway is closely related to gemcitabine tolerance in

pancreatic cancer and the activated NF-κB pathway enhances the tolerance of PC

cells to gemcitabine. NF-κB pathway inhibitors can enhance the sensitivity of PC

cells to chemotherapy and can reverse the acquired resistance to gemcitabine (Lo J

et al. 2015, Davoudi Z et al. 2014).

Recent studies have shown that 3,3-dindolylmethane (DIM), a natural compounds

derived from cruciferous vegetables, kills PC cells by down-regulating NF-κB and

its downstream target genes (e.g., survivin, Bcl-xL, XIAP and cIAP). Further

analysis revealed that DIM treatment resulted in loss of phosphorylated p65,

down-regulation of NF-κB activity and its downstream genes in these tumors.

These results also indicated that DIM can eliminate the activation of NF-κB

induced by chemotherapeutic agents (cisplatin, gemcitabine and / or oxaliplatin) in

animal models, leading to reversal of chemotherapy tolerance in pancreatic cancer

(Sakuma Y et al. 2014, Galvani E et al. 2015).

Thus, NF-κB-specific inhibitors and other molecular therapies targeting the NF-κB

9

pathway can provide an effective treatment for pancreatic cancer. Many chemical

prophylactic agents, such as beta-lappa ketone, phenylephrine and sulindac are

reported to inhibit NF-κB activation and subsequently affect the expression levels

of genes regulated by NF-κB (Raninga PV et al. 2016, Sui H et al. 2016). In

addition to inhibition of NF-κB by natural compounds, the use of NF-κB -specific

siRNA can also improve the sensitivity of PC cells to chemotherapy. Pan et al.

inhibited the expression of p65 / relA by a siRNA and the result revealed that the

inhibition of NF-κB induced cell apoptosis and further increased the sensitivity of

PC cells to gemcitabine (Huang H et al. 2012, Nadiminty N et al. 2013).

Based on the above findings, NF-κB might be taken as a potential target for PC

treatment. These studies showed that inhibition of NF-κB pathway can reduce drug

resistance of PC. Targeting a single molecule or pathway may not be sufficient to

overcome drug resistance in PC. However, treatments targeting multiple pathways

may have a better effect in the treatment of PC.

1.2.3 HSP90 and MDR in pancreatic cancer

Heat shock proteins (HSP) are a family of proteins that are produced by cells in

response to prevent protein aggregation after environmental stress (Yan C et al.

2013). Numerous studies have shown that the disorders of heat shock response

leads to overexpression of HSP. A group of HSPs has recently been reported to

related with tumor MDR (Shapiro RS et al. 2012, Ahn JY et al. 2015).

10

HSP90 is a group of highly conserved, constitutive proteins. HSP90, as an

important molecular chaperone, assists in protein folding, assembly, maturation

and maintaining the stability of a variety of signaling proteins relevant to cell

growth and survival (Acquaviva J et al. 2014). Hsp90 forms complexes with other

molecules such as Hsp70, Hsp40, Hop, p23 and cdc37 (Koizumi H et al. 2012).

Then, the complexes binds to many of the "client proteins" in the cell, facilitating

the proper folding and assembly of these proteins to achieve their active

conformation and enhance their stability (Ma W et al. 2013). Recently, over 200

client proteins of Hsp90 have been identified, including tyrosine kinase (AKT,

MEK, etc.), transcription factors (AR, ER and p53, etc.) and structural proteins

(microtubules and microfilaments) (Zhang Z et al. 2015). Most of them are

oncoproteins and play a crucial role in regulating the signal transduction pathways

that were involved in the regulation of tumor proliferation, tissue invasion,

metastasis, angiogenesis and other landmark event in tumor occurrence (Robbins

N et al. 2012, Centenera MM et al. 2015). Hsp90 is mainly in a silent state in the

normal cells (Liu X 2016). However it was activated in the tumor cells and the

activated Hsp90 forms a complex with client proteins or secondary molecular

chaperones to protect client proteins from proteasome degradation, which further

promotes tumor development and even produces multidrug resistance (Liang W et

al. 2015).

HSP90 was consistently overexpressed in pancreatic cancer cells and involved in

the regulation of tumor proliferation, invasion, metastasis and angiogenesis by

11

regulating a variety of oncogene proteins (Gillis JL et al. 2013). Wang et al.

reported that the serum levels Hsp90 in PC patient was remarkably higher than

healthy people and individuals with chronic pancreatitis, which confirm the

findings of previous study (Wang M et al. 2016). The mRNA level of HSP90 in

human PC tissue was significantly increased compared with normal tissue from

the same patient. Moreover, the expression level of HSP90 in plasma was found to

be positively correlated with the tumor growth rate. (Ciocca DR et al. 2013).

It was confirmed that the overexpressed Hsp90 are closely related to the

development of chemotherapeutic tolerance and appear to protect tumor against

chemotherapy (Centenera MM et al. 2015). After tumor metastasis, the expression

level of Hsp90 in metastases site is significantly higher than that of primary tumor

tissue, suggesting that Hsp90 promotes tumor metastasis, supports tumor cells to

adhesion or adapt to the adverse environment (Fogliatto G et al. 2013). Recent

reports have also reported that heat shock proteins are crucial in the regulation of

tumor stem cell functions and are relevant to self-renewal, infection and

metastasis of cancer stem cells. (Wang YQ et al. 2016).

Since Hsp90 simultaneously regulates a number of tumor signaling pathways, it is

viable to realize the inactivation and degradation of many cancer proteins at the

same time by blocking the molecular chaperone function of Hsp90, which would

effectively avoid and overcome the drug resistance (Ahn JY et al. 2015). Therefore,

the development of HSP90 inhibitor has become a research hot spot of antitumor

drugs. Studies have shown that inhibition of Hsp90 function leads to the

12

instability of the client protein and degradation by proteasome, which will cut off

the dependence of tumor on the branch signal, showing significant anti-tumor

effect (Acquaviva J et al. 2014). Further studies have shown that some of the

chemoresistant cells are still sensitive to Hsp90 inhibitors, suggesting that Hsp90

inhibitors can serve as a treatment strategy for drug resistance (Ciocca DR et al.

2013). The feasibility of targeted therapy aiming at heat shock proteins in the

treatment of cancer has been supported by numerous experimental data.

Therefore, due to the important role of HSP90 in the chemoresistance of PC,

inhibition of HSP90 can be used as a powerful strategy to overcome the

chemotherapy tolerance of PC.

1.2.4 Tumor angiogenesis and MDR in pancreatic cancer

Tumor chemotherapy tolerance and angiogenesis are the critical stages of tumor

development. Angiogenesis is essential for the occurrence and development of the

tumors (Sebens S et al. 2012).

Angiogenesis factors are overexpressed in tumor cells at both transcriptional level

or at the protein level. The abnormal gene overexpression in tumor cells leads to the

increased concentration of angiogenesis factors in tumor tissue, which will

stimulate angiogenesis, accelerate tumor cell growth and further lead to tumor

invasion and metastasis (Mirandola L et al. 2014, Hamdollah Zadeh MA et al.

2015). Therefore, the plasma level of angiogenic factors has not only become an

13

important factor in tumor prognosis, but also as a target for tumor therapy.

The current study suggests that tumor angiogenesis is a result of complex

interactions between multiple factors including cancer cells and endothelial cells.

Vascular endothelial growth factor (VEGF) is a major promoter of tumor

angiogenesis and is overexpressed in a variety of malignant tumors (Takeuchi S et

al. 2012, Rosas C et al. 2014). VEGF binds to the vascular endothelial growth

factor receptor (VEGFR), which causes tumor cell proliferation, migration and

induction of angiogenesis. At present, "VEGF-VEGFR" axis is the main target of

anti-angiogenesis therapy. As one of the most important angiogenic factors, VEGF

can promote the proliferation and invasion of cancer cells, and further result in the

drug resistance of cancer cells to affect the biological behavior of the tumor (Han

ES et al. 2013, Kinugasa Y et al. 2014). Thus, inhibition of VEGF and its receptors

has become the focus of anti-angiogenic therapy for tumors.

It has been reported that the growth of most tumors could be inhibited by blocking

tumor VEGF signaling pathway. However, the signal pathway can only be

temporarily suppressed, once the drug removed, the signal pathway will be

restarted. No matter chemical or biotechnology drugs, in the intermittent period of

treatment, tumor tissue can quickly establish blood supply and the growth rate is

much faster (Hartwich J et al. 2013).

Although some anti-angiogenic target drugs have been successfully on the market

or under development, the clinical efficacy of these drugs is poor and some still

have resistance problems. The reason might be attributed to the various regulatory

14

pathways of tumor angiogenesis (Schneider K et al. 2015). Studies have shown that

a single angiogenesis inhibitor cannot inhibit all angiogenic factors and their

downstream signaling pathways. Blocking the VEGF signaling pathway may lead

to the increase of other angiogenesis factors or enhancement of other related signal

pathways, resulting in acquired resistance (Dačević M et al. 2013, Smith BD et al.

2015).

Although it has been reported that a variety of cells and cytokines within the tumor

microenvironment was involved in anti-angiogenic resistance through different

pathways, the mechanism of drug resistance is not very clear (Nakazawa Y et al.

2015). In order to reduce the occurrence of drug resistance, we need to further

explore the changes of tumor microenvironment after anti-angiogenesis therapy to

find new targets for anti-angiogenic therapy and improve the clinical effect of

anti-angiogenesis therapy (Hida K et al. 2013).

Angiogenesis is a characteristic manifestation of PC development，which was also

a prerequisite and basis for early metastasis. It has been reported that VEGF was

overexpressed in PC, leading to hypoxia in PC tissue due to lack of angiogenesis,

and the presence of stromal cells leads to further resistance to both chemotherapy

and radiotherapy (Karashima T et al. 2103, Sharma B et al. 2013). Therefore,

anti-hypoxia and inhibition of stromal cells should also be considered as the

potential strategies to inhibit tumor angiogenesis in PC.

In summary, the mechanisms described above were involved in PC MDR in

different ways and exhibit potential targets for PC treatment. Some researchers use

15

them as a target to overcome the tolerance of PC to chemotherapy and the efficacy

were approved by animal experiments. Novel treatments are currently being

developed to overcome drug resistance in PC, including suppression of abnormal

gene expression, regulation of the signal transduction pathway, tumor

microenvironment and cancer stem cells (Huang Y et al. 2015). However, the

unique characteristic of PC is the dense connective tissue matrices, which

significantly affects the penetration and delivery of chemotherapeutic drugs,

leading to the insensitivity of PC patients to drugs in clinical trial (Rebbaa A et al.

2013). Therefore, in addition to development of therapeutic drugs with multiple

targets and mechanisms, how to improve the efficiency of drug delivery and reduce

drug resistance is the urgent task for PC treatment.

1.3 Celastrol as a potential drug for treatment of pancreatic cancer and the

impediment to its clinical application

Celastrol (CSL) is a quinone methyl triterpenoid monomer extracted from

Tripterygium wilfordii Hook.F., the structure was shown in Figure 1. Celastrol was

identified as the important active ingredient and main toxic ingredient in

Tripterygium wilfordii (Kapoor S et al. 2014). Celastrol has a variety of

pharmacological activity due to its specific chemical structure, such as

anti-inflammatory, immunosuppressive activity, antitumor activity, and

anti-neurodegenerative diseases (Chakravarthy R et al. 2013).

16

Figure 1 The chemical structure of Celastrol

Celastrol demonstrated strong antiproliferation activity on different kinds of cancer

cells, including human mononuclear leukocyte cancer cells, human prostate cancer

cells, melanoma cells, PC cells and the human lung cancer cells, suggesting the

broad-spectrum antitumor activity of celastrol (Lin L et al. 2016).

It is reported that celastrol can suppress tumor growth by multiple mechanisms.

Celastrol can inhibit the expression of HSP90 in tumor cells and reflect synergistic

effect with the traditional HSP90 inhibitor (Shao L et al. 2013). Celastrol can

reduce the expression level of P-gp to reverse the resistance of tumor cells to

chemotherapy (Lee JH et al. 2012). Moreover, celastrol has inhibitory effect on

NF-κB signaling pathway. Celastrol can inhibit the activation of NF-κB system by

inhibiting the phosphorylation and degradation of IKKβ kinase substrate IκBα.

Celastrol can suppress tumor growth by inhibiting tumor angiogenesis (Boridy S et

al. 2014). Studies showed that celastrol can inhibit the expression of VEGF by

inhibiting tumor necrosis factor alpha (TNF-α), suggesting that celastrol has the

ability to antagonize tumor metastasis. Celastrol can significantly suppress the cell

17

growth, migration and invasion of HUVEC induced by VEGF (Zhao X et al. 2014).

In addition, celastrol can also effectively inhibit tumor invasion and metastasis.

Kim et al. found that celastrol suppress the migration and invasion of breast cancer

cells by inhibiting NF-κB-mediated Matrix Metalloprotein 9 (MMP-9) expression

(Zanphorlin LM et al. 2014). Yadav VR found that celastrol inhibits the metastasis

of PC cells by downregulating the expression level of CXCR4 chemokine receptors

(Sharma S et al. 2015).

Celastrol has also expressed its excellent antitumor activity in a variety of tumor

animal models, including human prostate cancer animal model, human breast

cancer animal model, human pancreatic cancer animal model and human glioma

animal model (Yu X et al. 2015). In the preclinical study, celastrol was found to

induce melanoma cell apoptosis and inhibit the growth and metastasis of melanoma

(Tang WJ et al. 2015).

In summary, due to the strong antitumor activity and multiple antitumor

mechanisms, celastrol might be used as an alternative drug for the treatment of

cancer (Li HY et al. 2014). It is noteworthy that a large number of studies have

shown that celastrol could effectively suppress PC growth (Wang YL et al. 2015).

More importantly, celastrol could counteract the above chemoresistance-related

molecular mechanisms of PC, including decreasing the expression of

P-glycoprotein, suppressing the activity of HSP90 and inactivating the NF-κB

signaling, etc (Pazhang Y et al. 2016). Therefore, celastrol might be developed as a

potential drug for PC by aiming at chemoresistance. However, the poor water

18

solubility and toxicity to healthy tissues (e.g. liver) caused by non-specific

exposure are the major impediments to the clinical application of celastrol. Thus, it

is desirable to seek a strategy to facilitate CSL selectively targeting PC tissues or

cells and simultaneously reducing its exposure to healthy tissues (e.g. liver).

1.4 The application of aptamer in tumor targeted therapy

Aptamers are short, single-stranded DNA (ss DNA) or RNA oligonucleotides,

which can specifically recognize and bind to targets by distinct secondary and

tertiary structures (Lassalle HP et al. 2012). Aptamers can now be screened by an

in vitro selection process named SELEX, which could use different biomarkers as

target. To date, aptamers have attracted widespread attention and have been applied

as therapeutic agents or used as targeting ligand for targeted drug delivery

(Sundaram et al. P 2013).

Table 1 Antibody and aptamer comparision

Antibody Aptamer

Molecular weight High molecular weight Low molecular weight

Allergenicity Easily induces allergic reaction No immunogenicity

Selection process Complicated Simple

Synthesis and

modification Cannot be synthesized Easy to synthesize and modify

Storage condition Strict storage condition Good chemical stability

Manufacturing Complicated process, harsh

manufacturing conditions Relatively easy to industrialize

19

Aptamers possess several key advantages, including good chemical stability, low

molecular weight, low immunogenicity, stable physical and chemical properties,

and susceptibility to various chemical modifications (Hu M et al. 2013). Based on

these advantages, aptamers are likely to replace antibodies in many biological

applications (Table1). In 2005, the first aptamer drug Macugen from Pfizer and

Eyetech, was approved by the FDA, suggesting that the modification of small

molecule drugs with aptamers is a promising direction for the development of new

drugs (Scaggiante B et al. 2013). Recently, numerous aptamers have been

successfully obtained through SELEX, some of which have been proven valuable

in disease diagnosis and treatment (Table 2). A number of aptamers are under

pre-clinical or clinical trials and are gradually developing into a new drug class.

Table 2 Overseas Clinical Research of Aptamer Drugs

Name Nucleotide Target Condition Phase Company

ARC1905 RNA C5 Age-Related Macular

Degeneration Phase Ⅰ Ophthotech

NOX-E36 RNA CCL2 Type 2 Diabetes

Mellitus Phase Ⅰ

NOXXON

Pharma AG

AS1411 DNA Nucleolin Acute Myeloid

Leukemia Phase Ⅱ

Antisoma

Research

Nu172 DNA Thrombin Heart Disease Phase Ⅱ ARCA

Biopharma

NOX-A12 RNA CXCL12 Hematopoietic Stem

Cell Transplantation Phase Ⅰ

NOXXON

Pharma AG

ARC1779 DNA vWF Purpura, Thrombotic

Thrombocytopenic Phase Ⅱ Archemix

REG1

(RB006/RB007) RNA

Coagulation

Factor IX

Coronary Artery

Disease Phase Ⅱ

Regado

Biosciences

Pegaptanib RNA VEGF Macular Degeneration FDA

Approved Pfizer/Eyetech

E10030 DNA PDGF Age-Related Macular

Degeneration Phase Ⅱ Ophthotech

20

Several companies, including Achemix (USA), SomaLogic (USA) and Noxxon AG

(Germany) are exploring aptamer drugs and diagnostic reagents, with more than ten

aptamer drugs undergoing clinical trials in North America and Europe (Li X et al.

2013). In addition, the efficacy of Macugen has been proven in the clinical arena,

which has also demonstrated the advantages of aptamers in disease treatment

(Dassie JP et al. 2013).

In recent years, aptamer-drug conjugates have become a major focus in the research

of targeted therapies for tumors. Tumor cell targeted drug delivery is an effective

approach to simultaneously improve the efficacy of cancer treatment and reduce its

side effects (Reinemann C et al. 2014, Chen M et al. 2016). Aptamers are ideal

ligands that specifically bind to target cell surfaces. Thus, it is feasible to modify

drugs or nanoparticles with aptamers to provide a new direction for tumor-targeted

therapies (Zhu J et al. 2014, Dickey DD et al. 2015). Studies have shown that after

siRNA was linked to aptamers, the connected aptamers specifically transported

siRNA to targeted cells. In 2006, McNamara successfully realized the specific

delivery of siRNAs to cancer cells by using a PSMA-aptamers as a targeting

moiety. The author create a conjugate of a PSMA-specific aptamer and siRNAs to

target PLK1 and BCl2 and the efficacy was investigated in mouse model, it was

discovered that the expression levels of these oncogenes in PSMA-positive cells

and tumors were specifically down-regulated, however the PSMA-negative tissues

was not affected (Zhu H et al. 2015). In addition, aptamers can also be used for

targeted delivery of toxins, radioisotopes and chemotherapeutic drugs. Bagalkot

21

directly connected the PMSA aptamer (A10) to doxorubicin for cell-specific

delivery, which can induce apoptosis of cells expressing PSMA (Lao YH et al.

2015). Huang conjugated a DNA aptamer (sgc8c) with doxorubicin (Dox), the

conjugated sgc8c aptamer could facilitate Dox selectively targeting CEM cells and

subsequently result in the lower rate of cell proliferation. However, the

non-targeted cells were not affected, suggesting a high degree of selectivity

(Mathew A et al. 2015, Zhu G et al. 2015).

In conclusion, aptamers are excellent active targeting ligands for targeted delivery

of therapeutic agents in cancer therapy, which might realize the specific delivery of

celastrol in vivo.

1.5 Nucleolin aptamer as a moiety to target pancreatic cancer

Nucleolin is a protein that shuttle between cell surface, cytoplasm and nucleus. It

has been widely reported that expression level of nucleolin on the plasma

membrane of PC cells is remarkably higher than that of normal cells. So nucleolin

might be taken as an ideal cell surface receptor. Lan Peng’ group proved that

nucleolin was overexpressed in pancreatic ductal adenocarcinomas (PDAs) and

PDA cell lines ( Taghavi S V 2017).

Nucleolin-specific aptamer is an anti-proliferative G-rich phosphodiester

oligonucleotide that can bind to nucleolin with high affinity and specificity.

Preclinical distribution studies suggest that nucleolin aptamer could selectively

22

accumulate in the tumors of animal models (Wang C et al. 2016). It has been

successfully used for cell-specific delivery of various drugs or siRNAs in animal

studies. In addition, the safety of nucleolin aptamer has also been validated in

preclinical and clinical studies. The preclinical pharmacokinetics data concluded

that the major elimination route of nucleolin aptamer was renal and the β-phase

elimination half-life of nucleolin aptamer in plasma was 2 days as well as in whole

blood. Preclinical toxicology studies were performed to investigate the toxicity of

nucleolin aptamer and no obvious toxicity was observed (Dam DH et al. 2016).

The good safety of nucleolin aptamer was also confirmed by the phase I clinical

trials, which was inconsistent with the former animal study. The phase II clinical

trials continued proved that nucleolin-specific aptamer has minimal toxicity at the

dose achieved peak plasma concentration and be close to the IC50 concentration

identified for renal cancer cell lines in vitro (Zhu J et al. 2014, Dickey DD et al.

2015).

Then, nucleolin aptamer may facilitate celastrol selectively targeting PC cells after

it was linked to celastrol as a target ligand. Accordingly, it is necessary to link

nucleolin aptamer to celastrol.

1.6 Research Hypothesis

Based on the above information, we have the following hypothesis: After nucleolin

aptamer was linked to celastrol, the conjugated nucleolin aptamer may facilitate

celastrol selectively targeting pancreatic cancer cells to increase the drug

23

concentration within tumor tissue and improve the antitumor efficacy by

overcoming chemoresistance. Meanwhile, nucleolin aptamer may reduce the

distribution of celastrol to normal tissues to decrease the exposure to normal tissues

and reduce the side effect.

24

Chapter 2 Materials and Methods

2.1 Materials for chemical synthesis

Celastrol was purchased from Chengdu Biopurify Phytochemicals Ltd. Nucleolin

aptamer (sequence: 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-/3ammc7-r/ 3')

and CRO (sequence: 5'-TTTCCTCCTCCTCCTTCTCCTCCTCCTCC-/3ammc7-

r/-3') were synthesized by CHENGDU HitGen CO., LTD.

N,N-Diisopropylethylamine (DIPEA), N-Hydroxysulfosuccinimide sodium salt

(Sulfo-NHS), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-HCl (EDC) and

o-(7-Azabenzotriazol-1-yl) -N,N,N',N'-te-tramethyluronium hexafluoro-phosphate

(HATU) were purchased from Sigma-Aldrich (Shanghai, China). L-Alanine

methyl ester hydrochloride (L-Ala-OMe. HCl) was supplied by Chengdu

AstaTech Trading Co., Ltd. All reagents required for chemical synthesis,

separation and purification were reached analytical grade. Silica gel (200–300

mesh, purchased from Sigma) was used for separation and purification by column

chromatography. Thin layer chromatography (TLC) was performed using silica

gel precoated aluminum cards with fluorescent indicator visualizable at 254 nm

(Merck). The 1H NMR spectra was performed on Bruker AM-400 instruments

using CDCl3 as solvent and tetramethylsilane as an internal standard. LC-MS was

performed on a LC-MS-2020 Single Quadrupole Liquid Chromatograph Mass

Spectrometer. MS was performed on a Thermo LCQ Deca XP plus Mass

25

Spectrometer.

2.2 Preparative high performance liquid chromatography (Pre-HPLC)

analysis for purifying compounds

The residue was dissolved in methanol or acetonitrile and filtered through a

membrane filter into a glass vial, and then diluted with methanol or acetonitrile

(concentration of the residue about 60mg/mL). The solution of residue was

automatically injected, separated, purified and collected by Pre-HPLC, using

BondapakTM C18 column (80 mm* 500 mm) with a particle size of 10 μm. The

composition of the mobile phase is 60% methanol and 40% water. The flow rate

of the mobile phase is 60 mL/min and the temperature of column was 40°C.

Detection wavelength was 254 nm and injection volume is 1.5 mL. The

required fraction was collected, and the solvent was evaporated in vacuo to obtain

the pure product.

2.3 Nuclear magnetic resonance (NMR) analysis for indentifying compounds

1H NMR was used for the determination of the configuration of compounds. The

purified product was dissolved in chloroform and vigorous stirred. The

precipitated must be filtered and the filtrate was injected into NMR tube

(concentration of the compound ≥ 0.1mmol/mL). Tetramethylsilane (TMS) was

added as an internal standard. The solution was analyzed by a Bruker AM-400

26

spectrometer.

2.4 Reversed-phase high performance liquid chromatography (RP-HPLC)

analysis for indentifying compounds

The determination was carried on RP-HPLC, using C18 column (Xterra®), 0.1M

triethylammonium acetate (TEAA) solution as mobile phase A and acetonitrile as

mobile phase B. The acetonitrile concentration change per min (from an initial 5

to 10% acetonitrile concentration over 10 min, then from 10 to 60% acetonitrile

concentration over 5min) over 30 min was programmed as specified below:

Table 3 The experimental conditions of RP-HPLC

Time (min) %A %B Actual Acetonitrile (CAN) Concentration

0 95% 5% 5%

10 90% 10% 10%

15 40% 60% 60%

30 40% 60% 60%

The injection volume was 5 μL and the flow rate of was 1.0 mL/min. The

temperature of column was 40 °C. The detection wavelength was 254 nm.

2.5 Cell culture

PANC-1 cells purchased from ATCC were maintained in ATCC-formulated

27

DMEM Medium added with 10% FBS and 1% PS. Cells was incubated in 37℃

with 5% CO2 and 95% humidity. The medium was changed every other day. When

the cells covered 80% of the culture dish, cells were passaged at a dilution of 1:3.

2.6 Cell proliferation study

In order to investigate the antitumor activity of NACC on PANC-1 in vitro, the

CCK-8 assay was performed. According to the provided protocol, PANC-1 cells

were seeded in 96-well plates with approximately 5000 cells in each well and

incubated overnight for adherence. Then, cells were incubated with CSL,

CACC and NACC at different concentrations (from 25nM to 400nM) for 24h and

48 h, respectively. Cells in the control group were treated with solvent. At the end

of the incubation, 10 µL of CCK8 solution was added to each well. After an

additional incubation for 3h, the plates were read using the microplate reader

(Thermo Fisher) by a spectrometer at 492 nm. The viability data were analyzed

using Graph Pad Software.

2.7 Animal handing

BalB/C nude mice (6–8 weeks old, female, 18-20 g body weight) used for

pancreatic tumor xenograft models and BalB/C mice (6–8 weeks old, female, 18-20

g body weight) used for liver toxicity study were purchased from Hong Kong

university. All mice were kept in the animal center of the School of Chinese

28

Medicine with the controlled temperature (25℃) and the humidity (40–60%). Free

diet and water were provided. Mice were used for the experiment after 2 weeks of

adaptive feeding. All the experimental procedures and animal models in this study

were in accordance with the regulations of CAEES of HKBU.

2.8 Xenograft tumor model of pancreatic cancer

BalB/C nude mice (6–8 weeks old, female, 18-20 g body weight) were lightly

anesthetized by intraperitoneal injection of chloral hydrate (200 mg/kg). Then

mice were inoculated with 100 μl of human pancreatic cancer cell line PANC-1

(1.5×107/ml) suspended in normal saline on the right frank. The tumor volume was

examined regularly and when the tumors reached 100 mm3, the mice could be used

for experiments.

Tumor volume measurement: tumor size was measured using vernier calipers. The

length is the longest diameter and the width is the shortest diameter. The tumor

volumes (V) were calculated using the following formula: V = [length ×

(width)2]/2 .

2.9 Flow cytometry

Human pancreatic cancer cell line (PANC-1) and normal human liver cell line

(L-02) were seeded into a 12-well plate at density of 1×106 cells per well. Either

Cy3-labeled NACC or Cy3-labeled CACC were incubated with PANC-1 or L-02

29

for 1h under dark conditions. Then, after washing with PBS twice, cells were

digested with Trypsin-EDTA solution. Cell suspension was collected and

centrifuged (400 x g, 4 minutes). In order to remove the unbound conjugates in the

culture medium, cells were washed two times with PBS (Ca2+, Mg2+ free). After

centrifugation, cells were resuspended with the binding buffer. Then, the mean

fluorescence intensity of the incubated cells was determined by flow cytometry.

2.10 Cellular uptake pathways

To observe the pathways for the cellular uptake of NACC by pancreatic cancer cells,

PANC-1 cells were seeded onto the coverslips used for confocal microscopy. After

all the cells were adhered onto the coverslips, PANC-1 cells were incubated with

Alexa Fluor 488–labeled choleratoxin, Alexa Fluor 488–labeled dextran or Alexa

Fluor 488–labeled transferrin for 30min, respectively. After washing with DMEM

twice, the coverslips were incubated with Cy3-labeled NACC for 1h. Afterward,

the coverslips were washed with PBS twice to remove the unbound conjugate.

Coverslips were fixed and mounted onto microscope slides. Then cells were

washed by serum free medium twice and incubated with DAPI for 1min. The

co-localization of Cy3-labled NACC with Alexa Fluor 488 in PANC-1 cells was

analyzed by confocal microscopy.

30

2.11 In vivo injection of Microfil and Micro CT

Mice was heparinized (100 IU/Kg ip) and anesthetized by intraperitoneal injection

of chloral hydrate (400 mg/kg). Then, the mice were fixed onto the board with the

ventral facing up. The chest was opened and a scalp needle was inserted into the left

ventricular. The inferior vena cava was cut to ensure the systemic circulation of the

infused solutions. After perfusion, the mice were placed in ice for solidification of

the contrast medium (Microfil; Flow Tech). The tumors were removed and fixed in

10% formalin for 24 hours. Then the tumors were scanned with a desktop micro-CT

unit (viva CT40, SCANCOMEDICAL, Switzerland). Image processing, tissue

density measurements and quantification of tumor vascularization were performed

with the software.

2.12 Western blot analysis

Frozen tissue (present in liquid nitrogen) was cut into several small pieces. One

piece of tissue was weighed and smashed into smaller pieces in dry ice. Then

tumor tissue was added into lysis buffer and homogenized on ice. The total

protein was extracted by a standard extraction protocol. The protein content was

determined using the Lowry protein assay. SDS-PAGE was utilized for separation

of equal amounts of protein. The protein was transferred to nitrocellulose

membranes. The immunoblots were probed with primary antibodies and β-actin

antibody. After two washing steps, membranes were incubated with a secondary

anti-body. The immunocomplexes were detected using chemiluminescence (ECL)

31

system and quantified using Image Lab Software.

2.13 Tissues and cellular distribution

To validate the tissues and cellular distribution of NACC in vivo, xenograft tumor

model of pancreatic cancer was established as previously described. Once the

tumors reached 100 mm3, the mice were randomly divided into 2 group with 12 in

each group, and respectively subjected to Cy3-labeled CACC and Cy3-labeled

NACC at the same dose of 0.5 mg/kg of CSL via tail vein injection. Mice in each

group were sacrificed at 2h and 4 h after the administration. Tumors and major

organs (hearts, lungs, livers, spleens and kidneys) were collected for biophotonic

imaging. The fluorescence imaging was performed using IVIS® Lumina XR

IVIS® Lumina XR. Excitation (λex=530-560nm) and Emission (λem= 570-590

nm) filters were used. Then all the organs were homogenized and transferred into

microplates for quantitative study. The fluorescence strength was measured by

microplate reader.

To investigate the cellular distribution of NACC within tumor and liver in vivo,

portions of livers and tumors were respectively placed in a 4% paraformaldehyde

for overnight after harvesting. The liver samples and tumor samples were placed in

30% sucrose for overnight, which were then embedded in O.C.T. and sectioned at 7

µm thickness. After washing with PBS, the sections were permeabilized with 0.2%

Triton X-100. All the sections were incubated with DAPI for 5 minutes. The

images were acquired with confocal microscope.

32

2.14 Blood biochemical analysis

Blood samples were obtained at predetermined time and centrifuged at 3000 rpm

for 20 min. The serum was separated for the detection of ALT and AST by

AEROSET (Automatic biochemical analyzer).

2.15 Histological analysis

Liver tissues and tumor tissues were removed after the mice were sacrificed by

euthanasia. The tissue were fixed and preserved in 10% neutral buffered formalin

for more than 24 h. Then, the tissues were embedded using paraffin and sectioned at

5 µm thickness. All the sections were stained with hematoxylin–eosin. The

morphology change of liver tissues was observed using microscopy. Tumor

sections were examined for the presence of lymphocytic and neutrophilic

infiltration of the tumor tissue.

2.16 Statistical analysis

All the variables were expressed as mean ± standard deviation. Student's t-test or

one-way analyses of variance (ANOVA) were performed in statistical evaluation.

P<0.05 was considered to be statistically significant.

33

Chapter 3 The effect of modification on the carboxylic acid group on the

antitumor activity of celastrol

3.1 Aim

To investigate whether the modification on the carboxylic acid group would affect

the antitumor activity of celastrol.

3.2 Design

Literature review: In order to explore the progress of the modification of various

chemical groups of celastrol, we have carried out the following literature research.

Celastrol possesses several sites that can be modified. However, some of these sites

are not suitable for chemical synthesis or generating unstable product. Abbas

research group modified the quinonemethide functional group or carboxylic acid

group of celastrol to form amides or esters，respectively. It was found that the

antiapoptosis activity of celastrol was not influenced after the carboxylic acid

group was modified, but the antiproliferation activity was significantly reduced

after the quinonemethide moiety was modified. Previous studies also suggested

that quinonemethide moiety in celastrol does not work for its chemical chaperone

activity, but is critical to the antitumor activity.

Sun et al. designed and synthesized new derivate of celastrol by modification on

the carboxylic group. The results showed that two analogues had stronger antitumor

34

activity than celastrol, with EC50 values less than 0.1 and 0.3 µM against SW1 cells,

respectively. In addition, further study showed that the antitumor activity was

improved after the carboxylic functionality was replaced by amide. As noted above,

the carboxylic acid functionality of celastrol might be the most reasonable position

for modification.

In order to investigate the influence of modification in carboxylic acid group of

CSL, we designed and synthesized amide derivatives by the condensation reaction

at the carboxylic acid group to provide celastrol analogues (Scheme 1). Then, the

antitumor activity of these derivatives (Derivative 1a, Derivative 1b and Derivative

1c) was evaluated by cell proliferation study.

3.3 Results

The results showed that both CSL and the celastrol analogues (Derivative 1a,

Derivative 1b and Derivative 1c) inhibited the cell viability of PANC-1 cells in a

concentration-dependent manner after incubation for 24h. No significant difference

was observed between the antitumor activity of celastrol, Derivative 1a and

Derivative 1b, whereas Derivative 1c demonstrated higher antitumor activity than

celastrol. The above experimental results suggested that the C-28 carboxylic acid

group of celastrol might not be responsible for its biological activity. The

modification on the C-28 carboxylic acid group of celastrol would not affect the

antitumor activity and the results of this experiment are in consistent with the

35

results of the previous studies

Scheme 1 Synthesis and structures of celastrol analogues

Figure 2 Cytotoxicity of different concentrations of CSL (IC50=400nM),

Derivative 1a (IC50=380nM), Derivative 1b (IC50=420nM) and Derivative 1c

(IC50=280nM) on PANC-1 cell line after incubated for 24h. Error bars indicate

mean±standard deviation. n=3.

Cel

l via

bilit

y (%

)

36

Chapter 4 The synthesis and characterization of Nucleolin Aptamer-Celastrol

Conjugate (NACC)

4.1 Aim

To prepare the celastrol derivative, Nucleolin Aptamer-Celastrol Conjugate

(NACC) and CRO Aptamer-Celastrol Conjugate (CACC).

4.2 Synthesis of the celastrol derivative

4.2.1 Experimental design

Scheme 2 Synthetic route. Reagents and conditions. (a) L-Ala-OMe. HCl, DIPEA,

HATU, THF, r.t..

A mixture of celastrol (0.45 g, 1.0 mmol), L-Ala-OMe. HCl (0.14 g, 1.0 mmol),

HATU (0.42 g, 1.1 mmol) and DIPEA (380 μL, 2.3 mmol) in Tetrahydrofuran (THF)

(20 mL) were stirred at room temperature and TLC was used to monitor the

37

reaction. After the celastrol disappeared, the solvent was removed under reduced

pressure and the residue was purified by column chromatography (PE: EA=3:1 then

1:1) to afford the product as a pink foamed solid (0.44 g, 82%). The structure of the

product was confirmed by LC-MS and 1H NMR (Figure 3).

4.2.2 Results

Figure 3 Red solid, 80% yield. (a) 1H NMR (400 MHz, CDCl3):δ 0.61 (s, 3H),

1.03 (d, J=14.0 Hz, 1H), 1.11 (s, 3H), 1.13 (s, 3H),1.26 (s, 4H), 1.44 (s, 3H),

1.47-1.69 (m, 8H), 1.84-2.05 (m, 6H), 2.11-2.16 (m, 1H), 2.21 (s, 2H), 2.42-2.47

a

b

38

(m, 3H), 3.38 (q, J= 5.6 Hz, 5H), 3.67 (s, 3H), 6.33 (d, J=7.2 Hz, 1H), 6.46 (s, 1H),

6.52 (s, 1H), 7.01 (d, J= 6.8 Hz, 1H) (b) MS (ESI): [M+H]+: calculated 536.3,

found 536.3. Chemical Formula: C33H45NO5.

4.3 Synthesis process of Nucleolin Aptamer-Celastrol Conjugate (NACC).

4.3.1 Experimental design

Scheme 3 Synthetic route. Reagents and conditions. (a) Sulfo-NHS, EDC, 0.5 M

Na2CO3/NaHCO3 buffer, DMF, dd-H2O, 37°C.

Sulfo-NHS (13.0 mg, 60.0 μmol) dissolved in dd-H2O (600 μL) was added to the

solution of celastrol (22.5 mg, 50.0 μmol) and EDC (12.4 mg, 65.0 μmol) in 1 mL

N,N-Dimethylformamide (DMF). The mixture was stirred at room temperature for

2h. Then, the activated celastrol was incubated with amino-modified nucleolin

aptamer. Nucleolin aptamer (1.7 mg, 0.2 μmol) was dissolved in 5 mL of 0.5 M

Na2CO3/NaHCO3 buffer (0.4 mL, pH 8.4) and then added with 800 μL celatrol

N-Hydroxysulfosuccinimide ester reaction solution. After 2 hours of reaction, 800

39

μL of the active ester reaction solution was added (1.6 mL, 50 μmol in total). The

reaction solution was mixed at 37°C overnight, then added dd-H2O/ethanol (V:

V=1:3, 2.4 mL in total) and it was centrifugated at 2°C for 30 minutes. Afterward,

RP-HPLC was utilized for residue purification. The desired fraction was collected

and lyophilized to obtain Nucleolin Aptamer-Celastrol Conjugate (NACC). The

product was characterized by Thermo LCQ Deca XP plus Mass Spectrometer

(Figure 4b).

4.3.2 Results

a

40

Figure 4. Characterization of NACC. (a) The HPLC spectrum of the NACC. (b)

MS (ESI): [M+H]+: calculated 8916.0, found 8917.2.

4.4 Synthesis process of the CRO Aptamer-Celastrol Conjugate (CACC)

4.4.1 Experimental design

CRO is a C-rich oligo aptamer with random sequence, which has no specificity and

served as control to nucleolin aptamer. Using the same method in Part 4.3.1, CRO

Aptamer-Celastrol Conjugate (CACC) was obtained as white spumous solid. The

product was characterized by Thermo LCQ Deca XP plus Mass Spectrometer

(Figure 5b).

b

41

4.4.2 Results

Figure 5 Characterization of CACC. (a) The HPLC spectrum of the CACC. (b)

MS (ESI): [M+H]+: calculated 9149.5, found 9147.7.

a

b

42

4.5 The water solubility and the stability of the NACC in vitro

4.5.1 Experimental design

To compare the water solubility of celastrol and NACC, 2µmol of Celastrol or

NACC was dissolved in 1ml of water respectively. Meanwhile, 2µmol of Celastrol

was dissolved in 1ml of 1% DMSO and served as a control. Then, the appearance of

the solutions was examined by visual observation.

To test the stability of the NACC in mouse serum, NACC solution was added to

mouse serum or phosphate buffer solution (PBS) and incubated for 24h. Then, free

celastrol was determined by HPLC after the samples were incubated at 37 for ℃

indicated times (1h, 4h, 8h, 12h and 24h).

4.5.2 Results

The reported solubility of celastrol in water is no more than 0.014 mg/mL

(approximately 30 µM), while the solubility of NACC in water is at least greater

than 17.8 mg/mL (2 mM). The results indicated that the water solubility of NACC

was significant improved compared to that of free celastrol (Figure 6a).

The results showed that only a small amount of free celastrol was found in plasma

throughout the incubation period until 24 hours. No significant difference was

found between the NACC group and the PBS group at each time point. The result

indicated that NACC could be stable within the mouse plasma at least for 24h

(Figure 6b).

43

0 5 10 15 20 250

10

20

30

40

50

Time (hour)

The

Pre

cent

of F

ree

Cel

astr

ol (%

)

PBS

Mouse plasma

Figure 6 (a) The water solubility of free celastrol and NACC. The scale bar

indicated is 3 mm. (b) In vitro stability curve of NACC in mouse plasma and PBS.

The result indicated that NACC could be stable within the mouse plasma as well as

in PBS (mean ± SD, n = 3).

2mM CSL 2mM CSL 2mM NACC Water in water in 1% DMSO in water

a

b

44

Chapter 5 The effect of the nucleolin aptamer modification on the cytotoxicity

of the conjugated CSL in NACC in vitro

5.1 Aim

To investigate the effect of celastrol, CACC and NACC on proliferation of PANC-1

cell line in vitro.

5.2 Experimental design

In order to investigate the antitumor activity of Celastrol, CACC and NACC on

PANC-1 in vitro, the CCK-8 assay was performed. Cells were incubated with

Celastrol, CACC and NACC at different concentrations (from 25nM to 400nM)

for 24 h and 48h, respectively. Cells in the control group were treated with solvent.

CCK-8 kit was used for cell viability determination according to the provided

protocol. The data were analyzed using Graph Pad Software.

5.3 Results

The results showed that, after incubation for 24h and 48h, the cell viability

decreased with the increased concentration of Celastrol, CACC or NACC.

Furthermore, the cell viability in the NACC incubation group was always lower

45

than that in Celastrol and CACC incubation group at the same concentration (from

25nM to 400nM) (Figure 7). The IC50 value of NACC on PANC-1 cells were

200nM (24h) and 70nM (48h), which were significantly lower than 450nM (24h)

and 200nM (48h) of Celastrol. It indicated higher antitumor activity of the NACC

on pancreatic cancer cell line than that of Celastrol in vitro. More importantly, the

result also revealed that the nucleolin aptamer modification on carboxylic acid

group of Celastrol improved the anti-tumor activity.

Figure 7 Cytotoxicity of different concentrations of Celastrol (CSL), CACC or

NACC on PANC-1 cell line after incubated for 24h (left) and 48h (right). The

concentration range of each compound was from 25 to 400 nM. Error bars indicate

mean±standard deviation. n=3.

Cel

l via

bilit

y (%

)

Cel

l via

bilit

y (%

)

46

Chapter 6 The effect of the nucleolin aptamer modification on the cellular

uptake and the cellular internalization of NACC in vitro

6.1 Aim

6.1.1 To investigate the selectivity of NACC to pancreatic cells and the cellular

uptake in vitro

6.1.2 To investigate the mechanisms of cellular uptake of NACC by pancreatic

cancer cells in vitro.

6.2 Experimental design

To examine the selectivity of NACC to pancreatic cancer cells and normal liver

cells, either Cy3-labeled NACC or Cy3-labeled CACC were incubated with human

pancreatic cancer cell line (PANC-1) and normal human liver cell line

(L-02), respectively. Then, the mean fluorescence intensity of the incubated cells

was measured by flow cytometry.

To examine the cellular uptake of CSL and NACC in pancreatic cancer cells, either

the Cy3-labeled CSL, Cy3-labeled NACC or Cy3-labeled CACC was incubated

with human pancreatic cancer cell line PANC-1. The fluorescence intensity was

observed by confocal microscopy.

To observe the pathways for the cellular uptake of NACC by pancreatic cancer

47

cell, PANC-1 cells were incubated with Alexa Fluor 488–labeled endocytosis

markers (transferrin, choleratoxin and dextran; green) for 30min, respectively.

Then, cells were washed with DMEM and fixed. The nucleus was counterstained

with DAPI. Coverslips were mounted onto microscope slides and the

co-localization of Cy3-labled NACC with Alexa Fluor 488-labled in PANC-1

cells were analyzed by confocal microscopy. A Pearson correlation coefficient

(PCC) was utilized to quantify the degree of colocalization between the

fluorescence of Cy3-labled NACC and the Alexa Fluor 488-labled endocytosis

markers. To further investigate the mechanisms of cellular uptake of NACC by

pancreatic cancer cell, PANC-1 was incubated with inhibitors of clathrin-mediated

endocytosis (dynasore), caveolae-mediated endocytosis (filipin) or

macropinocytosis (EIPA) for 30min, respectively. Afterward, cells were incubated

with Cy3-labeld NACC at 37°C for 1h and fixed with 1% formalin. Then the

microscope slides were covered with coverslips. The image were analysed by

confocal microscopy and relative fluorescence intensity of Cy3-labeld NACC was

quantified.

6.3 Results

The result showed that the fluorescence intensity in PANC-1 incubated with Cy3-

labeled NACC was remarkably higher than that in normal liver cells, whereas no

differences were found in the fluorescence intensity among the two cell types

48

incubated with CACC (Figure 8-1a). The above results revealed that the binding

ability of NACC to pancreatic cancer cells was higher than that to normal liver cells

in vitro.

In the cellular uptake study, cells treated with Cy3-labeled NACC showed higher

mean fluorescent intensity compared to cells treated with Cy3-labeled CSL and

Cy3-labeled CACC. However, no significant difference was observed between the

mean fluorescent intensity in cells treated with Cy3-labeled CSL and Cy3-labeled

CACC (Figure 8-1b). Taken together, the above two studies indicated that the

conjugated nucleolin aptamer could facilitate NACC selectively targeting PANC-1

cells in vitro.

A remarkable co-localization of Cy3-labeld NACC with Dextran (a marker for

macropinocytosis) in PANC-1 cells was observed. In contrast, a significant lower

co-localization of Cy3-labeld NACC with transferrin (a marker for

clathrin-mediated endocytosis) or choleratoxin (a marker for caveolae-mediated

endocytosis) were detected (Figure 8-2a), indicating that Cy3-labeld NACC was

mainly taken up by PANC-1 cells via macropinocytosis..

After treatment with inhibitors of clathrin-mediated endocytosis (dynasore),

caveolae-mediated endocytosis (filipin) or macropinocytosis (EIPA), the cellular

uptake of Cy3-labeld NACC in EIPA treated group was significantly reduced

compared with the untreated group. However, the cellular uptake of Cy3-labeld

NACC in dynasore and filipin-treated group was not substantially affected. The

result also confirm the findings from the confocal imaging (Figure 8-2b).

49

Figure 8-1 (a) The binding ability of Cy3-labeled NACC (left) or Cy3-labeled

CACC (right) to tumor cells and normal liver cells determined by flow cytometry.

(b) The cellular uptake of Cy3-labeled CSl, Cy3-labeled CACC and Cy3-labeled

NACC in PANC-1.

Light Cy3 DAPI Merge

Cy3

-labe

led

CSl

Cy3

-labe

led

CAC

C

Cy3

-labe

led

NAC

C

a

b

50

Alexa Fluor 488 transferrin

Alexa Fluor 488 choleratoxin

Alexa Fluor 488 dextran

Cy3-NACC

Cy3-NACC

Cy3-NACC

DAPI

DAPI

DAPI

Mac

ropi

nocy

tosi

s

Cav

eola

e P

athw

ay

Cla

thrin

Pat

hway

0.0

0.2

0.4

0.6

0.8

1.0

Pear

son’

s co

rrel

atio

n c

oeffi

cien

t

clathrin-mediated endocytosiscaveolae-mediated endocytosismacropinocytosis

**

DMSO 40 80 1200

20

40

60

80

100

120

Fluo

resc

ence

inte

nsity

norm

aliz

ed to

con

trol

(%)

dynasore (um)

NS

DMSO 40 80 1200

20

40

60

80

100

120

Fluo

resc

ence

inte

nsity

norm

aliz

ed to

con

trol

(%)

filipin (um)

NSe

c

d

a

b

51

Figure 8-2 (a) Representative images showing the co-localization of Cy3-labeled

NACC (red) and Alexa Fluor 488–labeled endocytosis markers (transferrin,

choleratoxin and dextran; green) in PANC-1 by confocal microscopy. The nucleus

was counterstained with DAPI (blue). Scale bars, 10 μm. (b) Pearson’s correlation

coefficient analysis of the colocalization between Cy3-labeled NACC and

endocytosis markers in PANC-1 cells by Image J Coloc2. Error bars indicate

mean±standard deviation. n= 6 per group. Each replicate is from one biological

experiment, quantified with 8 independent fields of view. (c, d, e) Chemical

inhibition of cellular uptake for NACC: The relative fluorescence intensity of

Cy3-labeld NACC was quantified after treatment with inhibitors of

clathrin-mediated endocytosis (dynasore), caveolae-mediated endocytosis (filipin)

or macropinocytosis (EIPA). The data were presented as the means ± standard

deviation. n= 6 for each group. ** P<0.01; * P<0.05; NS: no significant difference.

52

Chapter 7 The effect of the nucleolin aptamer modification on the distribution

of NACC in a xenograft mouse model of human pancreatic cancer

7.1 Aim

To investigate the distribution of Cy3-labled NACC and Cy3-labled CACC in

major organs and tumor tissues in vivo.

7.2 Experimental design

To evaluate the distribution of Cy3-labled NACC and Cy3-labled CACC in major

organs and tumor tissues in vivo, xenograft tumor model of pancreatic cancer was

established in BALB/c nude mice as previously mentioned. Mice were randomly

divided into 2 groups and injected with Cy3-labled NACC and Cy3-labled CACC

via tail vein injection, respectively, with CSL dose of 0.5 mg/kg. Mice in each

group were sacrificed and the major organs (heart, liver, spleen, lung, kidney and

tumor tissue) were collected at 2h and 4h after the injection. The fluorescence

signal of those organs in each group was detected using IVIS® Lumina XR

IVIS® Lumina XR. Then all the organs were homogenized for quantitative study.

The fluorescence strength was measured by microplate reader.

To evaluate the cellular distribution of Cy3-labled NACC and Cy3-labled CACC

in liver tissues and tumor tissues, the tumors and livers of mice were collected and

53

the cryosections were prepared for examining the co-localization of the

fluorescence signal from Cy3-labeled NACC (or Cy3-labeled CACC) by

immunofluorescence analysis. Then, the intensity of the fluorescence signal was

examined by confocal microcopy.

7.3 Results

We observed that the fluorescence signal of Cy3 within tumors in Cy3-labeled

NACC group was significantly higher than that in Cy3-labeled CACC group at 2h

and 4h after the injection. However, the fluorescence signal of Cy3 within liver

and kidney of Cy3-labeled NACC group were remarkably lower than Cy3-labeled

CACC group at 4h after injection (Figure 9a). Furthermore, the percentage of

injected dose of Cy3-NACC and Cy3-CACC in tumor and other major organs at

2h and 4h were also quantitatively analyzed. In consistency, the accumulation of

Cy3-NACC in tumor was significantly higher than that Cy3-CACC at both time

points, while the clearance of Cy3-NACC in liver and kidney at 4h was

considerably faster (Figure 9b).

To further confirm whether nucleolin aptamer could selectively deliver the NACC

into tumor cells, we examined the distribution of Cy3-labeled CACC and

Cy3-labeled NACC in both tumor tissue and liver tissue at cellular level. The

results were shown in Figure 9c, the fluorescence signal of Cy3 within tumor slice

in Cy3-labeled NACC group was significantly higher than that in Cy3-labeled

54

CACC group at 4h after the injection. On the contrary, the fluorescence signal of

Cy3 within liver slice in Cy3-labeled NACC group was remarkably lower than that

in Cy3-labeled CACC group.

Taken together, the above results suggested that the nucleolin aptamer could

facilitate NACC selectively targeting tumor tissue in vivo.

Cy3-CACC Cy3-NACC

Tumor

Heart

Liver

Lung

Spleen

Kidney

Cy3-CACC Cy3-NACC

2h after injection 4h after injection

a

55

tumor

heart

liver

lung

spleenkid

ney

tumor

heart

liver

lung

spleenkid

ney

Cy3-NACC Light

Cy3-CACC

Cy3-NACC

Cy3-CACC

DAPI

DAPI

DAPI

DAPI

Light

Light

Light

Merge

Merge

Merge

Merge

Panc

reat

ic tu

mor

tiss

ue

M

ice

liver

tiss

ue

b

c

56

Figure 9 The tissue distribution of Cy3-labled NACC and Cy3-labled CACC in

vivo. (a) The localization of Cy3 in major organs and tumor tissues of

tumor-bearing nude mice injected with Cy3-labled NACC and Cy3-labled CACC,

visualized by biophotonic imaging at 2h and 4h. (b) Percentage of injected dose

in tumor and major organs 2h (left) and 4h (right) after intravenous injection of

Cy3-labled NACC or Cy3-labled CACC evaluated by measuring the fluorescent.

Data were expressed as mean±SD, n=6. NS: not significant, *P<0.05. (c) The

representative fluorescent micrographs of the distribution of the conjugated

Cy3-labeled NACC and Cy3-labeled CACC in tumor and liver at tissue level 4h

after intravenous injection of Cy3-labeled NACC or Cy3-labeled CACC examined

by confocal microscopy.

57

Chapter 8 The effect of the nucleolin aptamer modification on the antitumor

activity of the NACC in a xenograft mouse model of human pancreatic cancer

8.1 Aim

8.1.1 To investigate the antitumor efficacy of NACC in xenograft tumor model of

pancreatic cancer.

8.1.2 To investigate the effect of NACC on angiogenesis of tumor

microenvironment.

8.1.3 To investigate the effect of NACC on expression level of NF-κB, HSP 90 and

P-glycoprotein in tumor tissue.

8.1.4 To investigate the effect of NACC on survival of tumor bearing mice.

8.2 Experimental design

To evaluate the antitumor efficacy of NACC in vivo, xenograft tumor model of

pancreatic cancer was established in BALB/c nude mice as previously mentioned.

For 28 days, mice were intravenously injected with NS, Celastrol, CACC and

NACC at a dosage of 1.11 μmol/kg (with the equivalent celastrol concentration of

0.5 mg/kg) twice a week, respectively. The body weight and tumor size were

monitored every four days. All the tumor tissues were removed for picture taken

58

and weighted at the end of the experiment. Then, tumors were fixed for

histopathology and vessel analysis.

To evaluate effect of NACC on angiogenesis of tumor microenvironment, blood

vessels of mice were perfused with a contrast agent (Microfil; Flow Tech). The

tumors were removed and fixed after solidification of the contrast agent. Then the

tumors were scanned with a desktop micro-CT unit (viva CT40,

SCANCOMEDICAL, Switzerland). Image processing, tissue density

measurements and quantification of tumor vascularization were performed with the

software.

To determine the expression level of NF-κB, HSP 90 and P-glycoprotein in tumor

tissue, after drug administration for 28 days, tumors were removed from mice

treated with NS, Celastrol, CACC and NACC, respectively. The protein expression

levels of NF-κB, HSP 90 and P-glycoprotein were determined by western blot

analysis.

To investigate the effect of NACC on survival of tumor bearing mice, xenograft

tumor model of pancreatic cancer was established in BALB/c nude mice as

previously described. Afterward, 40 mice were divided into four groups (n=10)

and intravenously injected with NS, Celastrol, CACC and NACC at a dosage of

1.11 μmol/kg (with the equivalent celastrol concentration of 0.5 mg/kg) for 60 days,

respectively. The body weight and tumor size were monitored every four days.

Mice were monitored for up to 100 days and when tumors exceeded 2cm in

diameter or visible necrosis was observed, mice were euthanized with chloral

59

hydrate. The survival analysis was also performed using the Kaplan Meier method.

8.3 Results

After drug administration for 28 days, free Celastrol, CACC and NACC

significantly reduced tumor volume (Figure 10-1a) and final tumor weight (Figure

10-2b) compared to NS group. NACC significantly inhibited tumor growth

compared to Celastrol and CACC. However, there was no significant difference

between the CACC-treated group and the Celastrol-treated group, which indicated

that the increased antitumor activity of NACC might be attributed to the

modification of nucleolin aptamer. The gross evaluation of tumor volumes was

shown in Figure 10-1c. The histopathology result was shown in Figure10-1d. For

H&E staining, the cytoplasm was stained by eosin in pink and the nuclei were

stained with hematoxylin in blue. In the NS group, the growth of tumor cells was

active and the obvious cell division was found. In the Celastrol and CACC treated

group, tumor cells grew slowly and the tumor tissue was infiltrated by few immune

cells. In the NACC-treated group, obvious tumor cell apoptosis and immunocytes

infiltration were observed. It indicated that the modification of nucleolin aptamer

on the carboxylic acid group of celastrol improved the antitumor efficacy to

pancreatic cancer in vivo.

Tumor angiogenesis was significantly reduced in drug-treated animals compared to

untreated animals. In addition, only small fragmented vessels were observed on the

60

surface of the solid tumor in the NACC-treated group (Figure 10-2a). The

quantification analysis results showed that both the vessel volume and vessel

density in NACC-treated group were remarkably lower than that in

Celastrol-treated group and CACC-treated group (Figure 10-2b).

We found that the protein expression levels of NF-κB, HSP 90 and P-glycoprotein

in NACC group were remarkably lower than those in NS group, Celastrol group

and CACC group. In contrast, no significant difference was found between the

Celastrol group and CACC group (Figure 10-3).

We also investigate the effect of NACC on survival of tumor bearing mice. Our

analysis indicated that, after drug treatment for 60 days, a significantly increase was

observed in the median survival time of mice treated with NACC when compared

to mice treated with Celastrol or vehicle control. There was no significant

difference between mice treated with CACC and Celastrol (Figure 10-4).

61

b

c

a

NS

Celastrol

CACC

NACC

62

Figure 10-1 (a) The tumor volume change of BALB/c nude mice in NS group,

Celastrol group, CACC group and NACC group. Data represent the mean ± SD

(n=6). Significance were assessed compared with Celastrol group, *p<0.05. (b) The

final tumor weight of BALB/c nude mice in NS group, Celastrol group, CACC

group and NACC group, significance were assessed compared with Celastrol group,

*p<0.05. (c) Gross evaluation of tumor volumes in NS group, Celastrol group,

CACC group and NACC group. (d) Representative H&E-stained sections of

tumors from NS group (A), Celastrol group (B), CACC group (C) and NACC

group (D) (n = 6; ×4 magnification on an Aperio ScanScope scanner)

NS Celastrol

CACC NACC

d

63

NS Celastrol

CACC NACC

NS

Celastr

ol

CACCNACC

Ves

sel V

olum

e (m

m3 )

NS

Celastr

ol

CACCNACC

VV/T

V (P

erce

nt)

a

b

64

Figure 10-2 (a) Three-dimensional volume rendering technique (VRT) images of

tumor angiogenesis after treatment with NS, Celastrol, CACC and NACC. (b)

Quantification of the vessel volume (VV) and vessel density (VV/TV) of tumors

after treatment with NS, Celastrol, CACC and NACC. Data represent the mean ±

SD (n=6), NS: not significant, *p<0.05.

Figure 10-3 Levels of NF-κB, HSP 90 and P-glycoprotein (P-gp) in tumor tissue

of mice treated with NS, celastrol (CSL), CACC and NACC determined by

western blot analysis. Upper panel: the representative electrophoretic bands.

Lower panel: the semi-quantitative data of the protein levels.

NF-κB

β-actin

HSP 90

β-actin

P-gp

β-actin

NS CSL CACC NACC

Rel

ativ

e Pr

otei

n Le

vels

65

Figure 10-4 The survival rate of tumor bearing mice in NS group, Celastrol group,

CACC group and NACC group.

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100

NS

Celastrol

CACC

NACC

Perc

ent S

urvi

val

Time (days)

66

Chapter 9 The effect of the nucleolin aptamer modification on the liver

toxicity of NACC

9.1 Aim

To evaluate the effect of the nucleolin aptamer modification on the liver toxicity of

NACC in vivo.

9.2 Experimental design

To examine the liver toxicity of continuous administration of NACC, female

BALB/c mice were randomly divided into 4 groups and intravenously injected with

NS, Celastrol, CACC and NACC at a dosage of 5.55 μmol/kg (with the equivalent

celasrol concentration of 2.5 mg/kg) twice a week for 60 days, respectively. 72

hours after the last administration, blood samples were collected from abdominal

aorta under deep anesthetization. Liver tissues were removed after the mice were

sacrificed by euthanasia. The content of ALT and AST from the blood samples was

examined. The liver tissue was excised in 10% neutral buffered formalin for

histopathological studies.

67

9.3 Result

As shown in Figure 11, compared to NS group, the serum level of ALT and AST in

the Celastrol-treated group were increased 3.1 and 9-fold, respectively (P<0.05).

However, the serum level of ALT and AST (Figure 11a) in NACC group was

lower than those in Celastrol and CACC treated group. The H&E-stained sections

of mice liver tissue were shown in Figure 11b. In the Celastrol-treated group,

lobule structural disorder, cell swelling and neutrophil infiltration were observed.

Hepatocyte necrosis and apoptotic hepatocytes appeared mainly around the central

vein, with increased eosinophility of hepatocytes with pyknotic or karyolitic nuclei.

In NACC-treated group, normal lobular architecture with central veins and

radiating hepatic cords was observed. Although spotty necrosis was observed, but

necrotic cells are quite rare. The incidence and severity of histopathological lesions

were significantly decreased compared with Celastrol-treated group. The above

result revealed that the hepatotoxicity of NACC is lower than Celastrol, which is

also consistent with the serum biochemical test results.

68

NS Celastrol

CACC NACC

NS

Celastr

ol

CACCNACC NS

Celastr

ol

CACCNACC

0

100

200

300 ***

a

b

69

Figure 11 Effects of NS, Celastrol, CACC and NACC on serum level of

biochemical markers and pathological changes in mice liver. (a) The serum level of

ALT (left) and AST (right). Data represent the mean ± SD (n=10), significance

were assessed compared with NACC group, *p<0.05, **p<0.01; (b) Representative

H&E-stained sections of mice liver from NS (A), Celastrol (B), CACC (C) and

NACC group (D) (n=10; ×10 magnification on an Aperio ScanScope scanner).

70

Chapter 10 Discussion

PC is one of the most devastating malignancies with median survival of 5~6 months

and a 5-year survival rate inferior to 6%. The prognosis of patients with pancreatic

cancer is extremely poor due to the rapid progress and the metastasis, although

efforts have been made to improve diagnosis and treatment over the past three

decades. Besides, the devastating prognosis of PC is also associated with a high

degree of resistance to systemic therapy. So it is necessary to explore new drugs

aiming at chemoresistant-cancer cells to provide an effective therapy for PC.

Celastrol demonstrated strong antiproliferation activity on different kinds of cancer

cells, including human mononuclear leukocyte cancer cells, human prostate cancer

cells, melanoma cells, PC cells and the human lung cancer cells, suggesting the

broad-spectrum antitumor activity of celastrol (Lin L et al. 2016). It is reported that

celastrol suppresses tumor growth by multiple mechanisms. Celastrol inhibits the

expression of HSP90 in tumor cells and reflect synergistic effect with the traditional

HSP90 inhibitor (Shao L et al. 2013). Celastrol reduces the expression level of P-gp

to reverse the resistance of tumor cells to chemotherapy (Lee JH et al. 2012).

Moreover, celastrol has inhibitory effect on NF-κB signaling pathway. Celastrol

inhibits the activation of NF-κB system by inhibiting the phosphorylation and

degradation of IKKβ kinase substrate IκBα. Celastrol also suppresses tumor growth

by inhibiting tumor angiogenesis (Boridy S et al. 2014). Studies showed that

71

celastrol inhibits the expression of VEGF by inhibiting tumor necrosis factor alpha

(TNF-α), suggesting that celastrol has the ability to antagonize tumor metastasis.

Celastrol significantly suppresses the cell growth, migration and invasion of

HUVEC induced by VEGF (Zhao X et al. 2014). In summary, due to the strong

antitumor activity and multiple antitumor mechanisms, celastrol might be used as

an alternative drug for the treatment of pancreatic cancer (Li HY et al. 2014).

Nucleolin aptamer is a G-rich phosphodiester oligonucleotide that can bind

nucleolin with high affinity and specificity. Previous studies showed that nucleolin

protein was overexpressed in pancreatic cancer cells compared with normal cells.

Tissue distribution studies suggested that nucleolin aptamer could selectively

accumulate in the tumors of animal models. It has been successfully used for

cell-specific delivery of various drugs or siRNAs in animal studies. In addition, the

safety of nucleolin aptamer has also been validated in preclinical and clinical

studies. The preclinical pharmacokinetics data concluded that the major elimination

route of nucleolin aptamer was renal and the elimination half-life of nucleolin

aptamer in plasma was two days as well as in whole blood. These results suggested

that nucleolin aptamer is an ideal target moiety. In our study, the in vitro results

indicated that the modification of nucleolin aptamer facilitates NACC selectively

binding to PC cells rather than normal liver cells. Besides, NACC was mainly

taken up by cancer cells through macropinocytosis, which is consistent with the

findings previously reported that macropinocytosis was the major endocytic

72

pathway of nucleolin aptamer.

In this study, Celastrol (CSL) was modified by nucleolin aptamer to form the

Nuleolin Aptamer-Celastrol Conjugate (NACC). The water solubility of NACC

was significant improved compared to that of free celastrol. The reported solubility

of celastrol in water is no more than 0.014 mg/mL (approximately 30 µM), while

the solubility of NACC in water is at least greater than 17.8 mg/mL (2 mM).

NACC demonstrated superior antitumor activity on pancreatic cancer both in vitro

and in vivo. The cell proliferation study showed that the antitumor activity of the

NACC on pancreatic cancer cell line was stronger than that of CSL in vitro, which

indicated that the antitumor activity of CSL was improved after it was conjugated

to nucleolin aptamer, which is consistent with the findings previously reported

that the carboxylic acid group of celastrol was not required for its apoptotic activity.

The in vivo study revealed that NACC could downregulate the expression level of

the molecular mechanisms related to chemoresistance of PC. It's worth noting that

the liver toxicity of NACC was remarkably lower than that of CSL. Thus, NACC

might be developed as a potential drug for PC by aiming at chemoresistance.

The in vivo distribution study revealed that the nucleolin aptamer modification

facilitated the accumulation of the NACC in tumor tissue rather than major organs.

Moreover, compared to CSL and CACC, NACC consistently demonstrated higher

antitumor activity and lower liver toxicity in vivo. The enhanced antitumor activity

73

of NACC might be explained by the following possible reasons. Firstly, the

conjugated nucleolin aptamer could facilitate CSL selectively accumulating in

tumor tissue to increase the drug concentration within the tumor microenvironment.

Secondly, NACC could be taken up by PC cells mainly through macropinocytosis,

which indicated that the conjugated nucleolin aptamer could increase the cellular

uptake of NACC by PC cells to increase the drug concentration in the tumor cells.

Tumor angiogenesis is crucial for rapid growth of a malignant tumor. Tumor blood

vessels provide sufficient nutrients for tumor growth and necessary conditions for

distant metastasis. Thus, inhibition of tumor angiogenesis has become a modern

approach to suppress tumor progression. It has been widely reported that celastrol

could effectively suppress tumor growth through inhibition of tumor angiogenesis

via multiple mechanism. In the present study, we found that NACC could

effectively inhibit tumor angiogenesis in xenograft tumor model of pancreatic

cancer and the efficacy was higher than that of CSL and CACC. The improved

anti-angiogenesis activity might be attributed to the selectivity of nucleolin

aptamer.

In summary, we have successfully conjugated the nucleolin aptamer to CSL by

chemical synthesis to form NACC. We found that the NACC consistently

demonstrated higher anti-proliferation activity on pancreatic cancer cell line in

vitro, compared with CSL. Moreover, NACC could selectively bind to pancreatic

74

cancer cell in vitro. The NACC can be taken up by PANC-1 cell line mainly via

macropinocytosis. Moreover, NACC could selectively accumulate in pancreatic

cancer tissue and reduce the distribution to liver tissue in vivo. In addition, NACC

also demonstrated higher antitumor activity and less liver toxicity in vivo. Taken

together, the above results revealed that the conjugated nucleolin aptamer could

facilitate CSL selectively targeting pancreatic cancer cells to achieve higher

antitumor activity and less liver toxicity.

Although the anti-tumor efficacy of NACC has been demonstrated both in vitro

and in vivo, there is still some distance between laboratory findings and clinical

applications. On the one hand, various animal models should be applied to

evaluate the anti-tumor efficacy of NACC in vivo. A multi-dose experiment

should be performed to evaluate the toxicity of NACC. On the other hand, to

promote the further application of NACC, it is necessary to improve the synthesis

methods of the aptamers in order to reduce the cost of NACC. While the synthesis

process and reaction conditions of NACC also need to be optimized in order to

obtain higher productivity of NACC.

To our knowledge, this was the first time that celatrol was directly linked to

nucleolin aptamer to create a single macromolecule. The modification of nucleolin

aptamer on celastrol has not only increased the water solubility, but also reduced

the liver toxicity, which successfully solved the two major bottlenecks that hinder

75

the clinical application of celastrol at the same time. So NACC may provide a new

treatment for pancreatic cancer patients and further promote the clinical translation

of celastrol. More importantly, we established an innovative approach to develop

smart compounds by modifying natural products with aptamers. Based on the

above studies, an innovative technology platform for exploring Aptamer-Drug

Conjugate was also established. The key technology in this platform might be

applied to develop more natural products with similar drawbacks to promote their

technical innovation. In addition, based on the aptamer screening technology of the

platform, tumor cell specific biomarkers could be utilized to screen more aptamers

for the modification of small molecule compounds.

76

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CURRICULUM VITAE

Academic qualifications of the thesis author, Mr. LIU Biao:

Received the degree of Bachelor of Engineering from HuBei University,

WuHan, China, July 2008.

Received the degree of Master of Science from HuBei University, WuHan,

China, July 2011.

August 2017