hong kong baptist university doctoral thesis a novel
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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