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The Use of Endogenous and Synthetic Cannabinoids in Prostate Cancer Therapy
by
Domenica Roberto
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto
© Copyright by Domenica Roberto, 2018
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The Use of Endogenous and Synthetic Cannabinoids in Prostate
Cancer Therapy
Domenica Roberto
Master of Science
Institute of Medical Science
University of Toronto
2018
Abstract
Cannabinoids are mainly used as an antiemetic and for cancer-related pain, however, studies
have implicated an anti-proliferative role in various cancer models. This thesis examines the
therapeutic potential of cannabinoids anandamide and WIN55,212-2 in prostate cancer.
In vitro studies on prostate cancer cells showed that the cannabinoids significantly reduce
proliferation, migration, invasion, and induce apoptosis in a dose-dependent manner. Inhibition
of cannabinoid receptor 2 resulted in a reversal of the anti-proliferative effects. Cell cycle
analysis revealed that WIN55,212-2 caused arrest in G1/2 phases, and mechanistic studies
demonstrated that these effects were mediated through alterations in key cell cycle regulators.
Based on these studies, the effect of WIN55,212-2 was assessed using a xenograft model,
resulting in a reduction in tumor volume compared to control.
Evidence from this thesis provides a framework for future studies and provides a more in-depth
understanding of the potential benefit of cannabinoid use in prostate cancer.
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Acknowledgments
First and foremost, I would like to thank my primary supervisor Dr. Vasundara Venkateswaran
and my co-supervisor Dr. Laurence Klotz. They accepted me into the lab with very little
experience and helped me to identify my passion for research. They have consistently mentored
and supported me throughout my degree, provided me with numerous opportunities to grow as a
scientist, and pushed me to reach my potential.
My sincere thanks must also go to my fellow lab members both past and present. Michelle
Mayer who introduced me to the lab, Dr. Natalie Venier for all her advice and detailed protocol
notes, Dr. Azik Hoffman who greatly contributed to my understanding of urology and provided
guidance throughout my degree, and Dr. Roman Bass for his positive influence towards the end
of my degree.
I would like to acknowledge the amazing people I have met at Sunnybrook for inspiring me and
making my experience as a graduate student a positive one. These individuals offered their time
and expertise so generously and have become great friends that I am truly lucky to have met.
I am grateful to my Program Advisory Committee Members Dr. Urban Emmenegger and Dr.
David Ma for lending me their expertise and intuition, instrumental for the completion of my
degree. I would like to recognize my examiners Dr. Stanley Liu, Dr. Marianne Koritzinsky, and
Dr. Sanjay Gupta who took the time to review and edit my thesis.
I would like to acknowledge the IMS department for awarding me the Entrance Award and the
Open Fellowship Award which has provided me with financial support throughout my degree.
I deeply thank and dedicate this thesis to my wonderful parents for their unconditional love and
endless patience. I have learned so much from them and I appreciate all of the sacrifices that they
have made for me. My brother, Adriano who has encouraged me to pursue my passion and who
has always supported me when times were challenging. My Nonna, for the knowledge and
values she has instilled in me; I know that she would be proud of the person I have become. I
cannot thank them enough for everything they have done for me and would not be where I am
today without them.
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At last, I would like to express my deepest gratitude to my best friend and soul mate, Kogulan
who has provided me continuous support, love and encouragement not only throughout my
degree, but throughout every aspect of my life. His positive outlook on life has given me the
strength to accomplish anything. I am blessed to have someone so kind, loving, and selfless in
my life; he has taught me so much about myself and continuously inspires me to become a better
person.
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Table of Contents
Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents .............................................................................................................................v
List of Abbreviations ..................................................................................................................... ix
List of Tables ................................................................................................................................. xi
List of Figures ............................................................................................................................... xii
Chapter 1 Introduction .....................................................................................................................1
Introduction .................................................................................................................................1
1.1 Prostate Anatomy .................................................................................................................1
1.2 Prostate Cancer Epidemiology .............................................................................................2
1.3 Prostate Cancer Pathophysiology ........................................................................................2
1.4 Prostate Cancer Detection and Diagnosis ............................................................................4
1.5 Treatment Strategies ............................................................................................................6
1.5.1 Active Surveillance ..................................................................................................6
1.5.2 Surgery .....................................................................................................................6
1.5.3 Androgen Deprivation Therapy ...............................................................................7
1.5.4 Radiotherapy ............................................................................................................7
1.5.5 Adjuvant and Neo-Adjuvant Therapies ...................................................................8
1.5.6 Chemotherapy ..........................................................................................................8
1.5.7 Chemopreventive and Novel Agents .......................................................................9
1.6 Cannabinoids......................................................................................................................10
1.6.1 Phytocannabinoids .................................................................................................11
1.6.2 Endocannabinoids ..................................................................................................12
1.6.3 Synthetic Cannabinoids .........................................................................................17
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1.7 Molecular Targets of Cannabinoids ...................................................................................19
1.7.1 Endocannabinoid System .......................................................................................19
1.7.2 Cannabinoids and Non-Cannabinoid Receptors ....................................................21
1.7.3 Endoplasmic Reticulum Stress Response ..............................................................25
1.7.4 Oxidative Stress .....................................................................................................28
1.7.5 Rho GTPase Signalling ..........................................................................................32
1.7.6 Apoptosis ...............................................................................................................35
1.7.7 Cell- Cycle Regulation ...........................................................................................37
1.8 Preclinical Models of Prostate Cancer ...............................................................................46
1.8.1 In Vitro Models ......................................................................................................46
1.8.2 In Vivo Models .......................................................................................................48
1.8.2.1 Xenograft Mouse Models ........................................................................48
1.8.2.2 TRAMP Mouse Model ............................................................................49
1.8.2.3 Lady Transgenic Model ...........................................................................50
1.8.2.4 The Phosphatase and tensin homolog deleted on chromosome ten
(PTEN) Model .........................................................................................50
1.8.2.5 c-MYC Model .........................................................................................51
1.8.2.6 NK3 Homeobox (1NKX3.1) Model ........................................................51
Chapter 2 Rationale, Hypothesis, and Aims .................................................................................52
Rationale, Hypothesis, and Aims ..............................................................................................52
2.1 Rationale ............................................................................................................................52
2.2 Hypothesis..........................................................................................................................52
2.3 Aims ...................................................................................................................................53
Chapter 3 Materials and Methods .................................................................................................54
Materials and Methods ..............................................................................................................54
3.1 Cell Culture ........................................................................................................................54
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3.2 Chemicals ...........................................................................................................................54
3.3 MTS Cell Proliferation Assay ............................................................................................54
3.4 Wound Healing (Scratch) Assay ........................................................................................55
3.5 Matrigel Invasion Assay ....................................................................................................56
3.6 Flow Cytometry .................................................................................................................57
3.6.1 Cell Cycle Distribution ..........................................................................................57
3.6.2 Apoptosis ...............................................................................................................58
3.7 Western Blot Analysis .......................................................................................................58
3.8 Xenograft Studies...............................................................................................................59
3.8.1 Animals and Housing .............................................................................................59
3.8.2 Establishment of Xenografts ..................................................................................59
3.8.3 Administration of WIN 55,212-2 ...........................................................................60
3.9 In Vitro Mitogenicity Assay...............................................................................................62
3.10 Statistical Analysis .............................................................................................................62
Chapter 4 Results ...........................................................................................................................63
Results .......................................................................................................................................63
4.1 Differential growth inhibitory effect of anandamide on prostate cancer cell lines ............63
4.2 Treatment with WIN-55,212-2 reduces prostate cancer cell proliferation ........................67
4.3 Anandamide and WIN 55,212-2 treatment reduces the migration and invasion
capacity of prostate cancer cells ........................................................................................71
4.4 Anandamide treatment does not significantly alter the cell cycle distribution in
DU145 and LNCaP cells ....................................................................................................75
4.5 WIN 55,212-2 treatment causes cell cycle arrest in DU145 and PC3 cells .......................78
4.6 Anandamide reduces proliferation and induces apoptosis in LNCaP cells but not in
DU145 cells .......................................................................................................................81
4.7 WIN 55,212-2 significantly induces apoptosis in PC3 and DU145 cells but not in
LNCaP cells .......................................................................................................................84
4.8 Cannabinoid receptor 2 antagonist AM630 does not alter prostate cancer cell growth ....88
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4.9 Treatment with CB2 antagonist AM630 abrogates the anti-proliferative effects of
anandamide in DU145 and LNCaP cell lines ....................................................................90
4.10 Treatment with CB2 antagonist AM630 abrogates the anti-proliferative effects of
WIN 55,212-2 in prostate cancer cell lines ........................................................................92
4.11 WIN 55,212-2 treatment alters expression of pRb, Cdk4, and p27 in PC3 cells ...............94
4.12 WIN 55,212-2 treatment reduces tumor growth in a mouse xenograft model ..................96
4.13 Serum containing WIN 55,212-2 reduces PC3 cell proliferation ......................................99
Chapter 5 Discussion ...................................................................................................................103
Discussion ...............................................................................................................................103
Chapter 6 Future Directions and Overall Conclusion ..................................................................108
Future Directions and Overall Conclusion ..............................................................................108
6.1 Potential In Vivo Studies ..................................................................................................108
6.2 Current and Potential Clinical Trials ...............................................................................109
6.3 Overall Conclusion ..........................................................................................................110
References ....................................................................................................................................112
Appendix ......................................................................................................................................133
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List of Abbreviations
ACPA: Arachidonoyl cyclopropamide
ADT: Androgen Deprivation Therapy
AEA: Anandamide
2AG: 2-arachidonoyl glycerol
ANOVA: One-Way Analysis of Variance
AOM: Azoxymethane
AR: Androgen Receptor
BCL-2: B-cell lymphoma 2 (oncogene)
BPH: Benign prostatic hyperplasia
BrdU: BromodeoxyUridine
CB: Cannabinoid Receptor
CBC: Cannabichromene
CBD: Cannabidiol
CBDA: Cannabidiolic acid
CBG: Cannabigerol
CBL: Cannabicyclol
CBN: Cannabinol
COX: Cyclooxygenase
CRC: Colorectal cancer
CRPC: Castration-Resistant Prostate Cancer
DAG: Diacylglycerol
DAGL: Diacylglycerol lipase
DMSO: Dimethyl Sulfoxide
DR5: Death Receptor 5
DRE: Digital Rectal Examination
DSS: Dextran sulfate sodium
DU145: Human Prostate Cancer Cell Line
EAU: European Association of Urology
EDTA: Ethylenediaminetetraacetic Acid
ER: Endoplasmic Reticulum
FAAH: Fatty Acid Amide Hydrolase
FITC: Fluorescein Isothiocyanate
FBS: Fetal Bovine Serum
GGG: Gleason Grade Group
GPCR: G-Protein Coupled Receptor
GW: GW405833
HCl: Hydrochloric Acid
HGPIN: High-Grade Prostatic Intraepithelial Neoplasia
5-HT: 5-hydroxytryptamine
LHRH: Luteinizing Hormone Releasing Hormone
LNCaP: Lymph Node Carcinoma of the Prostate
MAGL: Monoacylglycerol lipase
MAPK: Mitogen-activated Protein Kinase
MET: Methanandamide
Met-F-AEA: 2-methyl-2’-F- anandamide
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MMP: Metalloproteinase
mTOR: Mammalian Target of Rapamycin
NAPE: N-arachidonoyl-phosphatidylethanolamine
NAPE-PLD: NAPE-specific phospholipase D
NAT: N-acyl transferase
NCCN: National Comprehensive Cancer Network
NPC: Nutrition Prevention of Cancer Trial
OEA: N-oleoylethanolamide
p53: Tumor Suppressor Protein 53
PARP: Poly (ADP-ribose) polymerase
PBS: Phosphate Buffered Saline
PC3: Human Prostate Cancer Cell Line
PCa: Prostate cancer
pCB: Phytocannabinoid
PDX: Patient-derived
PEA: N-palmitoylethanolamide
PI: Propidium Iodide
PIN: Prostatic Intraepithelial Neoplasia
PIP2: Phosphatidylinositol-4,5-bisphosphate
PKA: Protein kinase A
PLC: Phospholipase C
PPAR: Nuclear Peroxisome Proliferator-Activated Receptor
PSA: Prostate Specific Antigen
PTEN: Phosphatase and tensin homolog deleted on chromosome ten
QOL: Quality of life
Rb: Retinoblastoma (tumor suppressor gene)
ROS: Reactive Oxygen Species
SC: Synthetic Cannabinoid
SD: Standard deviation
SDS: Sodium Dodecyl Sulfate
SELECT: Selenium and Vitamin E Cancer Prevention Trial
SRI: Sunnybrook Research Institute
∆8-THC: delta-8-tetracannabinol
THC: delta-9-tetrahydrocannabinol
TNF: Tumor necrosis factor
TRAMP: Transgenic Adenocarcinoma Mouse Prostate
TRB3: Tribbles Homolog 3
TRP: Transient Receptor Potential
TRPV1: Transient Receptor Potential cation channel Vanilloid Type 1
TRUS: Transrectal Ultrasound
UPR: Unfolded Protein Response
WIN: WIN 55,212-2
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List of Tables
Table 1: Effects of cannabinoids on cell viability, migration, and invasion in prostate and various
cancers.
Table 2: General characteristics of common immortalized human prostate cancer cell lines.
Table 3: Summary of in vitro results.
Table 4: Summary of in vivo results.
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List of Figures
Figure 1: The prostate anatomy.
Figure 2: The process of carcinogenesis.
Figure 3: The biosynthesis of anandamide.
Figure 4: The biosynthesis of 2-arachidonoylglycerol.
Figure 5: The chemical structure of anandamide.
Figure 6: The chemical structure of WIN 55,212-2.
Figure 7: Cannabinoid receptor localization.
Figure 8: Proposed endoplasmic reticulum stress signalling pathway.
Figure 9: Proposed oxidative stress signalling pathway.
Figure 10: Proposed RhoA GTPase signalling pathway.
Figure 11: Proposed cell cycle regulation pathway.
Figure 12: MTS cell proliferation assay.
Figure 13: Wound healing (scratch) assay.
Figure 14: Matrigel invasion assay.
Figure 15: Xenograft establishment and group assignment.
Figure 16: Xenograft experiment timeline.
Figure 17: Effect of anandamide treatment on proliferation of PC3 cells.
Figure 18: Effect of anandamide treatment on proliferation of LNCaP cells.
Figure 19: Effect of anandamide treatment on proliferation of DU145 cells.
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Figure 20: Effect of WIN 55,212-2 treatment on proliferation of PC3 cells.
Figure 21: Effect of WIN 55,212-2 treatment on proliferation of LNCaP cells.
Figure 22: Effect of WIN 55,212-2 treatment on proliferation of DU145 cells.
Figure 23: Effect of 24hr treatment of anandamide on DU145 cell migration and invasion.
Figure 24: Effect of 24hr treatment of WIN 55,212-2 on DU145 cell migration and invasion.
Figure 25: Effect of 24hr treatment of WIN 55,212-2 on PC3 cell migration and invasion.
Figure 26: Effect of anandamide treatment on cell cycle distribution in DU145 cells.
Figure 27: Effect of anandamide treatment on cell cycle distribution in LNCaP cells.
Figure 28: Effect of WIN 55,212-2 treatment on cell cycle distribution in DU145 cells.
Figure 29: Effect of WIN 55,212-2 treatment on cell cycle distribution in PC3 cells.
Figure 30: Effect of anandamide on proportion of live versus apoptotic DU145 cells using
Annexin V flow cytometry.
Figure 31: Effect of anandamide on proportion of live versus apoptotic LNCaP cells using
Annexin V flow cytometry.
Figure 32: Effect of WIN 55,212-2 on proportion of live versus apoptotic PC3 cells using
Annexin V flow cytometry.
Figure 33: Effect of WIN 55,212-2 on proportion of live versus apoptotic DU145 cells using
Annexin V flow cytometry.
Figure 34: Effect of WIN 55,212-2 on proportion of live versus apoptotic LNCaP cells using
Annexin V flow cytometry.
Figure 35: Effect of Cannabinoid receptor 2 antagonist AM630 on viability of prostate cancer
cell lines.
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Figure 36: Effect of anandamide after treatment with cannabinoid receptor 2 antagonist AM630
on proliferation of DU145 and LNCaP cell lines.
Figure 37: Effect of WIN 55,212-2 after treatment with cannabinoid receptor 2 antagonist
AM630 on cell proliferation.
Figure 38: Effect of WIN 55,212-2 on expression of cell cycle regulator proteins.
Figure 39: WIN 55,212-2 significantly reduces tumor growth rate.
Figure 40: Lack of effect of WIN 55,212-2 treatment on animal weight.
Figure 41: Representative images of mice tumors before and after excision.
Figure 42: Effect of WIN 55,212-2 containing serum on proliferation of PC3 cells.
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Chapter 1 Introduction
Introduction
1.1 Prostate Anatomy
The prostate is a gland of the male reproductive system (Figure 1), located anterior to the rectum
and inferior to the bladder. It is the size and shape of a walnut but increases in size when men
reach their late forties and early fifties. It surrounds the urethra, the tube that carries urine and
semen through the penis.
The main functions of the prostate are to produce prostatic fluid for semen and to contribute to
urinary continence. Prostatic fluid is rich in enzymes, proteins and minerals that help protect and
nourish sperm. Hormones, including testosterone and those made by the adrenal and pituitary
glands, help control the function of the prostate gland.
Figure 1: The Prostate Anatomy. The location of the prostate gland below the bladder and
surrounding the urethra.
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1.2 Prostate Cancer Epidemiology
Prostate cancer (PCa) is the second most commonly diagnosed cancer, with 1.1 million new
cases reported globally (Zhou et al., 2016). It is one of the leading causes of morbidity and
mortality worldwide and thus considered as an important public health issue. Risk factors for
prostate cancer include age, family history, race, diet, and certain genetic polymorphisms,
amongst many others (Fradet et al 2009). The incidence rates of PCa vary by more than 50-fold
worldwide, with highest rates observed in Australia, North America, and Western Europe, and
lowest in South Central Asia and China (Wong et al., 2016). The practice of prostate specific
antigen (PSA) screening and subsequent biopsy in developed countries has led to a rise in PCa
incidence rates. However, earlier diagnosis comes at the expense of potentially treating a
significant proportion of men who are at little risk of symptom development or associated
disease complications during their lifetime (Fradet, Klotz, Trachtenberg, & Zlotta, 2009).
In comparison to incidence and detection, the trends in mortality are less clear. With over
300,000 deaths worldwide, PCa is the fifth leading cause of death from cancer in men (Wong et
al 2016). PSA testing substantially increases prostate cancer incidence rates. However, the effect
of this detection tool on mortality reduction is less clear. There is a ten-fold variation in mortality
rates worldwide (Ferlay et al., 2013). The lowest mortality rates reported are in Asia and North
Africa, and the highest death rates are seen in the Caribbean (Wong et al 2016). The variation in
PCa mortality rates can be attributed to various factors. It is accounted for by differences in
genetic predisposition, as well as variations in accuracy of recording mortality
causes and differences in treatment strategies (Wong et al 2016).
1.3 Prostate Cancer Pathophysiology
As is the case with other cancers, PCa development depends on cardinal characteristics such as
sustained proliferative signalling, evasion of growth suppressors, avoiding immune destruction,
resistance to cell death, replicative immortality, promotion of inflammation, angiogenesis,
invasion and migration, genome instability and deregulation of cellular energetics (D Hanahan &
Weinberg, 2000)(,Douglas Hanahan & Weinberg, 2011).
Prostate cancer development occurs in three stages (Figure 2). In the initiation stage, normal cells
acquire irreversible mutations. Overtime, these mutations accumulate, further deregulating cell
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growth and leading to a tumor large enough to become detectable. Eventually, cells enter the
promotion phase, where they can undergo biological effects without metabolic activation. In the
final stage of progression, tumors acquire a malignant phenotype. These may remain localized to
the prostate, or evolve the ability to secrete proteases and other mediators of invasion that allow
infiltration and metastasis (Abel & DiGiovanni, 2011),(Pitot, 1993).
Figure 2: The Process of Carcinogenesis. Prostate cancer develops in three stages: initiation,
promotion and progression. Figure was adapted from (Siddiqui, Sanna, Ahmad, Sechi, &
Mukhtar, 2015).
Greater than 95% of prostate cancers are classified as adenocarcinoma. Within this classification,
there is profound molecular and phenotypic heterogeneity. This heterogeneity underlies the
distinction between latent and clinical disease, as well as the correlation between PCa
progression and aging. Prostate cancer develops commonly with age. It is ubiquitous in aging
men, and common in autopsies on young men dying of other unknown causes. This supports the
view that prostatic carcinogenesis is initiated early in life (Yatani, Kusano, Shiraishi, Hayashi, &
Stemmermann, 1989). High-grade prostatic intraepithelial neoplasia (HGPIN), defined as a
neoplastic growth of epithelial cells within pre-existing prostatic acini or ducts, is considered to
be a precursor for PCa development, with more than 40% of HGPIN patients clinically
diagnosed with PCa within 3 years of HGPIN diagnosis (S. H. Lee et al., 2016). Prostatic
intraepithelial neoplasia (PIN) spreads through the prostatic ducts causing cell proliferation and
cytologic changes similar to those of cancer. PIN is associated with progressive phenotypic and
genotypic abnormalities that are an intermediate between normal prostate epithelium and
prostate cancer, with more than 36 genetic and molecular alterations reported (Klink,
Miocinovic, Galluzzi, & Klein, 2012). A current area of uncertainty is whether high grade,
aggressive cancer develops in most cases from low grade cancer, or has a separate phylogeny
derived directly from PIN.
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1.4 Prostate Cancer Detection and Diagnosis
Throughout the developed world there are efforts to improve the outcome of prostate cancer
through early detection. The concept is to detect the cancer at a point where it is more amenable
to cure, resulting in improved survival and quality of life (QOL). Currently, the main screening
measures that exist to help detect PCa at an earlier stage include serum concentration of prostate-
specific antigen, digital rectal examination (DRE), and the transrectal ultrasound (TRUS)-guided
biopsy (Tenke, Horti, Balint, & Kovacs, 2007).
PSA is a protein produced by the cells of the prostate and is secreted into seminal fluid in high
concentrations. Trace amounts of PSA can be detected in circulating blood of healthy men,
which allows PSA to be measured using a blood test. Although the adoption of PSA screening
since the early 1990s has dramatically increased the detection of PCa, this has also contributed to
presumptive overdiagnosis and overtreatment, resulting in unfavourable effects on patient’s QOL
(E. H. Kim & Andriole, 2015).
DRE is a procedure in which a healthcare professional inserts a gloved finger into the rectum to
check for abnormalities in the size and shape of the prostate. DRE has low sensitivity when used
alone. However, when used together with PSA testing, the detection rate can be improved. Of the
three, TRUS guided biopsy is the most invasive and is associated with higher costs. The DRE
aids not only in diagnosing PCa but also in determining its clinical stage.
TRUS-guided biopsy has become the standard way to obtain material for histopathologic
examination. Web based nomograms allow for the integration of multiple risk factors such as
PSA level, age, family history, positive DRE, among others, to provide a risk prediction of the
likelihood cancer considered to be of significant nature. This level of risk drives the decision to
perform a biopsy. In this procedure, a biopsy needle is inserted into the prostate gland and small
samples of tissue are removed for pathological examination. In general, for a prostate gland
volume of 30-40ml, 10-12 cores should be sampled (Heidenreich et al., 2011). The cores are then
examined to evaluate microscopic features of potential cancer cells and the Gleason grade(s) of
the tumor are reported, as well as extent of cancer, presence of lymphovascular or perineural
invasion, if any.
The Gleason system remains one of the most powerful prognostic predictors in prostate cancer,
5
and accurate grading is crucial for predicting a patient’s prognosis and treatment options (Epstein
et al., 2016). Depending on morphological PCa growth features, a score between 1-5 is applied,
with higher numbers indicating worse prognosis. In the current application, Gleason patterns of 1
and 2 are no longer assigned on needle core biopsy, however, consist of single closely-packed
glands with well-defined edges (Gleason 1), and simple, round, loosely-packed glands (Gleason
2). Gleason pattern 3 consists of well-formed, individual glands of varying sizes. Gleason
pattern 4 includes poorly-formed glands, fused into chords or chains. Gleason pattern 5 consists
of sheets of tumor, individual cells, and cords of cells with no glandular differentiation
(Gordetsky & Epstein, 2016).
The Gleason score is a combination of two tissue grades; the primary grade, and the secondary
grade. The primary grade is assigned to the dominant pattern of the tumor, whereas the
secondary grade is assigned to the next-most frequent pattern. The sum of the primary and
secondary grades results in the final Gleason score, which ranges from 6-10. Based on these
grading scores, tumors can be categorized into groups that show similar biologic behavior.
Scores of 6 (3+3) are now referred to as Gleason grade group (GGG) 1, Gleason scores 3+4 are
GGG II, Gleason score 4+3 is GGG III, Gleason score 4+4 is GGG IV, and Gleason scores of 9-
10 are GGG V. This new classification makes it easier for counselling patients on disease status
in comparison to the Gleason risk stratification groups (≤6, 7, 8–10) (Pierorazio, Walsh, Partin,
& Epstein, 2013).
Aside from Gleason score, tumor staging is another useful technique to predict the pathological
stage of prostate cancer. Currently, the TNM staging is the most widely used system for prostate
cancer staging. It aims to determine the extent of the primary tumor (T), the absence or presence
of regional lymph node (N) involvement, and the presence or absence of distant metastases (M).
Once the T, N, and M are determined, a stage of I, II, III, or IV can be assigned, with stage I
being early disease and stage IV being advanced disease (Cosma et al., 2016).
After diagnosis with prostate cancer, the National Comprehensive Cancer Network (NCCN),
recommends further classification of the cancer into one of four risk categories- very low risk,
low risk, intermediate risk, or high risk. As with staging and grading, several factors are taken
into consideration when determining risk category, including PSA level, size of the prostate,
biopsy results, and stage of disease. Very low risk prostate cancers include stage T1c, PSA less
6
than 10 ng/mL, and Gleason score 6 or less. Low risk cancers include stage T1c or T2a, PSA less
than 10 ng/mL, and Gleason score 6 or less. Patients classified as intermediate risk include stage
T2b-T2c, or PSA 10 to 20 ng/mL, or Gleason score 7. Lastly, prostate cancers classified as high
risk include stage T3a, PSA 20 ng/mL or higher, and Gleason score 8 or higher (Clinical,
Clinical, Guidelines, & Guidelines, 2009).
1.5 Treatment Strategies
Treatment choices for patients with prostate cancer depend on a combination of patient and
tumor characteristics. In the following section, various management and treatment modalities
will be discussed.
1.5.1 Active Surveillance
Many patients with prostate cancer are estimated to have a prolonged natural history of disease
which poses little threat during their lifetime. In such cases, patients are placed on active
surveillance in an effort to limit unnecessary treatments (Dall’Era et al., 2012). Under active
surveillance, patients with low-risk disease are carefully observed with repeated PSA
assessments, biopsies, and other tests to identify early signs of progression to more advanced
PCa. At the first signs of progression, these patients are placed on an appropriate treatment
regime within a timely window for opportunity in curing the disease (Cooperberg, Carroll, &
Klotz, 2011; Klotz, 2005). Active surveillance for prostate cancer offers an opportunity to delay
active treatment and its associated morbidities until evidence of clinical cancer progression is
discovered, which is never the case in a majority of low risk patients (Klotz et al., 2015).
1.5.2 Surgery
Treating patients with radical prostatectomy, in which the prostate gland and its surrounding
tissue is surgically excised, is a common primary treatment option for patients with intermediate
to high-risk prostate cancer. In a clinical trial comparing radical prostatectomy with observation
in the management of early prostate cancer, results show that radical prostatectomy reduced the
risk of death due to PCa by 50% and the risk of distant metastasis by 37% (Bill-Axelson et al.,
2005, 2011). The primary limitations of surgery include associated risk of incontinence and
erectile dysfunction amongst others, as well as positive surgical margins in patients with locally
advanced disease.
7
1.5.3 Androgen Deprivation Therapy
Androgens play a key role in prostate cancer development and progression. As such, targeting
the androgen receptor signalling pathway has been considered an effective strategy in PCa
management. Androgen deprivation therapy (ADT) is a treatment strategy wherein the
production or effects of testosterone and other male hormones are blocked. ADT can be achieved
by either surgical removal of both testicles or by medications which inhibit testosterone
production. Surgical castration is rarely used due to its permanent and irreversible nature,
combined with patients’ requests to avoid this procedure. The medications include anti-
androgens, which block ligand binding to the androgen receptor; luteinizing hormone releasing
hormone (LHRH) agonists or antagonists, which reduce luteinizing hormone secretion by the
pituitary, resulting in inhibition of gonadal synthesis of androgens; or new agents such as
Abiraterone, an inhibitor of Cyp 17, a crucial enzyme for androgen production. In addition to the
well-established role of ADT in treating patients with metastatic disease (Fizazi et al., 2012;
Scher et al., 2012), it is also used to treat patients with increasing PSA levels after local
treatment, and acts as an adjuvant therapy for men with localized disease undergoing radiation
therapy (Sharifi, Gulley, & Dahut, 2005).
1.5.4 Radiotherapy
Radiation therapy is a curative and effective therapy for localized prostate cancer. Although
there is a great deal of variability between risk group and treatment centers, a fairly large
percentage of men with localized PCa receive external beam radiation and brachytherapy
(Cooperberg, Broering, & Carroll, 2010). Radiation interacts with DNA in normal and malignant
cells, causing genetic damage and eventual cell death. With the development of highly conformal
techniques to limit the amount of normal tissues exposed to radiation, large cumulative doses can
be delivered in small daily fractions spread out over several weeks of treatment (Martin &
D’Amico, 2014). The conventional regime for prostate radiotherapy includes 70-80 Gy given in
2-2.5 Gy daily fractions. Alternative regimes have been used, including the delivery of fewer,
larger fractions, also known as hypofractionation. In addition to its economic and logistic
advantages, hypofractionation offers great tumor control and reduced normal tissue toxicities
(Stein, Boehmer, & Kuten, 2007). With brachytherapy, multiple radioactive seeds are implanted
into the tumor site under ultrasound guidance. This technique has also yielded favourable results,
8
with reports of ten-year disease-specific survival rates in favorable patients of greater than 95%
of treated patients (Stock, Cesaretti, & Stone, 2006).
1.5.5 Adjuvant and Neo-Adjuvant Therapies
Adjuvant therapy is often used after primary treatment, such as surgery, in order to reduce the
risk of cancer recurrence. Some of the main modalities used as adjuvant therapies include,
chemotherapy, hormone therapy, and radiation therapy (A V Bono, 2004).
Neo-adjuvant therapy is the administration of therapeutic agents after diagnosis but before
primary treatment with the goal of enhancing the effectiveness of local therapy, down staging
disease and eliminating micrometastasis. Neoadjuvant therapy not only provides the benefit of
assessing the efficacy of a particular treatment in the patient and gauging prognosis, but also
helps to increase the number of patients eligible for local therapy (Kent & Hussain, 2003). There
are several studies that have demonstrated the reduced incidence of positive disease margins with
the use of neo-adjuvant hormone therapy and surgery as treatment for PCa, with no reported
improvement in PSA recurrence rates (bNED survival) (Aldo V. Bono et al., 2001; Soloway et
al., 1995, 2002). Additionally, studies investigating neoadjuvant hormone therapy with
brachytherapy in locally advanced disease have reported improved biochemical outcomes in
high-risk patients (Merrick, Butler, Galbreath, Lief, & Adamovich, 2003).
1.5.6 Chemotherapy
Patients with advanced PCa initially respond to ADT in 90% of cases. However, a significant
proportion of patients develop castrate resistant prostate cancer (CRPC) over the course of time,
leading to the increased use of cytotoxic chemotherapy for management. CRPC is characterized
by a continuous rise in serum PSA levels, or other signs of PCa progression, despite castrate
testosterone levels (Saad & Hotte, 2010). According to the European Association for Urology
(EAU) guidelines on PCa, patients with asymptomatic or mildly asymptomatic metastatic disease
typically should be treated with enzalutamide or abiraterone plus prednisone, whereas patients
with aggressive metastatic CRPC should receive docetaxel in combination with prednisone, at
three week intervals (Heidenreich et al., 2014). Additionally, cabazitaxel is also considered for
its significant increases in overall survival in patients with docetaxel-resistant metastatic CRPC
(Hotte & Saad, 2010). Radium 223 is a bone seeking radioisotope for men with bone metastases
only offering a survival benefit when compared to best supportive care.
9
1.5.7 Chemopreventive and Novel Agents
Chemoprevention is the use of specific agents to hinder or delay the process of carcinogenesis
(described in prostate cancer pathophysiology), thus preventing the development of advanced
disease (Syed, Khan, Afaq, & Mukhtar, 2007). PCa is considered an ideal disease candidate for
chemoprevention given its extended duration of time for disease inception to clinical diagnosis,
combined with a relatively slow growth rate. Thus, minor delays in the development of PCa may
result in a substantial reduction in the incidence of clinically relevant disease.
Dietary agents, including lycopene, vitamins D, green tea polyphenols, and capsaicin have the
potential to be chemopreventive agents. Several other potentially interesting micronutrients have
failed in prospective phase 3 trials to demonstrate benefit, or have caused harm, despite pre-
clinical evidence supporting their use. These agents interact with a variety of oncogenic
pathways, including cell cycle processes, apoptosis, androgen metabolism, and oxidative stress
(Venkateswaran & Klotz, 2010). The benefits of selenium, for example, were demonstrated in
vitro and in vivo studies, showing activation of apoptosis and inhibition of PCa development (Hu
et al., 2006; S. O. Lee et al., 2006). The Nutrition Prevention of Cancer Trial (NPC) showed a
50% reduction in PCa incidence among men supplemented with selenium. However, the
Selenium and Vitamin E Cancer Prevention Trial (SELECT) demonstrated no benefit from
selenium and found an increase in the rates of diabetes in the selenium arm (Duffield-Lillico et
al., 2003; Lippman et al., 2009). Lycopene has also been shown to reduce the amount of
oxidative DNA damage in cell and animal studies (Matos, Capelozzi, Gomes, Mascio, &
Medeiros, 2001), along with phase II studies demonstrating significant decreases in PSA levels
over one year (Barber et al., 2006). An abundance of evidence has also depicted the role of
vitamin D in the inhibition of proliferation, invasion and PCa metastasis (Kubota et al., 1998;
Donna M Peehl, Krishnan, & Feldman, 2003). This also included epidemiological data
portraying decreases in PCa incidence and PSA levels in patients taking vitamin D
supplementation (Colli & Colli, 2006). Vitamin E is also a potent intracellular antioxidant,
recognized for its ability to inhibit lipid peroxidation and regulate the cell cycle through DNA
synthesis arrest (Israel, Sanders, & Kline, 1995). Vitamin E, which was evaluated in the
SELECT trial, was associated with an increased the risk of PCa development (Lippman et al
2009). In comparison, green tea, with its high polyphenolic content, has been shown to be an
effective chemopreventive agent for a variety of cancers. Studies have shown its potential to
10
inhibit cell growth, induce apoptosis and inhibit tumor growth, as well as PSA secretion (Vaqar
M Adhami, Ahmad, & Mukhtar, 2003; Vaqar Mustafa Adhami, Siddiqui, Ahmad, Gupta, &
Mukhtar, 2004). However, two small clinical trials which tested green tea in patients with high
grade PIN and advanced prostate cancer reported minimal clinical activity against PCa (Bettuzzi
et al., 2006; Brausi, Rizzi, & Bettuzzi, 2008; Choan et al., 2005). Capsaicin has been shown to
reduce proliferation and induce apoptosis in prostate cells by a mechanism involving reactive
oxygen species (ROS) generation and the dissipation of the mitochondrial membrane potential
(Mori et al., 2006; A. M. Sánchez, Sánchez, Malagarie-Cazenave, Olea, & Díaz-Laviada, 2006),
in addition to its radio-sensitizing properties through the inhibition of NFκB signalling (Venier,
Colquhoun, et al., 2015). Additionally, in vivo studies have demonstrated a significant reduction
in the metastatic burden of mice treated with capsaicin compared to control (Venier, Yamamoto,
et al., 2015). A prospective phase II trial (CAPSAICIN) has been launched to evaluate the
efficacy and safety of capsaicin treatment in men on active surveillance for localized prostate
cancer (ClinicalTrials.gov Identifier: NCT02037464).
1.6 Cannabinoids
Cannabis has been used for medicinal purposes, with its origin dating back more than 5000
years. In 1839, William O’Shaughnessy, a British physician and surgeon discovered the
analgesic, antiemetic, muscle relaxant and anticonvulsant properties of cannabis. These
observations quickly prompted an expanded medical use of cannabis (Ben Amar, 2006). In the
early 1990’s, the cannabinoid receptors and their endogenous ligands were discovered, leading to
a volume of research on the physiology and therapeutic benefit of cannabinoids (Díaz-Laviada,
2011).
Cannabinoids can be classified into three groups based on the source of their production;
phytocannabinoids, endogenous cannabinoids, and synthetic cannabinoids. Cannabinoids exert
their effects by binding to two G-protein coupled receptors: the cannabinoid receptor 1 (CB1),
identified by Devane et al in 1988 (Devane, Dysarz, Johnson, Melvin, & Howlett, 1988), and the
cannabinoid receptor 2 (CB2), discovered by Munro et al in 1993 (Munro, Thomas, & Abu-
Shaar, 1993).
The endogenous cannabinoids, their receptors and the enzymes responsible for their synthesis,
transport and degradation, make up the endocannabinoid system. This system is crucial for
11
neuromodulation, control of cardiovascular tone, energy metabolism, and immunity, thus making
it a promising target for the management of a variety of diseases (Roberto, Klotz, &
Venkateswaran, 2017).
1.6.1 Phytocannabinoids
Phytocannabinoids (pCBs) are lipid-soluble phytochemicals occurring naturally in the plant,
Cannabis sativa L, and include the main psychoactive constituents, delta-9-tetrahydrocannabinol
(THC) and cannabidiol (CBD). Other cannabinoids present in the plant include delta-8-
tetracannabinol (∆8-THC), cannabinol (CBN), cannabicyclol (CBL), cannabichromene (CBC),
and cannabigerol (CBG). However, these molecules are present in much smaller quantities and
possess no significant psychotropic effects as compared to THC (Ben Amar 2006). THC acts as a
partial agonist at the CB receptors, with most of its psychoactive effects being mediated by
activation of the CB1G-protein coupled receptor. CBD, on the other hand, is not a ligand for the
two CB receptors, but has shown cannabimimetic characteristics attributed to its antioxidant
properties, including inhibiting the degradation of the endogenous cannabinoid anandamide
(ElSohly & Slade, 2005). The therapeutic potential of the rest of the cannabinoids found in C.
Sativa is poorly explored.
The palliative effects of pCBs in the inhibition of nausea and emesis associated with
radiotherapy or chemotherapy, appetite stimulation, mood elation, pain relief, and insomnia in
cancer patients has been recognized for centuries (Pacher et al 2006). Several pCBs have been
reported to bind to and interact with CB receptors at high affinities, appearing as promising
candidates for drug development and cancer therapeutics (Patil, Goyal, Sharma, Patil, & Ojha,
2015). Research has suggested that CBD exerts some of its pharmacological activity through the
inhibition of fatty acid amide hydrolase (FAAH), which subsequently increases the levels of
endogenous cannabinoids. FAAH plays an important role in the endocannabinoid system and the
progression of prostate cancer. Data has shown elevated expression of the FAAH enzyme in
prostate tumor biopsies, corresponding to increasing Gleason grade and poor disease-specific
survival (Endsley et al., 2008).
Sativex, a pharmaceutical product composed of controlled amounts of THC and other plant-
derived cannabinoids, has been approved for its use in controlling nausea in cancer patients
undergoing chemotherapy and is also used as an appetite stimulant and in pain management in
12
cancer patients (Velasco, Sánchez, & Guzmán, 2012). This drug is currently available as an oral
mucosal spray with a one to one ratio of THC to CBD. Phase I and II clinical trials have recently
been launched for glioblastoma multiforme, whereby Sativex is used in combination with
temozolomide (a standard chemotherapeutic agent in the treatment of brain cancer). In part one
of this study, 6 patients with recurrent glioblastoma multiforme were treated with Sativex adjunct
to dose-intense temozolomide to assess safety of the combination. Part two involved 20 patients
receiving either an individualized dose of Sativex or placebo plus temozolomide. Results of this
two part safety and exploratory study were recently published in the Journal of Clinical
Oncology (Twelves, Short, & Wright Stephen, 2017), and has shown no Grade 3 or 4 toxicities
associated with use of the drug. Phase II results showed a median survival in the placebo group
of 369 days compared to 550 days in the Sativex treatment group, as well as a 1-year survival of
83% in the Sativex group compared to 56% in the placebo group. As the first study to examine
cannabinoids in combination with chemotherapy, this research highlights the lack of potential
side effects or toxicities associated with cannabinoid use, and their interactions with
chemotherapeutic agents, thus paving the way for their use in a variety of cancers.
1.6.2 Endocannabinoids
Endocannabinoids are compounds produced in our body that bind to CB receptors. They act as
neuromodulators, affecting the release of various neurotransmitters in the periphery and play a
vital role in inflammation and fat/energy metabolism (Christie & Vaughan, 2001). Traditional
compounds with cannabinoid activity include the following molecular characteristics; a phenolic
hydroxyl group, a lipophilic side chain and an appropriately oriented carbocyclic ring system
(Razdan, 1986). Several non-classical compounds have been discovered, which contain differing
chemical characteristics, but still bind to the CB receptors. These endocannabinoids are usually a
lipid derived from long chain polyunsaturated fatty acids bound to ethanolamine or glycerol. The
ethanolamine-bound fatty acids include anandamide (AEA), the earliest discovered
endocannabinoid, N-oleoylethanolamide (OEA), and N-palmitoylethanolamide (PEA). While
AEA has a high affinity for the classical cannabinoid receptors, OEA and PEA are structurally
similar, however mainly interact with the transient receptor potential cation channel vanilloid
type 1 (TRPV1) and the nuclear peroxisome proliferator-activated receptor (PPAR) (Alger &
Kim, 2011). These two ethanolamides potentiate the effect of endocannabinoids but bind poorly
to the cannabinoid receptors and are thus referred to as “cannabinoid-like substances”.
13
Cannabinoids can also bind to glycerol and include 2-arachidonoyl glycerol (2-AG) and 2-
arachidonoyl glycerol ether. These compounds are highly abundant throughout the central
nervous system, the gastrointestinal tract, the spleen, and the pancreas, binding with a high
affinity to the cannabinoid receptors (Hanus et al., 2001).
Several studies have depicted the role of endocannabinoids in cancer through inhibition of cell
proliferation both in vitro and in vivo (Izzo & Camilleri, 2009). In colon cancer, treatment with
endocannabinoids have resulted in the inhibition of colonic inflammation, with this effect
reversed by the deletion of CB receptors (Storr et al., 2008; D. Wang et al., 2008). This
dysregulation in cannabinoid receptors possibly suggests their involvement in the malignant
transformation of the colon. Despite limited clinical use of endocannabinoids due to rapid
metabolism, it remains useful to uncover the dynamics between the endocannabinoid system and
a variety of disease states (Roberto et al 2017).
Two of the best-studied endocannabinoids are AEA and 2-AG. 2-AG is prevalent at relatively
high levels in the nervous system and is stored in intracellular compartments. AEA, on the other
hand, is present at very low levels throughout the body due to high metabolic breakdown rates,
thus is produced on demand rather than stored intracellularly.
Endocannabinoids are typically produced biosynthetically from phospholipids. AEA is produced
through the transfer of arachidonic acid from phosphatidylcholine to the nitrogen atom of
phosphatidylethanolamine by the enzyme N-acyl transferase (NAT), which results in the
formation of N-arachidonoyl-phosphatidylethanolamine (NAPE). NAPE is then converted into
AEA in a one-step hydrolysis reaction (Figure 3). 2-AG is synthesized through the hydrolysis of
phosphatidylinositol-4,5-bisphosphate (PIP2) with arachidonic acid to form diacylglycerol
(DAG). Subsequently, DAG is hydrolyzed to 2-AG by the enzyme diacylglycerol lipase (DAGL)
(Figure 4). The endocannabinoids are metabolized through an enzyme catalyzed hydrolysis
reaction to arachidonic acid, by fatty acid amide hydrolase for AEA, and by monoacylglycerol
lipase (MAGL) for 2-AG.
Endocannabinoids can be enzymatically transformed by other enzymes, such as cyclooxygenase
(COX), lipoxygenase, epoxygenase, or hydroxylases, to generate derivatives including
prostaglandins, prostacyclins, thromboxanes, and leukotriene eicosanoids (Khanapure, Garvey,
Janero, & Letts, 2007). Cytochrome P450 4X1, which is found in the prostate, efficiently
14
metabolizes anandamide into a single monooxygenated product (Stark, Dostalek, & Guengerich,
2008).
Figure 3: The Biosynthesis of Anandamide. AEA biosynthesis is initiated by the formation of
N-arachidonoyl phosphatidylethanolamine (NAPE), which is formed by the transfer of
arachidonic acid from phosphatidylcholine to phosphatidylethanolamine by N-acyltransferase
(NAT). NAPE is converted to AEA through a hydrolysis reaction catalyzed by NAPE-specific
phospholipase D. Figure was adapted from (Bambang et al., 2010).
15
Figure 4: The Biosynthesis of 2-Arachidonoylglycerol. 2-AG is produced by the hydrolysis of
phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC) and subsequent cleavage of the
generated diacylglycerol (DAG) by diacylglycerol lipase. Figure was adapted from (Fonseca,
Costa, Almada, Correia-Da-Silva, & Teixeira, 2013).
16
Anandamide (cis-5,8,11,14-eicosatetraenoylethanolamide) was the first endogenous ligand of the
CB1 receptor to be discovered and isolated in 1992 (Devane et al., 1992). Its structure is an
ethanolamide with a tetraenic twenty-carbon fatty acid, as depicted in Figure 5. In earlier studies,
it appeared that AEA also exhibited some effects that were not mediated by the cannabinoid
receptors. To date, at least two different G-protein-coupled AEA receptors have been suggested
to exist in the brain and vascular endothelium. However, these receptors have yet to be
characterized (V. Di Marzo, De Petrocellis, Fezza, Ligresti, & Bisogno, 2002). The only
reasonably well characterized, non-cannabinoid site of action for AEA is the transient receptor
potential vanilloid receptor 1, which is a non-selective cation channel gated by capsaicin, protons
and heat.
Figure 5: The Chemical Structure of Anandamide. The structure of anandamide contains an
ethanolamide and a twenty-carbon fatty acid with four double bonds. Structure was obtained
from (Fonseca et al., 2013).
17
1.6.3 Synthetic Cannabinoids
Synthetic cannabinoids (SC) have been extensively used as research tools to gain insight into the
endogenous cannabinoid system and to assess therapeutic use. In vitro and in vivo studies have
shown that the analgesic, anti-inflammatory and anticancer growth effects of SCs are
approximately two to one hundred times more potent than their phytocannabinoid counterparts
(Castaneto et al., 2014). This is possibly due to their higher binding affinity to the cannabinoid
receptors in comparison to phytocannabinoids. Several studies focus on CP55940, a non-
classical cannabinoid, WIN 55,212-2, an aminoalkylindole, as well as JWH cannabinoids
(synthesized by the John W Huffman research group at Clemson University). This section will
discuss WIN 55,212-2 in more detail below.
CP55940 is an SC that mimics the effects of THC and is currently being used to study the
endocannabinoid system. It was created by Pfizer in 1974 and acts as a full agonist at both the
CB1 and CB2 receptors. Studies have demonstrated the effect of CP55940 on inducing apoptosis
in gastric cancer cells and inducing changes in the cells’ morphology (Ortega et al., 2016).
JWH cannabinoids such as JWH-007, JWH-015, JWH-018, and JWH-030 are from the
naphthoylindole family and act as selective CB receptor agonists (Romero-Sandoval & Eisenach,
2007). Each of these cannabinoids were created to explore the effect of differing binding
affinities for the cannabinoid receptors.
WIN 55,212-2 (WIN) also mimics the effects of THC; however, it has a different chemical
structure and a much higher affinity for the CB2 receptor compared to THC (see Figure 6). This
cannabinoid was synthesized by the Sterling Research Group in New York in the late 1980s
(D’mbra et al., 1992). WIN 55,212-2 is an aminoalkylindole; a member of one of the four
distinct classes of cannabinoid receptor agonists, including fatty acid derivative such as the
endocannabinoids anandamide and 2-AG, the classical cannabinoids such as THC, and the non-
classical cannabinoids such as CP55940. The structure of WIN contains a polar amine, a central
indole ring system, and a lipophilic naphthalene group. Early work with the cannabinoid WIN
55,212-2 showed that this SC produced the typical tetrad effects similar to those elicited by
THC, i.e. hypothermia, hypolocomotion, analgesia, and catalepsy, but does so at lower dosages,
18
indicating much greater potency (Compton, Gold, Ward, Balster, & Martin, 1992; Fan,
Compton, Ward, Melvin, & Martin, 1994).
To date, there is very little information about the pharmacological properties of synthetic
cannabinoids. Generally, they share similarities in liposolubility, nonpolarity, volatility, and
contain a side chain with a range of four to nine saturated carbons. Unlike phytocannabinoids,
SCs are stable in their active form and can be rapidly absorbed via inhalation.
Figure 6: The Chemical Structure of WIN 55,212-2. The structure of WIN contains a polar
amine, a central indole ring system, and a lipophilic naphthalene group. Structure was obtained
from (Ortega et al., 2015).
19
1.7 Molecular Targets of Cannabinoids
Many investigators have attempted to elucidate the molecular mechanisms through which
cannabinoids alter tumorigenesis. The following sections will explore proposed mechanisms that
have been brought to light, including those related to endoplasmic reticulum stress, oxidative
stress, Rho GTPase signalling, apoptosis and cell cycle regulation (summarized in Table 1, found
at the end of this section).
1.7.1 Endocannabinoid System
The endocannabinoid system is a biological system composed of cannabinoid receptors, which
are endocannabinoids expressed throughout the central and peripheral nervous systems, and their
endogenous ligands, the endocannabinoids.
The endocannabinoid system is involved in regulating a variety of physiological and cognitive
processes including appetite, energy metabolism, pain and inflammation, mood, memory,
learning, fertility, and in mediating the pharmacological effects of cannabis. The realization of
the complexity of the endocannabinoid system in physiological and pathological conditions has
led to the exploration of its association with conditions such as pain and inflammation,
immunological disorders, neurological diseases, obesity, cardiovascular disorders,
gastrointestinal conditions and cancer (Vincenzo Di Marzo, 2008).
Although the biosynthesis of endocannabinoids by the healthy human prostate tissue has not
been established, several studies have demonstrated the importance of the endocannabinoid
system in prostate function and physiology.
Cannabinoid receptors have been identified in human prostate and in biopsy samples from
patients with benign prostatic hyperplasia (BPH) and prostate cancer. In normal prostate tissue,
the CB1 receptor has been localized in two areas; the parasympathetic afferent nerves and the
acini epithelium. Activation of this receptor results in the inhibition of prostate contraction, as
well as the regulation of prostatic secretory activity (Gratzke et al., 2010; Tokanovic, Malone, &
Ventura, 2007).
The first evidence suggesting a dysregulation of the endocannabinoid system in prostate cancer
was reported in 2005, where it was shown that CB1 expression was higher in cancer-derived cell
20
lines compared to normal human prostate epithelial cells (Sarfaraz, Afaq, Adhami, & Mukhtar,
2005). Later it was shown that the expression of this receptor was correlated with degree of
malignancy in prostate cancer tissues, with higher expression of CB1 in the most aggressive
samples of PCa as compared to normal prostate tissue (Orellana-Serradell et al., 2015).
A study conducted on a total of 399 human prostate cancer samples demonstrated that the
expression levels of the CB1 receptor were significantly higher in patients with metastases at the
time of diagnosis and those with Gleason scores of 8-10 (Chung et al., 2009). This suggests that
high expression of CB1 in prostate tumor is associated with prostate cancer severity and poor
clinical outcome.
FAAH, the enzyme responsible for the metabolic breakdown of anandamide, is expressed in
normal prostate tissue and is upregulated after puberty (Dhanasekaran et al., 2005). This suggests
that androgens are involved in the regulation of its expression, and that the endocannabinoid
system may be important in the development and growth of the prostate. Additionally, AEA,
PEA, and OEA have all been detected in seminal fluid, where they regulate the fertilizing
potential of human sperm (Schuel et al., 2002).
In the human prostate cancer cell lines PC3, DU145, and LNCaP, endocannabinoids, including
2-AG, as well as several enzymes involved in the synthesis of cannabinoids, including NAPE-
specific phospholipase D (NAPE-PLD) are produced at high concentrations (Endsley et al.,
2007; J. Wang et al., 2008). Additionally, there is also evidence of upregulation of the
cannabinoid receptors CB1 and CB2 (M. G. Sánchez, Ruiz-Llorente, Sánchez, & Díaz-Laviada,
2003). Several receptor agonists, including WIN,55-212-2 and JWH015 have been shown to
inhibit prostate cancer cell growth, induce cell cycle arrest and increase apoptotic rates (Olea-
Herrero, Vara, Malagarie-Cazenave, & Díaz-Laviada, 2009; Sarfaraz et al., 2005). Studies
focusing on the CB2 receptor are of interest, as this receptor limits the psychotropic activity
associated with cannabinoid use and may be more suitable for pharmacological targeting in the
PCa context. The following section will discuss the more commonly known cannabinoid
receptors and briefly describe the non-conventional cannabinoid receptors.
21
1.7.2 Cannabinoids and Non-Cannabinoid Receptors
The CB1 and CB2 receptors are members of the G protein-coupled receptor (GPCR) family that
were identified almost 30 years ago. These receptors mediate the effects of the primary
psychoactive component of marijuana, THC, as well as the endogenous cannabinoids
anandamide and 2-AG, and numerous synthetic cannabinoids.
The CB1 receptor was initially characterized in rat brains by Devane and colleagues in 1988 and
was later cloned from the human testis. The CB1 receptor is mainly distributed throughout the
central nervous system, particularly the hippocampus, cerebral cortex, cerebellum and basal
ganglia (Galiegue et al., 1995). Expression of this receptor has also been found on peripheral
neurons and in non-neuronal tissues such as the adrenal glands, lungs, testis, ovary, uterus,
prostate and vascular tissues (Liu et al., 2000; Roger G. Pertwee, 1999). The CB1 receptor is
thought to modulate central functions including motor activity, learning and memory,
motivation, emotion, and energy homeostasis (R. G. Pertwee et al., 2010). The CB1 receptor
inhibits adenylyl cyclase and activates mitogen-activated protein kinase (MAPK) by signalling
through Gi/o proteins, although it may also be coupled to Gs proteins or ion channels. This
receptor can also mediate the activation of potassium currents and the inhibition of calcium
currents (Howlett, 2005).
The G-protein coupled cannabinoid receptor CB2 was cloned from rat spleen macrophages. It
was also identified in B cells and natural killer cells, spleen and tonsils, where CB2 modulates
immune cell migration and cytokine release (Galiegue et al 1995). Apart from the immune
system, CB2 receptor is also expressed in the male and female reproductive systems, the
gastrointestinal system, bone, and adipose tissue (Patel, Davison, Pittman, & Sharkey, 2010).
There is also some evidence that the CB2 receptor is expressed on some neurons, both within and
outside of the brain, where it modulates neurotransmitter release (Morgan, Stanford, &
Woodhall, 2009). As with CB1 receptors, CB2 couples to Gi/o proteins to inhibit adenylyl cyclase,
however, does not seem to be coupled to potassium or calcium ion channels. The expression of
the cannabinoid receptors throughout the body can be visualized in Figure 7 below.
Accumulating evidence has demonstrated that the cannabinergic effects of cannabinoids cannot
only be attributed to activation of CB1 and CB2. GPR55 was originally isolated in 1999 as an
22
orphan GPCR (Sawzdargo et al., 1999), belonging to group of the rhodopsin-like receptors,
and not sharing significant similarities in gene sequence to the CB1 or CB2 receptors, particularly
in the ligand binding site (McPartland, Matias, Di Marzo, & Glass, 2006). Studies have
demonstrated that several cannabinoids, including anandamide, 2-AG, THC, CBD, and the CB1
antagonist AM251, activate GPR55 and cause downstream effects such as ERK1/2
phosphorylation, calcium mobilization, and RhoA activation (Pertwee et al 2010). Additionally,
studies in mice models of colorectal cancer have shown that GPR55 and CB1 play differential
roles in colon carcinogenesis, where GPR55 acts as an oncogene, and CB1 acts as a tumor
suppressor (Hasenoehrl et al., 2017). Due to inconsistency in pharmacological data, the efficacy
of other cannabinoids at this receptor remains unknown. Hence, the classification of GPR55 as a
cannabinoid receptor remains indefinite. Several other orphan receptors including GPR23,
GPR18, GPR120, and GPR84 have also been discovered. However, their role in the
endocannabinoid system remains unknown. Thus, there is a need to test for their responsiveness
on a broad range of cannabinoids in order to help clarify their pharmacological profiles and
physiological roles within the endocannabinoid system.
There is further evidence to demonstrate that CB1 receptor antagonists, rimonabant and
taranabant, including the endocannabinoids AEA and 2-AG, can bind to some types of
adrenergic, dopamine, opioid, adenosine, melatonin, 5-hydroxytryptamine (5-HT), angiotensin,
tachykinin, and prostanoid receptors (Christopoulos & Wilson, 2001; T. M. Fong et al., 2009;
Lane, Beukers, Mulder-Krieger, & IJzerman, 2010) . Their potency at these receptors is
significantly less than the potency at which they bind to the CB1 and CB2 receptors.
Additionally, some of these reactions appear to be allosteric in nature, thus no convincing
evidence can implicate any of these receptors as novel cannabinoid receptors.
The transient receptor potential (TRP) family of cation channels includes six subfamilies:
canonical, vanilloid (TRPV), melastatin (TRPM), polycystin, mucolipin, and ankyrin (TRPA).
Each of these channels contain six-transmembrane domain integral membrane proteins. TRP
channels are involved in the transduction of a range of stimuli, including light, taste, electrical
charge, temperature, mechanical and osmotic stimuli (Venkatachalam & Montell, 2007). Five of
these types of TRP channels have been suggested to interact with cannabinoids: TRPV1,
TRPV2, TRPV4, TRPM8 and TRPA1.
23
TRPV1 was the first TRP channel to be cloned as a receptor for capsaicin, the component in hot
chili peppers responsible for their pungency (Caterina et al., 1997). This receptor is activated by
stimuli such as temperature, protons, and other natural toxins and colocalizes with the CB1 and
CB2 receptors, suggesting the potential for intracellular crosstalk (Vincenzo Di Marzo &
Cristino, 2008). It has been well established that endocannabinoids, including anandamide, N-
arachidonoyl dopamine, but not 2-AG, act as full agonists to both human and rat TRPV1
channels. Furthermore, phytocannabinoids that do not bind to the cannabinoid receptors and
synthetic CB1 and CB2 ligands, act as full agonists at TRPV1 receptors (Ligresti et al., 2006).
Thus, there is sufficient basis for the classification of TRPV1 as an “ionotropic cannabinoid
receptor”.
Aside from TRPV1, five other TRP channels have been discovered and cloned. TRPV2, -3, and -
4 are all involved in high-temperature sensing and nociception, whereas TRPV5 and -6 are
involved in calcium absorption and reabsorption. There is evidence to show that THC,
cannabinol and CP55940 interact with TRPV2 (Qin et al., 2008), and that anandamide and 2-AG
activate TRPV4 via the formation of cytochrome P450 metabolites of arachidonic acid
(Watanabe et al., 2003). There is insufficient evidence to deduce conclusions about whether or
not TRPV2 and TRPV4 can be considered cannabinoid receptors.
TRPM8 and TRPA1 are both involved in thermosensation, however belong to a different
subfamily than that of the capsaicin (TRPV1) receptor. It has been shown that anandamide, N-
arachidonoyl dopamine, and several nonpsychotropic phytocannabinoids can antagonize the
stimulatory effect of TRPM8 agonists (L. De Petrocellis et al., 2008; Luciano De Petrocellis et
al., 2007). Additionally, TRPA1 has been shown to be activated by phytocannabinoid CB1 and
CB2 agonists, THC, CBN, and the synthetic cannabinoid WIN 55,212-2 (Akopian, Ruparel,
Patwardhan, & Hargreaves, 2008; Jordt et al., 2004). In order to definitively classify TRPA1 and
TRPM8 as cannabinoid receptors, further research is warranted to investigate how these
receptors are able to mediate the pharmacological effects of cannabinoids.
24
Figure 7: Cannabinoid Receptor Localization. Cannabinoid receptor 1 is primarily located in
the brain, central nervous system, and many other parts of the body. The cannabinoid receptor 2
is found throughout the body on cells associated with the immune system. Figure was adapted
from (Sharma, Murumkar, Kanhed, Giridhar, & Yadav, 2014).
25
1.7.3 Endoplasmic Reticulum Stress Response
The endoplasmic reticulum (ER) is an organelle responsible for the synthesis, folding
and modification of secreted, membrane-bound and organelle-targeted proteins. In order to
achieve optimum protein folding, several factors are required, including intraluminal calcium
concentrations, ATP availability and an oxidizing environment for disulphide-bond formation. A
range of physiological and pathological conditions such as exposure to anticancer agents,
calcium depletion, and viral infections, may lead to an imbalance between ER protein folding
load and capacity. This causes ER stress, which is an accumulation and aggregation of unfolded
proteins in the ER lumen (Verfaillie, Salazar, Velasco, & Agostinis, 2010). Cells have evolved
strategies to protect against the deleterious effects of ER stress, in which protein translation and
genetic transcription are temporarily halted. This strategy is commonly referred to as the
unfolded protein response (UPR). The UPR is considered a pro-survival response initiated to
reduce the accumulation of unfolded proteins, thereby restoring normal ER functioning
(Schröder & Kaufman, 2005). However, if this transcriptional program fails to re-establish,
persistent ER stress can cause a switch to a pro-apoptotic response.
Recent literature has suggested that cannabinoids exert their anticancer effects through
activation of apoptosis. It is postulated that the production of ceramide may induce ER stress and
initiate apoptosis. The inability to return to ER homeostasis may result in cell death by a
mechanism involving mammalian target of rapamycin (mTOR) pathway inhibition, and
subsequently, autophagy. Studies have demonstrated that activation of the CB2 receptor by the
synthetic cannabinoid JWH-015 induces synthesis of ceramide in PC3 cells, inhibiting the Akt-
mTOR pathway and activating initiation factors involved in autophagy regulation and the ER
stress response (Olea-Herrero et al., 2009). The authors showed that this effect was dependent on
CB2 activation, as combined treatment with CB2 antagonist SR144528 resulted in the prevention
of cell death and a decrease in the synthesis of intracellular ceramide. Additionally, studies have
shown that THC induces ceramide accumulation, activating an ER stress response that promotes
autophagy through tribbles homolog 3 dependent (TRB3-dependent) inhibition of the mTORC1
axis in human and mouse cancer cells (Salazar et al., 2009). An increase in ceramide levels and
ER stress may trigger activation of the caspase cascade leading to apoptosis. The proposed
pathway through which this may occur is depicted below in Figure 8. This idea was
demonstrated in a study where primary cultures of prostate cancer cells treated with the
26
endocannabinoids 2-AG, AEA and methanandamide (MET) showed increased levels of active
caspase-3, and decreased expression of Bcl-2 and Akt (Orellana-Serradell et al., 2015). This
study suggests that the inhibition in Akt may contribute to the activation of anti-proliferative
pathways, and unlike Olea-Herrero et al., these effects were CB1 dependent, as combination
treatment with the CB1 antagonist SR141716 prevented apoptosis in these cells. Additionally,
studies by Carracedo and colleagues (Carracedo et al., 2006) have shown that THC upregulates
the stress-regulated protein p8, which then mediates its apoptotic effect via the upregulation of
ER stress related genes, including ATF-4, CHOP, and TRB3. This pathway activation is limited
to tumor cells, and does not become activated in nontransformed cells, supporting the notion
previously described by Guzman (Guzman, 2003).
Future studies are warranted to clarify the role of the cannabinoid receptors in the activation of
ER stress related pathways and to elucidate the link between CB receptors and downstream
targets in the ER stress response. Identification of the pathways involved will help to clarify the
molecular events that lead to activation of ER-stress mediated cell death by cannabinoids and
may contribute to the design of novel therapeutic strategies for inhibiting tumor growth and
progression.
27
Figure 8: Proposed Endoplasmic Reticulum Stress Signalling Pathway. Cannabinoid binding
to cannabinoid receptors results in the accumulation of ceramide and alters the expression of ER
stress related proteins downstream, inducing apoptosis.
28
1.7.4 Oxidative Stress
The term reactive oxygen species (ROS) is used to describe a number of reactive molecules and
free radicals derived from molecular oxygen. These molecules, produced as byproducts during
the mitochondrial electron transport of aerobic respiration, are generated during every day
metabolic processes in normal cells and play a vital role in cell signalling, including apoptosis,
gene expression, and the activation of cell signalling cascades. They have the potential to cause a
number of deleterious effects. Excessive production of ROS or an inadequate antioxidant defense
system may lead to a phenomenon known as oxidative stress, which has been associated with the
initiation and development of a variety of cancers, including prostate cancer (Khandrika, Kumar,
Koul, Maroni, & Koul, 2009). Oxidative free radicals caused by modulation of androgens,
vitamin D, inflammation, tumor suppressor protein 53 (p53), and antioxidants may initiate
prostate cancer. Specifically, in men with prostate cancer, serum androgens may promote ROS
production and its accumulation in prostate cancer cells. (Minelli, Bellezza, Conte, & Culig,
2009). Supporting evidence has suggested that increasing ROS production in prostate cancer
cells are associated with aggressive phenotype, thus, targeting ROS production might offer a
potential approach in preventing cancer development (Kumar, Koul, Khandrika, Meacham, &
Koul, 2008).
Paradoxically, oxidative stress occurring at the intracellular level can have chemopreventive
effects and thus oxidative stress induction may be used as an anticancer strategy triggering
apoptosis in malignant cells. Various studies have reported that chemopreventive agents work in
some part by generating ROS and disrupting redox homeostasis (Ling, Liebes, Zou, & Perez-
Soler, 2003; Sikka, 2003). Studies have reported that ROS may act as secondary messengers
influencing mitochondrial function, mediating the elevation of intracellular calcium, and thus
activating the caspase cascade. In addition, ROS production may induce pro-apoptotic signals
leading to the release of proteins from the mitochondrial intermembrane space into the cytosol,
thereby promoting apoptosis (Paradies, Petrosillo, Pistolese, & Ruggiero, 2002).
Studies have shown that cannabinoids may induce apoptosis in cancer cells through the
production of ROS (Figure 9). It was reported that intracellular calcium levels were elevated and
ROS production was activated in LNCaP cells after treatment with CBD (De Petrocellis et al
2013). This suggests that both oxidative stress and ER stress are contributing factors in the pro-
29
apoptotic effect of CBD. These results were also seen in non-AR expressing cells, DU145 and
PC3, indicating that CBD increases oxidative stress and ER stress independent of p53 status or
androgen receptor status. It is speculated that ROS is necessary for the increase in the AMP/ATP
ratio, which mediates the activation of AMPK by cannabinoids, leading to cell death. Studies
have proposed that ROS production by cannabinoids activates a positive feedback loop. Here,
electron transport chain inhibition leads to NADH accumulation and the subsequent inhibition of
oxidative phosphorylation, amplifying the production of ROS (Dando et al., 2013). However, it
is important to note that differing non-THC cannabinoids might produce different effects on the
cell cycle and apoptosis, so an all-encompassing, definitive mechanism may not be possible to
describe.
Cannabinoid-mediated ROS production may also trigger the release of pro-apoptotic proteins
such as cytochrome c, caspase-9, apoptosis inducing factor, and Smac/DIABLO from the inner
mitochondrial membrane space into the cytosol, leading to the activation of apoptosis.
Researchers showed that the endocannabinoid, AEA induced cell death through a pathway
involving mitochondrial uncoupling and cytochrome c release, potentially mediated by oxidative
stress and ROS production through activation of TRP (vanilloid) receptors (Maccarrone,
Lorenzon, Bari, Melino, & Finazzi-Agro, 2000). Similarly, it was reported that CBD exposure to
human glioma cells resulted in an induction of ROS production with a time course preceding
caspase-8 and -9 activations (Massi et al., 2006). This time course suggests that the concomitant
activation of both caspase 8 and 9 is the cause, rather than consequence, of caspase 3 activation,
and that both intrinsic and extrinsic pathways of apoptosis are involved in CBD-related death.
How cannabinoids induce ROS accumulation remains controversial. It has been suggested that
CBD is able to induce apoptosis in a cannabinoid and vanilloid receptor-independent
mechanism, through intercalating into the cell membrane, or by possibly binding to an
unidentified cannabinoid receptor (Roger G. Pertwee, Thomas, Stevenson, Maor, & Mechoulam,
2005). Moreover, due to CBD’s ability to act as a potent modulator of intracellular calcium,
researchers have suggested a role of calcium in driving some aspects of tumor cell death
(Drysdale, Ryan, Pertwee, & Platt, 2006). Another working theory is that CBD signalling may be
mediated by a membrane lipid raft domain, causing apoptosis and triggering a complete caspase
cascade (Sarker & Maruyama, 2003).
30
Despite increasing evidence associating cannabinoid treatment to increased ROS production and
oxidative stress, the conflicting findings regarding the benefit and/or harm of ROS production
cannot be ignored. Thus, an in-depth analysis of these pathways involving oxidative stress and
ROS production is warranted to develop a deeper understanding of the future use of
cannabinoids as anticancer treatment modalities.
31
Figure 9: Proposed Oxidative Stress Signalling Pathway. Upon binding to and activating the
cannabinoid receptors, cannabinoids increase ROS production, which induces a decrease in the
mitochondria membrane potential, releasing cytochrome C from the mitochondria into the
cytosol, and results in the activation of the caspase cascade.
32
1.7.5 Rho GTPase Signalling
Cell migration is an integral process that controls inflammation, wound healing, embryogenesis
and morphogenesis. It is a highly complex process, involving numerous compartments of the
cell, including signalling elements, surface receptors, and the cytoskeleton. Cell migration plays
an essential role in the delivery of protective immune responses to tissues, and aberrant cell
migration is associated with many disease states, including autoimmune syndromes,
developmental defects, chronic inflammation, and cancer invasion and metastasis (KS, 2010;
Wells & Parsons, 2011). Under pathological conditions such as tumor invasion and metastasis,
cells become detached from the primary tumor and enzymatically degrade the extracellular
matrix or basement membrane of tissues to become established in a new location.
A variety of intracellular signalling molecules have been implicated in cell migration and
invasion, including phospholipases, Tyr kinases, lipid kinases, Ser/Thr, and MAPK cascades. Of
these, the protein family most pivotal to the regulation of cell migration and invasion is the Rho
GTPases. The most well studied and highly conserved Rho GTPases include Rho, Rac, and
Cdc42.
Rho family GTPases play key roles in coordinating the cellular responses required for cell
migration. They regulate cell migration through the assembly of actin/myosin filaments, cell
adhesion and spreading, and the establishment of cell polarity (Lambrechts, Van Troys, & Ampe,
2004; Anne J. Ridley, 2015). There exist molecular crosslinks between Rho family proteins and
the actin cytoskeleton, where they act to regulate actin polymerization, depolymerization, and the
activity of actin-associated myosins. Additionally, Rho proteins affect the organization of the
microtubule and intermediate filament networks important for cell migration (A J Ridley, 2001).
Critical downstream components in Rho-GTPase signalling and actin binding proteins have been
linked to metastasis in vivo. In prostate carcinoma cells, activity of RhoA is amplified and
corresponds to an increase in cell migration and invasion. The amplification in RhoA is induced
by the stimulation of multiple G protein coupled receptors for thrombin and thromboxane A2
(Nie et al., 2008; Somlyo et al., 2000).
33
Stemming from this, other studies have explored the ability of cannabinoid receptor activation to
repress RhoA activity, thereby providing a novel mechanism to diminish migration and invasion
of aggressive prostate carcinoma cells (Figure 10). It was reported that activation of CB1 with
endogenous agonists AEA and 2-AG resulted in the suppression of RhoA activity in PCa cells,
contributing to the suppression of cell migration. This loss of RhoA activity was accompanied by
the loss of actin/myosin microfilaments, reduced cell migration, and decreased cell adhesion
(Nithipatikom et al., 2012). Similarly, studies on highly aggressive breast cancer cells MDA-
MB-231 reported a CB1 mediated inhibition in GTPase activity of RhoA (Laezza, Pisanti,
Malfitano, & Bifulco, 2008; Pillé et al., 2005). These results would suggest that the inhibition of
RhoA by cannabinoids mitigate Rho’s ability to promote invasion by causing a disruption in
RhoA membrane localization, necessary for its interaction with several signalling components.
Several other studies have shown a CB1 dependent inhibition of adenylyl cyclase and protein
kinase A, resulting in a reduction of RhoA activity, and subsequent decreases in prostate and
breast cancer cell invasion (Nithipatikom et al., 2004; Takeda et al., 2012).
A deeper understanding of signalling events that cause CB receptor dependent alterations in Rho
GTPase activity is warranted, despite conclusive evidence regarding CB receptor mediated
reductions in RhoA activity. This knowledge will help to elucidate how RhoA is targeted by
cannabinoid receptor stimulation and whether this pathway is responsible for cannabinoid
induced inhibition of cell migration and invasion.
34
Figure 10: Proposed RhoA GTPase Signalling Pathway. Cannabinoids binding to the
cannabinoid receptor result in the inhibition of RhoA activity, which causes a loss of actin and
myosin microfilaments, resulting in a reduction in cell migration.
35
1.7.6 Apoptosis
Apoptosis or programmed cell death is a key regulator of physiological growth and tissue
homeostasis. Most anticancer strategies currently utilized in clinical oncology involve the
activation of apoptosis signal transduction pathways. Thus, understanding the molecular
mechanisms that regulate apoptosis in response to anticancer treatment is crucial to the
development of a more rational approach to drug therapies.
The mechanisms of apoptosis are highly complex and involve an energy-dependent cascade of
molecular events. Research indicates that there are two main apoptotic pathways: the intrinsic
(mitochondrial) pathway, and the extrinsic (death receptor) pathway. Both these pathways
converge onto the same end goal; cleavage of caspase 3, formation of apoptotic bodies, DNA
fragmentation, and lastly, the uptake by phagocytic cells (Elmore, 2007).
The intrinsic mechanism of apoptosis is characterized by a diverse array of non-receptor
mediated stimuli which produce intracellular signals that act directly on targets within the cell.
This mechanism involves mitochondria-initiated events, ROS and excess intracellular calcium.
Stimuli including the absence of certain growth factors or hormones, or the presence of toxins,
free radicals, or viral infections cause changes in the inner mitochondrial membrane, which
results in the loss of the mitochondrial transmembrane potential and the release of cytochrome c
from the intermembrane space into the cytosol (Saelens et al., 2004). This initiates the caspase-
dependent mitochondrial pathway, ultimately leading to apoptosis.
The majority of studies examining the pro-apoptotic effects of cannabinoids on prostate and
other malignant cell lines have reported apoptosis linked to intrinsic mechanisms due to an
increase in ROS. It has been demonstrated that the interaction of cannabinoids with TRPV
receptors causes the activation of overlapping mechanisms, including the mitochondrial
apoptotic pathway, accompanied by an increase in the level of ROS with consequent oxidative
stress. This was shown in a study, where CBD increased the generation of ROS and reduced
mitochondrial membrane potential, released cytochrome c to the cytosol and ultimately led to the
activation of the intrinsic apoptotic pathway in breast cancer cells (Shrivastava, Kuzontkoski,
Groopman, & Prasad, 2011). Similarly, in various experimental tumor models, high ROS levels
induced ER stress (as demonstrated by increases in ER-stress mediators such as p8, CHOP, and
TRIB3), which in turn, led to the activation of mitochondrial intrinsic apoptosis (Malhotra &
36
Kaufman, 2007). In PC3 prostate cancer cells, the synthetic cannabinoid JWH-015 induced
cytochrome c release into the cytosol, and activated caspase 9, confirming the involvement of
apoptosis, and pointing towards an activation of the intrinsic apoptotic pathway. JWH-015 also
induced an increase in ceramide, leading to ER stress, and activation of autophagy, suggesting
this pathway may also be involved in cannabinoid-induced prostate cell death (Olea-Herrero et
al., 2009).
The extrinsic mechanism of apoptosis is characterized by transmembrane receptor-mediated
interactions. These involve death receptors, which are members of the tumor necrosis factor
(TNF) receptor gene superfamily. To date, the best characterized death receptors include
TNFR1, TRAIL, and DR (Locksley, Killeen, & Lenardo, 2001). These receptors can be activated
upon contact or insult by death signalling molecules, leading to downstream caspase-mediated
apoptosis (Khan, Blanco-Codesido, & Molife, 2014).
In the past decade, only a few studies have reported apoptosis by cannabinoids through extrinsic
mechanisms. In human leukemia cells, THC lead to caspase 8 activation, an event typically
associated with the extrinsic apoptotic pathway (Herrera et al., 2006). In immune cells, it was
demonstrated that treatment with the synthetic cannabinoid JWH-015 resulted in extrinsic
apoptosis activation, as demonstrated by caspase 8 activation (Lombard, Nagarkatti, &
Nagarkatti, 2007). This study suggested a possible cross-talk between the two pathways of
apoptosis, i.e. extrinsic and intrinsic, whereby the caspase 8 inhibitor could almost completely
block JWH-015 induced apoptosis, suggesting that the extrinsic pathway of apoptosis plays a
crucial role in cannabinoid-induced apoptosis. However, the precise death receptor/ligands
involved remains unclear. In hepatocellular carcinoma cells, WIN 55,212-2 treatment sensitized
cells to TRAIL- induced apoptosis, mediated by ER stress proteins p8 and CHOP and Death
Receptor 5 (DR5). The upregulation of TRAIL death receptor DR5 contributed to the
amplification of the caspase cascade, promoting extrinsic apoptosis (Pellerito et al., 2010). To
date, there have been no reports on extrinsic mediated apoptosis in prostate cancer cells.
37
1.7.7 Cell- Cycle Regulation
Regulation of the cell cycle involves processes that are crucial to the survival of a cell, including
the detection and repair of genetic damage and the prevention of uncontrolled cell division. Loss
of this regulatory cell-cycle control is a hallmark of neoplastic cells.
Common cell cycle abnormalities in PCa involve the RB1 pathway (Figure 11), wherein cyclin
dependent kinase inhibitor, p27 is activated and binds to cyclin D, inhibiting the catalytic activity
of Cdk4. This prevents Cdk4 from adding phosphate residues to the retinoblastoma protein, thus,
preventing pRb from releasing the transcription factor E2F1. In order for progression of a cell
through G1 and S phase, Rb inactivation by phosphorylation is necessary. However, once
phosphorylation of this tumor suppressor gene is inhibited, Rb can directly bind to E2F1 and
actively repress transcription, preventing the progression of cells from G1 into S phase (Harbour
& Dean, 2000).
In addition, several metalloproteinases (MMPs) contain E2F binding sites, thus inhibition of
E2F1 activity may also be linked to the impairment of migration and invasion through the
transcriptional inactivation of MMPs, although this exact mechanism remains unclear and was
not explored in this thesis (Johnson et al., 2012; Z. Wang et al., 2017).
Cannabinoids have been shown to cause alterations in cell cycle distribution or cell cycle arrest
in various cancer cell lines. Anandamide was shown to arrest the proliferation of MDA-MB-231
human breast cancer cells in the S phase of the cell cycle (Laezza, Simona Pisanti, Crescenzi, &
Bifulco, 2006). Additionally, the phytocannabinoid THC inhibited breast cancer cell
proliferation by arresting the progression of cells from the G2 to M phase in a CB2 receptor-
dependent manner (Caffarel, Sarrió, Palacios, Guzmán, & Sánchez, 2006). THC administration
was also shown to elicit G0/G1 cell cycle arrest in glioblastoma cells through suppression of
E2F1 (Galanti et al., 2008). In AGS and MKN-1 human gastric cancer cells, WIN 55,212-2
caused cells to arrest in the G0/G1 phase through the mechanism described in Figure 11 (Park et
al., 2011).
Studies within the field of prostate cancer have demonstrated that WIN 55,212-2 causes a dose-
dependent accumulation of LNCaP human prostate cancer cells in the G0/G1 phase of the cell
cycle through an induction of p27 and a dose-dependent decrease in pRb and E2F1 levels
38
(Sarfaraz, Afaq, Adhami, Malik, & Mukhtar, 2006). In DU145 cells, treatment with CBD
resulted in the inhibition of G1/S phase transition of the cell cycle, however no changes in G1/S
phase transition were observed in LNCaP cells (Luciano De Petrocellis et al., 2013).
Figure 11: Proposed Cell Cycle Regulation Pathway. Cannabinoids act to inhibit cell cycle
progression in the G0/G1 phase. Upregulation of p27 inhibits Cdk4, thus inhibiting
retinoblastoma phosphorylation and E2F1 activation. Inhibition of E2F1 may also lead to
reductions in migration and invasion through inhibition of MMPs, although the exact mechanism
remains unclear.
39
Table 1: Effects of cannabinoids on cell viability, migration, and invasion in prostate and
various cancers.
Cell Lines Cannabinoid Anticancer Effect Mechanism of Action References
PC3
prostate
cancer cells
WIN55212-2
Decrease in cell
motility
Activation of CB1 results
in repression of RhoA
activity (suppression of
cell migration)
Nithipaticom
et al 2012
PC3,
DU145,
LNCaP
prostate
cancer cells
JWH-015
MET
Decrease in cell
viability
IN VIVO:
Reduction in tumor
growth
CB2 activation by JWH-
015 inhibits Akt-mTOR
pathway and activates
eIF2α (induction of ER
stress- proapoptotic
effect)
Olea-Herrero
et al 2009
PC3,
DU145
prostate
cancer cells
2AG Inhibition of invasion 2AG activates CB1
receptor, inhibits
adenylyl cyclase and
decreases activity of
PKA (inhibition of
invasion)
Nithipaticom
et al 2004
PC3, and
primary
cultures of
prostate
cancer and
benign
prostatic
AEA, 2-AG,
MET
Decrease in viability
Increase in apoptosis
Activation of CB1
receptor results in
activation of apoptotic
pathway without
modification in cell cycle
or necrosis
Orellana-
Serradell et al
2015
40
hyperplasia
tissue Endocannabinoids
modulate AKT and ERK
pathways
LNCaP
prostate
cancer cells
WIN, CBD Inhibition of
proliferation
WIN and CBD activate
PARP cleavage and
induce apoptosis
WIN effects are CB
receptor independent and
CBD effects are CB
receptor dependent
Sreevalsan et
al 2011
DU145 and
LNCaP
prostate
cancer cells
CBD Inhibition of viability CBD induces ER stress
and production of ROS
DePetrocellis
et al 2013
LNCaP
prostate
cancer cells
WIN Inhibition of
neuroendocrine
differentiation
Inhibition of PI3K/Akt
pathway results in
activation of mTOR and
inhibition of AMPK
Role of CB2 implicated
but not fully elucidated
Morell et al
2016
LNCaP
prostate
cancer cells
WIN G0/G1 phase cell
cycle arrest
Upregulation of p27,
inactivation of pRb and
E2F1
Safaraz et al
2006
41
DU145
prostate
cancer cells
CBD G1/S phase cell cycle
arrest
Increased expression of
p27 and p21
DePetrocellis
et al 2013
Primary
cultures of
brain tumor
cells;
cortical
astrocytes
THC Induction of apoptosis
IN VIVO:
Reduction in tumor
growth and increased
apoptosis
Ceramide-dependent
upregulation of stress
protein p8 and several
downstream targets
(ATF-4, CHOP, TRB3)
related with ER stress
proapoptotic pathway
IN VIVO:
Upregulation of p8 in
tumors
Carracedo et
al 2006
Primary
cultures of
brain tumor
cells;
cortical
astrocytes
THC Induction of
autophagy; increased
apoptosis
IN VIVO:
Reduction in tumor
growth by 50%
Upregulation of the p8-
TRB3 pathway leads to
ceramide synthesis and
eIF2 phosphorylation,
promoting autophagy.
Inhibition of the
Akt/mTORC1 axis
IN VIVO:
Increases in TRB3
expression; increases in
LC3 and LC3-II, and
increases in active
caspase 3
Salazar et al
2009
42
U87 human
glioma
cells; Glial
primary
cultures
CBD Induction of apoptosis Induction of oxidative
stress (through early
production of ROS),
depletion of glutathione,
concomitant activation of
caspase 8 and 9, and the
cleavage of caspase 3
Massi et al
2006
Human
neuroblasto
ma
CHP100
and
lymphoma
U937 cells
AEA Induction of apoptosis Increases in intracellular
calcium, mitochondrial
uncoupling, and
cytochrome c release,
leading to apoptosis
Maccarrone
et al 2000
MDA-MB-
231 human
breast
cancer cells
CBDA, CBD Inhibition of cell
migration
PKA inhibited, which in
turn leads to decreased
levels of phosphorylated
RhoA.
Laezza et al
2005
Human
breast
carcinoma
cell line
MDA-MB-
231
Met-F-AEA Inhibition of cell
migration
Inhibition in the GTPase
activity of RhoA, which
induced a delocalization
of RhoA from cell
membrane to the
cytoplasm, leading to the
disruption of skeleton
actin stress fibers.
RHOA/ROCK signalling
could be involved in the
maintenance of actin
Pille et al
2005
43
organization and
induction of migration in
the MDA-MB-231 cells.
Human
leukemia
Jurkat cells
THC Induction of apoptosis Mitochondrial intrinsic
pathway activation.
Activation of caspase 8
leading to induction of
apoptosis.
Stimulation of the
extracellular signal-
regulated kinase, c-Jun
N-terminal kinase and
p38 mitogen-activated
protein kinase.
Herrera et al
2006
Human
hepatocellu
lar
carcinoma
cells
HepG2
WIN Induction of apoptosis Early activation of p8
and CHOP cause up-
regulation of TRAIL
receptor DR5, sensitizing
the cells to TRAIL.
Increases in PPAR leads
to down-regulation of
survival factors (pAkt,
Bcl-2, Survivin)
contributing to cell death.
Pellerito et al
2010
C57BL/6
Mice T and
B cells
JWH-015 Induction of apoptosis Activation of both
extrinsic and intrinsic
apoptotic pathways.
Lombard et al
2007
44
JWH-015 induced a loss
of inner mitochondrial
membrane potential and
lead to activation of
caspase 8, 9, and 3.
MDA-MB-
231 human
breast
cancer cells
CBD Induction of apoptosis
and autophagy
Induction of ER stress,
followed by LC3-II
accumulation.
Generation of ROS.
Inhibition of
AKT/mTOR signalling.
Activation of caspase-8,
the generation and
translocation of t-BID to
the mitochondria, the
release of cytochrome c
and SMAC into the
cytosol, and increased
levels of Fas-L
(mitochondria-mediated
apoptosis).
Shrivastava et
al 2011
MDA-MB-
231 human
breast
cancer cells
AEA Arrest in S phase of
cell cycle
Loss of Cdk2 activity,
upregulation of p21
Laezza et al
2006
45
Human
breast
cancer cells
THC Blocking progression
in the G2/M phase of
the cell cycle
Downregulation of Cdc2
via CB2 receptor
Caffarel et al
2006
Glioblasto
ma cells
THC G0/G1 cell cycle
arrest
Suppression of E2F1 Galanti et al
2008
AGS and
MKN-1
human
gastric
cancer cells
WIN Cell cycle arrest in
G0/G1 phase
Induction of p27,
decreased Cdk4,
decreased pRb and E2F1
expression
Park et al
2011
Abbreviations: CB: Cannabinoid Receptor; MET: Methanandamide; eIF2: Eukaryotic
Initiation Factor 2 Alpha; mTOR: Mammalian Target of Rapamycin; ER: Endoplasmic
Reticulum; 2AG: 2-arachidonoyl glycerol; PKA: Protein Kinase A; AEA: Anandamide; ERK:
Extracellular Signal-Regulated Kinases; WIN: WIN 55,212-2; CBD: Cannabidiol; PARP: Poly
(ADP-ribose) Polymerase; ROS: Reactive Oxygen Species; AMPK: 5' Adenosine
Monophosphate- Activated Protein Kinase; pRb: phospho- Retinoblastoma Protein; THC:
Tetrahydrocannabinol; ATF-4: Activating Transcription Factor 4; TRB3: Tribbles Homolog 3;
LC3: Light Chain 3; CBDA: Cannabidiolic Acid; Met-F-AEA: 2-Methyl-2'-F-Anandamide;
TRAIL: TNF-Related Apoptosis-Inducing Ligand; DR5: Death Receptor 5; Bcl-2: B Cell
Lymphoma 2; SMAC: Second Mitochondria-Derived Activator of Caspase
46
1.8 Preclinical Models of Prostate Cancer
Valid experimental models of prostate cancer that accurately reflect disease progression are
crucial in ensuring a proper experimental design and increasing our collective understanding of
the biology of the disease. Recently, more suitable models derived from human specimens have
been developed and are available for use. Although it is unlikely that a single model system
holistically reflects the process of prostate cancer development and progression, the use of these
models permits the study of significant aspects of cancer progression and they are essential tools
for the development of new therapies.
1.8.1 In Vitro Models
The most commonly used in vitro model available in prostate cancer research is cell culture.
Primary and immortalized cell lines are the two main types of cell systems predominately used in
vitro.
Primary cell lines are isolated and cultured from primary tumors and normal prostate tissue and
can be passaged a limited number of times. The use of primary cultures has become more
widespread, as cultures of normal prostate epithelium can be compared to established prostate
cancer cell lines to identify cancer-specific traits (D. M. Peehl, 2005).
Immortalized cell lines, commonly mentioned throughout this thesis, have been continually
passaged over long periods of time and have acquired the ability to proliferate indefinitely. The
first human prostatic tumor epithelial cell lines to become established were the Lymph Node
Carcinoma of the Prostate Cell Line (LNCaP), PC3, and DU145, which remain the most
commonly used PCa cell lines in research (Sampson, Neuwirt, Puhr, Klocker, & Eder, 2013).
While LNCaP cells were derived from a supraclavicular lymph node of a patient whose prostate
cancer was exhibiting androgen independent growth. LNCaP behave like androgen-dependent
cells. PC3 cells were derived from bone metastases of a patient with castration-resistant prostate
cancer, and DU145 cells were derived from a brain metastasis of an untreated prostate cancer.
Out of the three cell lines, LNCaP cells express significant levels of androgen receptor (AR) and
produces PSA, whereas DU145 and PC3 cells are considered androgen receptor negative and do
not produce PSA. For several years, LNCaP cells were the only ones available for the study of
AR signalling in vitro. However, several additional AR positive cell lines have now been
47
established and are used to investigate the mechanisms underlying castration resistance (Navone
et al., 1997). The table below was adapted from a review by Cunningham and You in 2015 and
provides a summary of general characteristics of common immortalized prostate cancer cell
lines. While these in vitro cell models do not recapitulate all aspects of human prostate cancer,
their use in basic science research increases knowledge about molecular mechanisms underlying
the development and progression of PCa and, with further development, can provide more
biologically relevant platforms for mechanistic studies and drug discovery.
Table 2: General Characteristics of Common Immortalized Human Prostate Cancer Cell
Lines
General Characteristics of Common Immortalized Human Prostate Cancer Cell Lines
LNCaP PC3 DU145
Source Lymph node Vertebral metastasis Brain metastasis
Doubling Time 28-60 hours ~33 hours ~34 hours
PSA Protein Yes No No
AR Status Positive Negative Negative
p53 Wild-type with point
mutation
Null Mutated
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1.8.2 In Vivo Models
In order to explore the complex biology of prostate cancer metastasis, pre-clinical in vivo models
are the best available approach, as they can be used to study the biological behavior of tumor
tissue in an environment that cannot be easily mimicked in an in vitro setting. The most widely
used animal models in prostate cancer research include human xenograft mouse models and
transgenic mouse models, each of which will be discussed in the following sections.
1.8.2.1 Xenograft Mouse Models
The human prostate cancer xenograft mouse model functions as an extremely useful alternative
approach for exploring the biology of prostate cancer, including potential interactions between
molecularly and genetically altered tumor cells and their microenvironment (Park et al., 2008;
Park, Kim, McCauley, & Gallick, 2010). Studies have shown that direct comparison of patient
tumor biopsy tissue with xenografts demonstrate a high concordance in gene expression and
similar alterations in the genome when tumors are cultivated in mice (Whiteford et al., 2007).
Additionally, xenograft mouse models are widely used for the investigation of new drugs and
therapeutic strategies. Although the predictive value of xenograft models is rather variable, these
models have been relatively accurate in identifying clinically active agents and effective drug
combinations (Peterson & Houghton, 2004), and have been shown to provide use in predicting
Phase II clinical trial performance of cancer drugs under the right framework (Voskoglou-
Nomikos, Pater, & Seymour, 2003).
Xenograft mouse models are established by inoculating a predetermined number of human tumor
tissues, cell lines or primary cell cultures into immunodeficient mice. Subcutaneous xenograft
mouse models, in which the cells are injected into the flank area, allow tumors to be easily
identified and measured, however lack the ability to metastasize. Orthotopic prostate cancer
xenograft models are developed by injecting human prostate cancer cells directly into the
prostate of the mouse. This model allows for the study of genetic and molecular changes in the
tumor cells and their organ microenvironment, as well as lymph node metastasis (Park et al.,
2008). One drawback of orthotopic models is their failure to lead to spontaneous metastasis to
bone, the most frequent metastatic site in men with prostate cancer. However, orthotopic intra-
tibial implantation of metastatic PCa cells produces homogenous cohorts of tumors in bone, and
allows for the study of these microenvironment interactions (S. J. Kim et al., 2006). In some
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situations, tumor xenograft models are established by directly transplanting human tissues into
mice, thus limiting the potential for molecular and epigenetic changes to occur during long
periods of in vitro growth. This approach has shown greater predictability for clinical tumor
responses to various drugs compared to more conventional cell line xenograft models (Garber,
2009; Kerbel, 2003). An emerging model, primarily used to study various aspects of PCa biology
including angiogenesis, the identification of castrate resistant stem-like cells, and the effect of
antiandrogen therapies, is a patient-derived model (PDX) (E. L. S. Fong et al., 2014). PDX
models are established by directly implanting surgically removed tumor tissue from the patient
into an immunocompromised mouse, without in vitro manipulation. However, given the high
cost, rare access to patient tissue, and variable engraftment rates, PDX models are not widely
employed in prostate cancer research, despite their ability to retain more reproducible tumor
features (Siolas & Hannon, 2013; Tentler et al., 2012).
1.8.2.2 TRAMP Mouse Model
The transgenic adenocarcinoma mouse prostate (TRAMP) model was first publicized in 1995 for
its ability to form prostate cancer (Greenberg, 1996) and was later explored for its metastatic
potential (Gingrich et al., 1996). This model was established by transgenic expression of SV40-
Tag early genes, under the prostate-specific rat Probasin promoter. The Probasin promoter
regulates the expression of the both the large and small simian virus (SV40) t antigen, specially
limited to the dorsolateral and ventral lobes of the prostate (Gingrich et al 1996). The large t
antigen inhibits tumour suppressor genes, p53 and Retinoblastoma (Rb), simultaneously, the
small t antigen terminates the function of protein phosphatase 2. Thus, the SV40-Tag oncogene
was selected as a target due to its role in inducing oncogenic progression by binding to and
activating tumor suppressors (Colvin, Weir, Ikin, & Hudson, 2014). TRAMP mice typically
develop epithelial hyperplasia after eight weeks of age, PIN at 18 weeks, and lymph node
metastasis by 28 weeks of age (Rea et al., 2016). The TRAMP model has been used to study the
effect of ADT on the progression of PCa by surgical castration of TRAMP mice, the effects of
several chemopreventive agents (including green tea, retinoic acid, vitamin E, dietary
restriction), and immunotherapy, among others (Zhang, Wang, Zhang, & Lu, 2013). TRAMP
mice have rarely been used to study metastases to distant organs of greater clinical relevance,
including bone, as these types of metastases are quite rate in this model (Hsieh et al., 2007).
50
However, the TRAMP model provides considerable promise in understanding aspects of PCa
development, and for testing novel therapies.
1.8.2.3 Lady Transgenic Model
The Lady transgenic mouse model was developed in 1998 and, similarly to the TRAMP model,
the Probasin promoter drives SV40 T antigen expression. However, in order to avoid the
problem of variable expression levels of the large and small simian virus (SV40) t antigen in the
TRAMP model, the Lady models target only the large-T antigen (Kasper et al., 1998). In this
model, PIN and high glandular proliferation develop by ten weeks of age, followed by high-
grade epithelial dysplasia and poorly undifferentiated adenocarcinoma by twenty weeks, with
metastasis to the lymph nodes, liver, and lung, reported in several transgenic lines (Masumori et
al., 2001). Although the Lady model is less aggressive than the TRAMP model, they share
several histopathological similarities and have also been used to study progression from initial
androgen-dependent regression to androgen-independent relapse following castration (Kasper et
al., 1998; Klein, 2005). Several studies using the Lady model have explored dietary effects of fat
and antioxidants on PCa progression (Venkateswaran, Fleshner, Sugar, & Klotz, 2004), while
others use this model to assess its interaction with TGF- signalling in promoting metastasis (Tu
et al., 2003). The Lady model more accurately mimics the majority of human PCa due to its slow
growth and mostly epithelial phenotype (Valkenburg & Williams, 2011). This model has helped
to move prostate cancer mouse modeling forward and has allowed researchers to better
understand the progression of the disease.
1.8.2.4 The Phosphatase and tensin homolog deleted on chromosome ten (PTEN) Model
PTEN is frequently lost in a variety of human cancers, including prostate, with deletions
occurring in approximately 23% of HGPIN, 69% of localized PCa, and 86% of metastatic CRPC
(Holcomb et al., 2009; Song, Salmena, & Pandolfi, 2012; Yoshimoto et al., 2006). It is a
significant tumor suppressor demonstrating numerous roles in cell metabolism, polarity, motility,
cancer “stem-ness,” and stromal-epithelial interactions (Cantley & Neel, 1999). Due to these
roles, various transgenic models targeting PTEN have been established. The homozygous
knockout of PTEN is lethal, however, the heterozygous knockout mice slowly develop tumors
and a spectrum of prostate phenotypes (Antonio Di Cristofano, Pesce, Cordon-Cardo, &
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Pandolfi, 1998; Podsypanina et al., 1999). Due to the slow progression of PCa in PTEN
knockout models, and the severe health issues they develop, some of these studies combined
PTEN knockout mice with other genes lost in PCa to accelerate disease progression. For
example, p27Kip1 knockout coupled with heterozygous PTEN deletion mice rapidly develop PIN
at 13 weeks and about 25% of mice develop invasive PCa (A. Di Cristofano, De Acetis, Koff,
Cordon-Cardo, & P Pandolfi, 2001).
1.8.2.5 c-MYC Model
c-MYC is a proto-oncogene and its overexpression is correlated with increasing tumor stage in
human prostate cancer samples (Gurel et al., 2008). Thus, targeting Myc over-expression to the
mouse prostate is a valid model of human PCa. The first studies to evaluate Myc overexpression
in the mouse prostate was performed by Ellwood-Yen et al, where both Lo-Myc and Hi-Myc
mice were developed under the control of the PB promoter. Hi-Myc mice progress from PIN at
13 weeks to adenocarcinoma with invasion by 26 weeks, while Lo-Myc mice progress slower
with both PIN and adenocarcinoma developing by about 30 weeks (Ellwood-Yen et al., 2003).
One of the major drawbacks of this model is its inability to progress following castration or its
inability to develop metastasis (Koh et al., 2010). However, due to the early onset of Myc
overexpression during PCa progression, the MYC model is an excellent starting point to assess
additional genetic alterations that drive PCa progression (Gurel et al., 2008).
1.8.2.6 NK3 Homeobox (1NKX3.1) Model
The NKX3.1 transcription factor plays a critical role in urogenital development and function and
is frequently lost in early human PIN and PCa samples (Bhatia-Gaur et al., 1999; Bowen et al.,
2000). Loss of NKX3.1 in mice will cause the development of hyperplasia and some dysplasia,
but its loss alone is not sufficient to induce prostate cancer or HGPIN in mice (Abdulkadir et al.,
2002; Tanaka et al., 2000). Combining NKX3.1 loss with PTEN deletion allows for accelerated
progression to HGPIN and early carcinoma (M. J. Kim et al., 2002).
Chapter 2 Rationale, Hypothesis, and Aims
Rationale, Hypothesis, and Aims
2.1 Rationale
PCa is the most commonly diagnosed cancer in men and is the second leading cause of cancer-
related death in Canadian men. In 2017, Statistics Canada reported that one in seven men will
develop PCa during their lifetime, and one in twenty-nine will die from the disease. Despite the
wide variety of treatment options available for patients, many of these treatments are associated
with adverse side effects that reduce an individual’s quality of life. Thus, there is a need for
novel treatment options which target cancerous cells as opposed to healthy ones, providing an
advantage over more conventional therapies. Given the anticancer properties that have been
demonstrated for cannabinoids both in vitro and in vivo, it is evident that there is a great need to
demonstrate if cannabinoids have the potential to inhibit varying types of prostate cancer cell
lines and to determine the potential mechanism of action involved. Since cannabinoids may have
psychoactive effects, it is crucial to determine the detailed role of the CB receptors in mediating
anti-cancer versus psychotropic effects. This may allow for approaches that reduce the
psychoactive effects while maintaining anti-cancer benefits. The present research will allow us to
explore the mechanism by which cannabinoids exert their anti-PCa effect and the role of the
cannabinoid receptors in targeting downstream pathways responsible for the inhibition of cell
proliferation, invasion, migration, and induction of apoptosis and cell cycle arrest. A comparison
of the endocannabinoid anandamide to synthetic cannabinoid WIN 55,212-2 will also be
explored, allowing for the discovery of how CB receptor affinity variations lead to alterations in
the anti-cancer properties.
2.2 Hypothesis
It is hypothesized that cannabinoids including Anandamide and WIN 55,212-2 reduce
proliferation, migration and invasion, and induce apoptosis and cell cycle arrest in preclinical
models of prostate cancer through the CB2 receptor mediated pathway.
52
53
2.3 Aims
The aim of this thesis is to explore the anticancer effect of cannabinoids on different prostate
cancer cell types. To achieve this, the following sub-aims were established.
1. To assess the anticancer properties of AEA and WIN on various prostate cancer cell lines
in vitro.
2. To investigate the mechanism of action of WIN treatment on prostate cancer cells in
vitro.
3. To determine the antitumor effect of WIN in a PC3 xenograft model of prostate cancer.
Chapter 3 Materials and Methods
Materials and Methods
3.1 Cell Culture
Three human prostate cancer cell lines (PC3, DU145, and LNCaP) were obtained from the
American Type Culture Collection (Rockville, Maryland, USA). DU145 and PC3 cells were
cultured in Dulbecco’s minimal essential medium/F12 (Invitrogen, Burlington, ON, Canada)
with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 0.3mg/ml l-glutamine and
100IU/ml penicillin and 100g/ml streptomycin (Invitrogen, Burlington, ON, Canada). LNCaP
cells were cultured in RPMI 1640 medium (Invitrogen, Burlington, ON, Canada) with 10% FBS
supplemented with 0.3mg/ml l-glutamine and 100IU/ml penicillin and 100g/ml streptomycin.
All cells were cultured under sterile conditions at 37C in a 5% CO2 incubator.
3.2 Chemicals
Health Canada approval was obtained for the use of controlled substances for research purposes.
Anandamide was obtained from Bio-Techne (Minneapolis, MN, USA) and was purchased pre-
dissolved in anhydrous ethanol. Stock solutions of 0.1mM were created and stored at -20C.
WIN 55,212-2 and AM630 were obtained from Cayman Chemical (Ann Arbor, MI, USA) and
were dissolved in dimethyl sulfoxide (DMSO; Sigma, USA) to create a stock concentration of
10mM, and stored at -20C. Working solutions of anandamide (5-40M), WIN 55,212-2 (1-
30M), and AM630 (1-10M) were diluted in appropriate medium and prepared fresh daily
prior to treatment. All compounds were prepared and stored with minimal exposure to light to
avoid oxidation. All other chemicals were purchased from Sigma unless otherwise specified.
3.3 MTS Cell Proliferation Assay
Cell proliferation was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation
(MTS) assay (Promega, Madison, WI) as depicted in Figure 12. Cells were plated in 96-well
micro-titre plates at a density of 5x103 (LNCaP) or 4x103 (PC3 and DU145) cells per well and
left to adhere at 37C for 24 hours. After adherence, cells were treated with a range of
54
55
anandamide concentrations (5, 10, 20, 40M), a range of WIN 55,212-2 concentrations (1, 5, 10,
20, 30M), or a range of AM630 concentrations (1, 5, 10M) for 24, 48, and 72hr time points to
establish dose standardization for each cell line. After evaluating the effect of single agent
treatment on the cell lines, CB2 receptor activation was blocked using CB2 antagonist AM630 in
addition to Anandamide or WIN 55,212-2. The following combinations were selected for
evaluation: 10M WIN 55,212-2 + 5M AM630, 10M WIN 55,212-2 + 10M AM630, 20M
WIN 55,212-2 + 5M AM630, 20M WIN 55,212-2 + 10M AM630, 20M Anandamide +
5M AM630, 20M Anandamide + 10M AM630, 40M Anandamide + 5M AM630, 40M
Anandamide + 10M AM630. Control wells were treated with vehicle alone (DMSO or ethanol
0.01%, respectively). After treatment for 24, 48, and 72hr, 20L of MTS dye was added to each
well and plates were incubated at 37C for 2 hours. The absorbance was recorded at 490nm
using a 96-well plate reader. Each experiment was carried out in triplicate wells and repeated at
least three times.
Figure 12: MTS Cell Proliferation Assay: Cells are plated in a 96-well plate and left to adhere
for 24 hours. Treatment is added into each well and cells are incubated for 24, 48, and 72-hour
time points. At the end of each time point, MTS dye is added to each well and the plate is
incubated for 2 hours at 37°C. Optical density is measured using a 96-well plate reader at an
absorbance of 490nm.
3.4 Wound Healing (Scratch) Assay
Cell migration was assessed in PC3 and DU145 cells using a wound healing assay as depicted in
Figure 13. A total of 5x104 cells were plated per well in a 24-well plate and grown until cells
reached approximately 90% confluence. Mitomycin C was prepared at a concentration of 1mg/L
and added to each well to temporarily inhibit cell proliferation, and cells were incubated for 1hr
at 37C. A vertical scratch was made across each well using a 100l pipette tip, followed by two
56
washes with phosphate buffered saline (PBS; Invitrogen, Burlington, ON, Canada). Reference
marks for imaging were made on the bottom of each well using a fine-tipped marker.
Anandamide (5, 10, 20, 40M) or WIN 55,212-2 (5, 10, 15, 20M) was then added to each well
and left for 24 hours. Images were captured at 2 different reference points along the wound in
each well at 0hr of treatment to establish a baseline measurement, and at the 24hr time point after
cells were left to migrate. AxioVision SE64 Rel. 4.9.1 software was used to measure the
percentage of wound closure over the 24hr time point.
Figure 13: Wound Healing (Scratch) Assay: Cells are plated in a 24-well plate and left to grow
for approximately 48hrs until 90% confluent. A vertical scratch is made across each well using a
100µL pipette tip. An image is captured of the wound, and cells are treated accordingly. After 24
hours, a second image is captured, and wound closure is quantified.
3.5 Matrigel Invasion Assay
Invasion of PC3 and DU145 cells was assessed using the BD BioCoat™ Matrigel™ Invasion
Chamber 8.0 Micron, obtained from BD Biosciences (Mississauga, ON, Canada). Matrigel was
diluted to a final concentration of 0.125g/L with PBS and added to the upper chamber of each
well, and incubated overnight at 37C. As depicted in Figure 14, 5x104 cells per well were
seeded onto the upper chamber using 6-well plates, and cultured for 24 hours at 37C.
Anandamide (20, 40M) or WIN 55,212-2 (1, 5M) treatment was then added to the bottom
wells, and cells were left to invade for 24hrs at 37C. Following incubation, non-invasive cells
were removed from the upper chamber using a cotton swab. The inserts were fixed in methanol
and stained with a 0.1% crystal violet solution. Cells were counted manually (cells per four
fields) using a microscope, and the number of invading cells were quantified. Each experiment
was carried out in duplicate wells and repeated three times.
57
Figure 14: Matrigel Invasion Assay: Cells are plated in the upper chamber of a transwell and
treatment is added to the lower chamber. Cells are allowed to invade through Matrigel layer for
24 hours. Non-invasive cells are removed from the upper chamber using a cotton swab, and
invasive cells are fixed in methanol, stained in crystal violet solution, and counted.
3.6 Flow Cytometry
3.6.1 Cell Cycle Distribution
Cell cycle distribution was determined by flow cytometry in LNCaP and DU145 cells treated
with anandamide and in PC3 and DU145 cells treated with WIN 55,212-2. Briefly,
asynchronously growing cells were plated at a density of 1×106 per 10cm petri dish and treated
with anandamide (20, 40M) for 24 hours or WIN 55,212-2 (10, 20M) for 48 hours. Control
plates were treated with vehicle alone (cell culture media or 0.01% ethanol). Cells were pulse
labeled with bromodeoxyuridine (BrdU) for 2 hours prior to harvesting. A no-BrdU control was
included. Following incubation with BrdU, cells were trypsinized, fixed in ice-cold 70% ethanol
and stored at -20C until further analysis. Cells were then washed in PBS buffer with 0.5%
Tween-20 and treated with 2N hydrochloric acid (HCl) for 20 min to denature and expose
labelled DNA. Cells were incubated in the dark on ice for 1 hour with anti-BrdU conjugated
FITC (DAKO, Burlington, ON, Canada). Cells were then washed, centrifuged and resuspended
in 1g/L propidium iodide (PI) and incubated for 30 minutes in the dark on ice. Samples were
filtered through a nylon mesh and cell cycle analysis performed on the FACSCalibur flow
cytometer using Cell Quest Pro software package (Becton-Dickinson, CA, USA). Ten thousand
events were counted for each experiment. Each experiment was carried out in duplicate tubes
and repeated three times.
58
3.6.2 Apoptosis
The proportion of apoptotic to live cells was assessed using the FITC Annexin V/Dead Cell
Apoptosis Kit (Invitrogen, cat# V13242). PC3, LNCaP and DU145 cells were plated at a density
of 1×106 per 10cm petri dish and treated with anandamide (20, 40M) or WIN 55,212-2 (10,
20M for PC3 and DU145; 20, 30M for LNCaP) for 24 hours. Control plates were treated with
vehicle alone (0.01% ethanol or 0.01% DMSO, respectively). After treatment, cells were
trypsinized, washed with cold PBS and centrifuged at 1200rpm for 5 min. The supernatant was
discarded, and the cells were resuspended in 1ml of cold PBS. The cells were counted with a
Neubauer chamber and diluted in binding buffer to a density of 1x106 cells/ml. Cells were
labelled with 2L fluorescein isothiocyanate (FITC) Annexin V and 1 μL of 100 μg/mL PI and
incubated at room temperature for 15 min. After the incubation period, 400L binding buffer
was added to each cytometry tube, samples were filtered through a nylon mesh and cell analysis
performed with the FACSCalibur flow cytometer using the Cell Quest Pro software package
(Becton Dickinson, San Jose, CA, USA). Each experiment was carried out in duplicate tubes and
repeated three times.
3.7 Western Blot Analysis
Cells were prepared for lysate collection by plating 1x106 cells per 10cm petri dish and allowed
to adhere for 24hrs at 37C. Following incubation, cells were treated with anandamide (20,
40M) and WIN 55,212-2 (10, 20M). Control plates were treated with vehicle alone (0.01%
ethanol or 0.01% DMSO, respectively). After 24hr treatment, cells were lysed using NP-40 lysis
buffer containing protease inhibitors (leupeptin/pepstatin, aprotinin and
phenylmethanesulfonylfluoride), sodium dodecyl sulfate (SDS), deoxycholate and
ethylenediaminetetraacetic acid (EDTA). Protein concentration was quantified using the Pierce
BCA Protein Assay Kit (Thermo Fischer Scientific) prior to loading into 10% SDS gels for
electrophoresis. Antibodies for phospho-retinoblastoma, Cdk4 and p27 were purchased from Cell
Signaling Technology (Beverly, Massachusetts, USA), and antibodies for β-actin were purchased
from Abcam (Cambridge Science Park, UK). ImageJ software (US National Institute of Health,
Bethesda, MA, USA) was used to semi-quantitatively determine protein expression levels,
relative to β-actin. Each experiment was carried out in duplicate wells and repeated three times.
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3.8 Xenograft Studies
3.8.1 Animals and Housing
Animal Ethics approval was obtained from the Sunnybrook Research Institute Ethics Board and
all work was conducted in accordance with established institutional guidelines including the Care
and Use of Experimental Animals guidelines of the Canadian Council. Athymic nude (nu/nu) six
to eight-week-old male mice were purchased from Taconic (Charles River Laboratories, USA)
and housed in a laminar airflow cabinet under pathogen-free conditions on a 12-h light/dark
schedule. Mice were provided access to food and water ad libitum. Animals were allowed to
acclimate for at least one week prior to the start of the experiments.
3.8.2 Establishment of Xenografts
PC3 cells were maintained in medium with 10% fetal bovine serum. Using a 7-gauge needle,
1.5 × 106 cells resuspended in 100μL Matrigel solution (BD Biosciences, CA, USA) were
subcutaneously inoculated into the left flank of mice anesthetized with isoflurane (induction with
5% for 5-15sec, maintenance with 1-5%). Mice were monitored daily for tumor growth. When
tumors achieved a volume of 100mm3, mice were randomly assigned to two treatment groups:
control (i.e. DMSO vehicle; n=5), and WIN 55,212-2 (n=5) (Figure 15). Tumor volume was
calculated using the formula: Volume= (Length x Width2) (/6).
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Figure 15: Xenograft Establishment and Group Assignment: Mice were subcutaneously
injected with 1.5 million PC3 cells in the flank. Mice were monitored for two weeks, allowing
tumors to reach a volume of 100mm3. Animals were then randomly assigned into two treatment
groups; control (i.e. DMSO vehicle) and 5mg/kg treatment with WIN 55,212-2.
3.8.3 Administration of WIN 55,212-2
WIN 55,212-2 was administered to mice three days per week by intraperitoneal injection at a
final dose of 5mg/kg body weight over the course of three weeks. WIN 55,212-2 in DMSO
(0.01M) was diluted fresh in saline solution prior to administration. Control animals received
only the vehicle. Mice were monitored daily and body weight and tumor measurements were
recorded thrice weekly. Mice with tumors exceeding the maximum permissible diameter of
17mm were euthanized, in accordance with the Canadian Council on Animal Care and Cancer
Endpoint Guidelines. At the experiment termination (21 days), blood was drawn from all mice
by direct cardiac puncture. Serum was separated, aliquoted, and stored at -80C for future
analysis. Tumors were excised, weighed, and processed for histopathologic studies. In addition,
liver samples were obtained and fixed for histological analysis by a pathologist to determine
potential toxicities associated with the study (data not shown). Methods overview is depicted in
Figure 16.
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Figure 16: Xenograft Experiment Timeline: Ten athymic nude mice were randomized and left
to acclimatize for one week at the animal facility in Sunnybrook Research Institute (SRI). Mice
were inoculated with PC3 cells and tumors were left to grow for approximately two weeks, until
they reached a volume of 100mm3. Mice were randomized to two groups and treated three times
per week with either WIN 55,212-2 (5mg/kg body weight) or DMSO vehicle control for a period
of 21 days. Tumor volume and body weight was monitored three days per week for the entirety
of the study. At the termination of the experiment, serum and tumors were collected and stored
for further analysis.
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3.9 In Vitro Mitogenicity Assay
For measurement of cell growth, the MTS method was employed as previously described. PC3
cells (4x103 cells/well) were plated in 96-well plates. After 24 hours, cells were washed twice
with PBS and treated with serum free media for an additional 24 hours. Following this, serum
free media was removed, and cells were treated for up to 72 hours with 10% animal serum in
DMEM/F12 (100L, filtered through a 0.2m syringe filter) obtained from animals in the
control and treatment groups. Treated cells, containing animal serum, were incubated with the
MTS dye for 2 hours. The ensuing formation of tetrazolium compounds was measured using a
plate reader. All experiments were conducted in duplicate wells and repeated three times per
animal.
3.10 Statistical Analysis
Statistical analysis was completed using Microsoft Excel 2016. All in vitro experiments were
assessed using two-tailed Student’s t-testing. Analysis of the in vivo results were performed
using either Student’s t-testing or repeated measures One-Way Analysis of Variance (ANOVA)
techniques. Statistical analysis was performed using SAS software, version 8 (SAS Institute Inc.,
Cary, NC, USA). Results were considered significant at the 5% level (p<0.05). The data shown
represents the mean standard deviation (SD).
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Chapter 4 Results
Results
4.1 Differential growth inhibitory effect of anandamide on prostate cancer cell lines
The MTS cell proliferation assay was used to determine the effect of anandamide treatment on
prostate cancer cell growth. Dose standardization experiments were completed on PC3, DU145,
and LNCaP cells in order to determine optimal doses for further experiments at time points of
24, 48, and 72 hours. Results revealed differences in growth inhibitory effects for each of the cell
lines. In PC3 cells, treatment of anandamide at a concentration of 5-40M did not affect cell
viability during the three time points tested (Figure 17). In the androgen- sensitive cell line,
LNCaP, treatment with 20M and 40M at 24 hours resulted in a 31% and 33% reduction in
proliferation, respectively. At 48 hours, LNCaP cell proliferation was inhibited by 25%, 26%,
and 30% upon treatment with 10M, 20M, and 40M, respectively (Figure 18). Treating
DU145 cells with 40M of anandamide resulted in a significant reduction in proliferation by
34%, 25%, and 16% at 24, 48, and 72 hours, respectively (Figure 19). Subsequent experiments
were conducted using DU145 and LNCaP cells at treatment concentrations of 20-40M
anandamide for 24 hours.
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Figure 17: Effect of anandamide treatment on proliferation of PC3 cells. PC3 cells were
treated with various concentrations of anandamide for A) 24, B) 48, and C) 72 hours and optical
density at 490nm was recorded.
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0.60
0.70
0.80
0.90
1.00
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
A)
B)
C)
Figure 18: Effect of anandamide treatment on proliferation of LNCaP cells. LNCaP cells
were treated with various concentrations of anandamide for A) 24, B) 48, and C) 72 hours and
optical density at 490nm was recorded. Differences in optical density relative to control that
reach significance (p<0.05) are denoted with an asterisk (*).
* *
* * *
* *
* * *
66
0.00
0.50
1.00
1.50
2.00
2.50
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Control 5 10 20 40
Op
tica
l Den
sity
(49
0nm
)
Anandamide Treatment Concentrations (µM)
A)
B)
C)
Figure 19: Effect of anandamide treatment on proliferation of DU145 cells. DU145 cells
were treated with various concentrations of anandamide for A) 24, B) 48, and C) 72 hours and
optical density at 490nm was recorded. Differences in optical density relative to control that
reach significance (p<0.05) are denoted with an asterisk (*).
*
*
*
*
*
67
4.2 Treatment with WIN-55,212-2 reduces prostate cancer cell proliferation
Using the MTS cell proliferation assay, the effect of WIN 55,212-2 on prostate cancer growth
was assessed. Results revealed that treatment with WIN 55,212-2 at a concentration of 1-30M
inhibited the growth of PC3, LNCaP, and DU145 cells in a dose-dependent manner. PC3 cells
displayed significant reductions in growth by 50%, 55%, and 64% at 5, 10, and 20M of WIN
55,212-2, respectively, at 24 hours (Figure 20). In DU145 cells, growth was significantly
inhibited by 46%, 51%, and 65% at 5, 10, 20M of WIN 55,212-2, respectively (Figure 22).
LNCaP cells showed a similar trend, however concentrations of 20M and 30M were needed in
order to significantly reduce cell proliferation (Figure 21). Subsequent experiments were
conducted in DU145 and PC3 cells using concentrations of 10-20M WIN 55,212-2, and in
LNCaP cells using concentrations of 20-30M WIN 55,212-2.
68
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
A)
B)
C)
Figure 20: Effect of WIN 55,212-2 treatment on proliferation of PC3 cells. PC3 cells were
treated with various concentrations of WIN 55,212-2 for A) 24, B) 48, and C) 72 hours and
optical density at 490nm was recorded. Differences in optical density relative to control that
reach significance (p<0.05) are denoted with an asterisk (*).
* *
*
*
*
*
*
*
*
69
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Control 5 10 20 30
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Control 5 10 20 30
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Control 5 10 20 30
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
A)
B)
C)
Figure 21: Effect of WIN 55,212-2 treatment on proliferation of LNCaP cells. LNCaP cells
were treated with various concentrations of WIN 55,212-2 for A) 24, B) 48, and C) 72 hours and
optical density at 490nm was recorded. The * symbol denotes significance (p<0.05) relative to
control; ** denotes significance (p<0.001) relative to control.
* *
* *
*
*
*
** **
** **
70
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Control 1 5 10 20
Op
tica
l Den
stiy
(49
0nm
)
WIN 55,212-2 Treatment Concentrations (µM)
A)
B)
C)
Figure 22: Effect of WIN 55,212-2 treatment on proliferation of DU145 cells. DU145 cells
were treated with various concentrations of WIN 55,212-2 for A) 24, B) 48, and C) 72 hours and
optical density at 490nm was recorded. The * symbol denotes significance (p<0.05) relative to
control; ** denotes significance (p<0.001) relative to control.
* * **
*
* *
**
*
** **
* * *
*
71
4.3 Anandamide and WIN 55,212-2 treatment reduces the migration and invasion capacity of prostate cancer cells
The effect of anandamide and/or WIN 55,212-2 treatments on cell migration was assessed using
a wound healing (scratch) assay. After treatment with 20-40M anandamide for 24 hours,
DU145 cells showed a significant decrease in cell migration compared to vehicle control, with
the maximal reduction in migration at 40M (p<0.001) (Figure 23A, B). Treatment with 15M
and 20M WIN 55,212-2 resulted in a significant reduction in migration of PC3 cells by 41%
and 37%, respectively (Figure 25A, B). As for WIN 55,212-2, treatment with 15M and 20M
resulted in a significant reduction in the migration of DU145 cells by 36% and 28%, respectively
(Figure 24A, B).
A Matrigel invasion assay was used to assess the ability of cells to penetrate an extracellular
matrix-like environment. Results from this experiment revealed that anandamide significantly
inhibited the invasion of DU145 cells at 20-40M (p<0.001) (Figure 23C). As for WIN 55,212-2
treatment, as little as 1-5M inhibited PC3 cell invasion by 34% and 39% (Figure 25C), and
DU145 cell invasion by 36% and 41%, respectively (Figure 24C).
72
0
20
40
60
80
100
120
Control 20 40
% W
ou
nd
Clo
sure
(Rel
ativ
e to
Co
ntr
ol)
Anandamide Treatment Concentrations (µM)
Migration Assay Quantification
0
10
20
30
40
50
60
70
80
90
Control 20 40
% In
vasi
on
Anandamide Treatment Concentrations (µM)
Invasion Assay Quantification
A)
B) C)
Figure 23: Effect of 24hr treatment of anandamide on DU145 cell migration and invasion.
A) Morphological representation of wound healing assay, where DU145 cells were treated with
anandamide (20M and 40M) for 24 hours; B) Quantification of wound healing assay; C)
Quantification of Matrigel invasion assay. The * symbol denotes significance (p<0.05) relative to
control; ** denotes significance (p<0.001) relative to control.
*
** ** **
73
0
20
40
60
80
100
120
Control 5 10 15 20
% W
ou
nd
Clo
sure
(R
elat
ive
to C
on
tro
l)
WIN 55,212-2 Treatment Concentrations (µM)
Migration Assay Quantification
0
20
40
60
80
100
120
Control 1 5
% In
vasi
on
WIN 55,212-2 Treatment Concentrations (µM)
Invasion Assay Quantification
A)
B) C)
Figure 24: Effect of 24hr treatment of WIN 55,212-2 on DU145 cell migration and invasion.
A) Morphological representation of wound healing assay, where DU145 cells were treated with
WIN 55,212-2 (5-20M) for 24 hours; B) Quantification of wound healing assay; C)
Quantification of Matrigel invasion assay. The * symbol denotes significance (p<0.05) relative to
control; ** denotes significance (p<0.001) relative to control.
* *
* **
74
0
20
40
60
80
100
120
Control 5 10 15 20
% W
ou
nd
Clo
sure
(R
elat
ive
to C
on
tro
l)
WIN 55,212-2 Treatment Concentrations (µM)
Migration Assay Quantification
0
20
40
60
80
100
120
Control 1 5
% In
vasi
on
WIN 55,212-2 Treatment Concentrations (µM)
Invasion Assay Quantification
A)
B) C)
Figure 25: Effect of 24hr treatment of WIN 55,212-2 on PC3 cell migration and invasion.
A) Morphological representation of wound healing assay, where PC3 cells were treated with
WIN 55,212-2 (5-20M) for 24 hours; B) Quantification of wound healing assay; C)
Quantification of Matrigel invasion assay. The * symbol denotes significance (p<0.05) relative to
control.
* *
* *
75
4.4 Anandamide treatment does not significantly alter the cell cycle distribution in DU145 and LNCaP cells
In order to determine whether anandamide treatment resulted in alterations in cell cycle
distribution, cells were treated with anandamide (20-40M), collected, fixed, and assessed for
cell cycle alterations at 24 hours. In DU145 and LNCaP cells, treatment with anandamide did not
significantly alter cell cycle distribution. Populations of cells in G1 phase, S phase, and G2/M
phase remained relatively consistent across treatment conditions (Figure 26-27).
76
0
10
20
30
40
50
60
70
Control 20 40
Pro
po
rtio
n o
f C
ells
(%
)
Anandamide Treatment Concentrations (µM)
G1 S G2
A)
B)
Figure 26: Effect of anandamide treatment on cell cycle distribution in DU145 cells. A) Cell
cycle histogram; B) Quantification. Cells were treated with anandamide, labeled with anti-
bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC) and propidium iodide (PI), and
fixed at 24 hours, and then subsequently analyzed by flow cytometry to determine the percentage
of cells in each phase of the cell cycle. Error bars represent the standard deviation (SD). All
experiments were carried out in triplicate.
77
0
10
20
30
40
50
60
70
Control 20 40
Pro
po
rtio
n o
f C
ells
(%
)
Anandamide Treatment Concentrations (µM)
G1 S G2
A)
B)
Figure 27: Effect of anandamide treatment on cell cycle distribution in LNCaP cells. A)
Cell cycle histogram; B) Quantification. Cells were treated with anandamide, labeled with anti-
bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC) and propidium iodide (PI), and
fixed at 24 hours, and then subsequently analyzed by flow cytometry to determine the percentage
of cells in each phase of the cell cycle. Error bars represent the standard deviation (SD). All
experiments were carried out in triplicate.
78
4.5 WIN 55,212-2 treatment causes cell cycle arrest in DU145 and PC3 cells
In order to determine whether WIN 55,212-2 treatment resulted in alterations in cell cycle
distribution, cells were treated with WIN 55,212-2 (10-20M), collected, fixed, and assessed for
cell cycle alterations at 48 hours. In both DU145 and PC3 cells, treatment with WIN 55,212-2
caused a decrease in the proportion of cells in S phase. In DU145 cells, the percentage of cells in
S phase dose-dependently decreased from approximately 10% in control to 6% and 4% in the
WIN treatment conditions. In addition, the percentage of cells in G1 increased from 46% in
control to 64% and 67% in the 10M and 20M treatment conditions, respectively (Figure 28).
This would indicate cell cycle arrest in G1 phase of the cell cycle. In PC3 cells, the percentage of
cells in S phase dose-dependently decreased from approximately 7% in control to 2% and 0.54%
in the WIN treatment conditions. The percentage of cells in G1 phase increased, and the
percentage of cells in G2 phase dose-dependently increased from 15.2% in control to 17.2% and
22.6% in the 10M and 20M treatment conditions, respectively (Figure 29). This would
indicate an arrest in G2/M phase of the cell cycle.
79
0
10
20
30
40
50
60
70
80
Control 10µM WIN 20µM WIN
Pro
po
rtio
n o
f C
ells
(%
)
WIN 55,212-2 Treatment Concentrations (µM)
G1 S G2
A)
B)
Figure 28: Effect of WIN 55,212-2 treatment on cell cycle distribution in DU145 cells. A)
Cell cycle histogram; B) Quantification. Cells were treated with WIN, labeled with anti-
bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC) and propidium iodide (PI), and
fixed at 24 hours, and then subsequently analyzed by flow cytometry to determine the percentage
of cells in each phase of the cell cycle. Error bars represent the standard deviation (SD). All
experiments were carried out in triplicate. The * symbol represents significance (p<0.05) relative
to control, the ** symbol represents significance (p<0.001) relative to control.
*
**
*
*
*
*
80
0
10
20
30
40
50
60
70
Control 10 20
Pro
po
rtio
n o
f C
ells
(%
)
WIN 55,212-2 Treatment Concentrations (µM)
G1 S G2
A)
B)
Figure 29: Effect of WIN 55,212-2 treatment on cell cycle distribution in PC3 cells. A) Cell
cycle histogram; B) Quantification. Cells were treated with WIN, labeled with anti-
bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC) and propidium iodide (PI), and
fixed at 24 hours, and then subsequently analyzed by flow cytometry to determine the percentage
of cells in each phase of the cell cycle. Error bars represent the standard deviation (SD). All
experiments were carried out in triplicate. The * symbol represents significance (p<0.05) relative
to control.
*
*
*
81
4.6 Anandamide reduces proliferation and induces apoptosis in LNCaP cells but not in DU145 cells
To analyze the effect of anandamide on prostate cancer cells, and the possibility of a pro-
apoptotic effect, a FITC Annexin V assay was used. As shown in Figure 30, there were no
significant differences in the proportion of apoptotic or live cells in the DU145 cells treated with
anandamide (20-40M) compared to vehicle control. In LNCaP cells, the number of live cells
significantly decreased from 57% to approximately 41% while the number of cells in the
apoptotic state significantly increased from 33% to approximately 49% in the anandamide
treatment conditions compared to untreated control (Figure 31).
82
0
10
20
30
40
50
60
70
80
Control 20 40
Pro
po
rtio
n o
f C
ells
(%
)
Anandamide Treatment Concentrations (µM)
Live Apoptotic
A)
B)
Figure 30: Effect of anandamide on proportion of live versus apoptotic DU145 cells using
Annexin V flow cytometry. A) Representative flow cytometry images; B) Quantification. Cells
were treated with anandamide for 24 hours, labelled with fluorescein isothiocyanate (FITC) and
propidium iodide (PI), and then subsequently analyzed by flow cytometry to determine the
percentage of live versus apoptotic cells.
83
0
10
20
30
40
50
60
70
Control 20 40
Pro
po
rtio
n o
f C
ells
(%
)
Anandamide Treatment Concentrations (µM)
Live Apoptotic
A)
B)
Figure 31: Effect of anandamide on proportion of live versus apoptotic LNCaP cells using
Annexin V flow cytometry. A) Representative flow cytometry images; B) Quantification. Cells
were treated with anandamide for 24 hours, labelled with fluorescein isothiocyanate (FITC) and
propidium iodide (PI), and then subsequently analyzed by flow cytometry to determine the
percentage of live versus apoptotic cells. The * symbol represents significance (p<0.05) relative
to control, the ** symbol represents significance (p<0.001) relative to control.
*
*
**
**
84
4.7 WIN 55,212-2 significantly induces apoptosis in PC3 and DU145 cells but not in LNCaP cells
A FITC Annexin V assay was used to analyze the possible pro-apoptotic effect of WIN 55,212-2
treatment on prostate cancer cells. In DU145 and PC3 cells, the proportion of cells in the
apoptotic state significantly increased in a dose-dependent manner after treatment with 10-20M
WIN 55,212-2, while the number of live cells remained statistically without variation (Figure 32,
33). On the contrary, there were no significant differences in the proportion of live or apoptotic
LNCaP cells upon treatment with 20-30M WIN 55,212-2 compared to vehicle control (Figure
34).
85
A)
B)
Figure 32: Effect of WIN 55,212-2 on proportion of live versus apoptotic PC3 cells using
Annexin V flow cytometry. A) Representative flow cytometry images; B) Quantification. Cells
were treated with WIN 55,212-2 for 24 hours, labelled with fluorescein isothiocyanate (FITC)
and propidium iodide (PI), and then subsequently analyzed by flow cytometry to determine the
percentage of live versus apoptotic cells. The * symbol represents significance (p<0.05) relative
to control.
0
10
20
30
40
50
60
70
80
90
100
Control 10 20
Pro
po
rtio
n o
f C
ells
(%
)
WIN 55,212-2 Treatment Concentrations (µM)
Live Apoptotic
* *
86
A)
B)
Figure 33: Effect of WIN 55,212-2 on proportion of live versus apoptotic DU145 cells using
Annexin V flow cytometry. A) Representative flow cytometry images; B) Quantification. Cells
were treated with WIN 55,212-2 for 24 hours, labelled with fluorescein isothiocyanate (FITC)
and propidium iodide (PI), and then subsequently analyzed by flow cytometry to determine the
percentage of live versus apoptotic cells. The * symbol represents significance (p<0.05) relative
to control.
0
10
20
30
40
50
60
70
80
90
Control 10 20
Pro
po
rtio
n o
f C
ells
(%
)
WIN 55,212-2 Treatment Concentrations (µM)
Live Apoptotic
* *
87
A)
B)
Figure 34: Effect of WIN 55,212-2 on proportion of live versus apoptotic LNCaP cells using
Annexin V flow cytometry. A) Representative flow cytometry images; B) Quantification. Cells
were treated with WIN 55,212-2 for 24 hours, labelled with fluorescein isothiocyanate (FITC)
and propidium iodide (PI), and then subsequently analyzed by flow cytometry to determine the
percentage of live versus apoptotic cells.
0
10
20
30
40
50
60
70
80
Control 20 30
Pro
po
rtio
n o
f C
ells
(%
)
WIN 55,212-2 Treatment Concentrations (µM)
Live Apoptotic
* *
88
4.8 Cannabinoid receptor 2 antagonist AM630 does not alter prostate cancer cell growth
To determine the role of the cannabinoid 2 receptor in reducing the proliferation of prostate
cancer cells, the cells were pre-treated with AM630, a cannabinoid 2 receptor antagonist, which
acts as an inverse agonist by binding to CB2. The effect of AM630 on cell proliferation was first
assessed using the MTS assay in order to confirm a postulated lack of change in cell viability
upon treatment. As depicted in Figure 35, treatment of AM630 at a concentration of 1-10M had
no effect on the viability of PC3, DU145, and LNCaP cells. This allowed us to move forward
with the use of this compound in combination with anandamide and/or WIN 55,212-2 to
determine their effects after inhibition of CB2.
89
A)
B)
C)
Figure 35: Effect of Cannabinoid receptor 2 antagonist AM630 on viability of prostate
cancer cell lines. A) PC3 cells; B) DU145 cells; C) LNCaP cells. Cells were treated with various
concentrations of AM630 for 24 hours and optical density at 490nm was recorded. All
experiments were carried out in triplicate.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Control 1 5 10
Op
tica
l Den
sity
(49
0nm
)
AM630 Treatment Concentrations (µM)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
Control 1 5 10
Op
tica
l Den
sity
(49
0nm
)
AM630 Treatment Concentrations (µM)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 1 5 10
Op
tica
l Den
sity
(49
0nm
)
AM630 Treatment Concentrations (µM)
90
4.9 Treatment with CB2 antagonist AM630 abrogates the anti-proliferative effects of anandamide in DU145 and LNCaP cell lines
Using the MTS cell proliferation assay, cell proliferation was assessed for cells treated with
anandamide after the inhibition of the CB2 receptor by AM630. In both DU145 and LNCaP cell
lines, treatment with anandamide and AM630 resulted in an abrogation of the anti-proliferative
effects of anandamide (Figure 36). Treatment with 40M anandamide and 5M AM630 resulted
in a significant increase by more than 200% in the proliferation of DU145 cells compared to
40M anandamide alone. Increasing the concentration of AM630 to 10M resulted in a similar
increase in proliferation by over 200% compared to anandamide alone. In LNCaP cells,
treatment with 20M anandamide and 5M AM630 resulted in a significant increase by
approximately 150% compared to 20M anandamide alone. Similarly, anandamide and 10M
AM630 resulted in an increase by approximately 140% compared to anandamide alone.
91
A)
B)
Figure 36: Effect of anandamide after treatment with cannabinoid receptor 2 antagonist
AM630 on proliferation of DU145 and LNCaP cell lines. A) DU145 cells; B) LNCaP cells.
Cells were pre-treated with CB2 antagonist AM630 and subsequently treated with anandamide
for 24 hours. Optical density was recorded at 490nm. The symbol * denotes significance
(p<0.05) relative to control, the symbol ** denotes significance (p<0.001) relative to control.
The symbol & denotes significance (p<0.05) relative to anandamide alone, the symbol &&
denotes significance (p<0.001) relative to anandamide alone.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Control 20µM AEA 20µM AEA+ 5µM AM630 20µM AEA+ 10µMAM630
Op
tica
l Den
sity
(49
0nm
)
Treatment Concentrations
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Control 40µM AEA 40µM AEA+ 5µM AM630 40µM AEA+ 10µMAM630
Op
tica
l Den
sity
(49
0nm
)
Treatment Concentrations
*
&
&
*
&& &&
**
&
&
92
4.10 Treatment with CB2 antagonist AM630 abrogates the anti-proliferative effects of WIN 55,212-2 in prostate cancer cell lines
Cell proliferation was assessed for cells treated with WIN 55,212-2 after inhibition of CB2 using
the MTS cell proliferation assay. In all three cell lines, blockage of the CB2 receptor by CB2
antagonist AM630 resulted in an abrogation of WIN 55,212-2’s anti-proliferative effect.
Treatment with 10M WIN 55-212,2 and 5M AM630 resulted in a significant increase in PC3
cell proliferation by 165% compared to WIN 55,212-2 alone. In DU145 cells, treatment with
10M WIN 55,212-2 and 5M AM630 resulted in an increase in cell proliferation by more than
180%. Following a similar trend, LNCaP cells treated with 30M WIN 55,212-2 and 5M
AM630 resulted in an increase in proliferation by approximately 150% compared to WIN
55,212-2 alone (Figure 37).
93
A)
B)
C)
Figure 37: Effect of WIN 55,212-2 after treatment with cannabinoid receptor 2 antagonist
AM630 on cell proliferation. A) PC3 cells; B) DU145 cells; C) LNCaP cells. Cells were pre-
treated with CB2 antagonist AM630 and subsequently treated with WIN 55,212-2 for 24 hours.
Optical density was recorded at 490nm. The symbol * denotes significance (p<0.05) relative to
control, the symbol ** denotes significance (p<0.001) relative to control. The symbol & denotes
significance (p<0.05) relative to WIN 55,212-2 alone.
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Control 10µM WIN 10µM WIN+ 5µMAM630
10µM WIN+10µM AM630
Op
tica
l Den
sity
(49
0nm
)
Treatment Concentrations
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Control 30µM WIN 30µM WIN+ 5µMAM630
30µM WIN+ 10µMAM630
Op
tica
l Den
sity
(49
0nm
)
Treatment Concentrations
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control 10µM WIN 10µM WIN+ 5µMAM630
10µM WIN+ 10µMAM630
Op
tica
l Den
sity
(49
0nm
)
Treatment Concentrations
*
*
*
**
&
&
& &
& &
94
4.11 WIN 55,212-2 treatment alters expression of pRb, Cdk4, and p27 in PC3 cells
Western blotting was used to investigate changes in the expression of downstream proteins in the
cell cycle pathway, including phosphorylated retinoblastoma protein, Cdk4, and p27 following
treatment with cannabinoid WIN 55,212-2. PC3 cells were treated with 10M and 20M WIN
55,212-2 for a period of 48 hours. Treatment with WIN 55,212-2 resulted in a dose-dependent
decrease in the expression of pRb and Cdk4, and a dose-dependent increase in the expression of
p27 compared to vehicle control (Figure 38). Quantification of the Western blot results are
provided in Figure 38.
95
0
0.2
0.4
0.6
0.8
1
1.2
Control 10µM WIN 20µM WIN
Rel
ativ
e D
ensi
ty
Densitometric Analysis of pRb
0
0.5
1
1.5
2
2.5
3
3.5
4
Control 10µM WIN 20µM WIN
Rel
ativ
e D
ensi
ty
Densitometric Analysis of p27
0
0.2
0.4
0.6
0.8
1
1.2
Control 10µM WIN 20µM WIN
Rel
ativ
e D
ensi
ty
Densitometric Analysis of Cdk4
A) B)
Figure 38: Effect of WIN 55,212-2 on expression of cell cycle regulator proteins. Western
Blot analyses demonstrate the changes in the expression of A) p27, Cdk4, and phosphorylated
retinoblastoma protein (pRb) in PC3 cells treated with WIN 55,212-2; B) Corresponding
densitometric analysis.
96
0
50
100
150
200
250
300
350
400
450
17 19 21 24 26 28 31 33 35 38
Tum
or
Vo
lum
e (1
00m
m3)
Days Post Inoculation
Treatment Control
4.12 WIN 55,212-2 treatment reduces tumor growth in a mouse xenograft model
The anti-tumor effect of WIN 55,212-2 was tested in vivo using the PC3 xenograft model. Tumor
size and body weight were measured thrice weekly during the duration of the study. A significant
difference in the overall tumor growth rates between each group was observed over time
(p<0.05). Mice in the WIN 55,212-2 treatment group had significantly smaller tumors and slower
tumor growth rate compared to the control group (Figure 39, 41). Mice had minor changes in
body weight, which leveled off towards the end of the study (Figure 40). WIN 55,212-2 was well
tolerated with no toxicities as assessed histologically (data not shown).
Figure 39: WIN 55,212-2 significantly reduces tumor growth rate. Variation in tumor volume
(mm3) measured over time in 2 groups; Control (vehicle alone) and WIN 55,212-2 (5mg/kg body
weight). WIN 55,212-2 was administered on the days indicated on the horizontal axis. Tumor
growth was monitored over time. Error bars represent standard deviation (SD). The symbol *
denotes significance (p<0.05) relative to control.
* * * * *
97
Figure 40: Lack of effect of WIN 55,212-2 treatment on animal weight. Average animal body
weight (grams) between control group and WIN 55-212-2 treatment group. Mice body weight
was measured thrice weekly for the duration of the study.
0
5
10
15
20
25
30
35
0 17 19 21 24 26 28 31 33 35 38
Wei
ght
(g)
Days Post Inoculation
Treatment Control
98
Figure 41: Representative images of mice tumors before and after excision. A, C) Control
group; B, D) WIN 55,212-2 treatment group.
A
B
C
D
99
4.13 Serum containing WIN 55,212-2 reduces PC3 cell proliferation
A mitogenicity experiment was conducted in which serum collected from control and WIN
55,212-2 treated mice was used to supplement DMEM/F12 media for PC3 cells. Using the MTS
cell proliferation assay, cells exposed to serum obtained from animals treated with WIN 55,212-
2 showed reductions in proliferation at 24 and 48 hours, but not at 72 hours. At 24 hours, cells
cultured in the serum from animals treated with WIN 55,212-2 displayed approximately 40%
reduction in proliferation, and a 55% reduction at 48 hours (p< 0.05) (Figure 42). A slight
increase in proliferation was seen at 72 hours.
100
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Control WINO
pti
cal D
ensi
ty (
490n
m)
Treatment Group
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Control WIN
Op
tica
l Den
sity
(49
0nm
)
Treatment Group
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Control WIN
Op
tica
l Den
sity
(49
0nm
)
Treatment Group
A)
B)
C)
Figure 42: Effect of WIN 55,212-2 containing serum on proliferation of PC3 cells. A) 24
hours; B) 48 hours; C) 72 hours. Cells were treated with mice serum from either control or WIN
55,212-2 group. Optical density was recorded at 490nm. The symbol * denotes significance
(p<0.05) relative to control.
*
101
Table 3: Summary of In Vitro Results.
Assay Anandamide WIN 55,212-2 Anandamide +
AM630
WIN 55,212-2 +
AM630
Proliferation
Decrease in
LNCaP and
DU145
Decrease in
LNCaP, DU145,
and PC3
Increase in
LNCaP and
DU145
Increase in
LNCaP, DU145,
and PC3
Wound Healing Decrease in
DU145
Decrease in
DU145, and PC3 - -
Invasion Decrease in
DU145
Decrease in
DU145, and PC3 - -
Cell Cycle Analysis No Changes
Cell cycle arrest
in G1 phase in
DU145 and
G2/M phase in
PC3
- -
Annexin V Flow
Cytometry
LNCaP:
Decrease in
proliferating
cells, increase
in apoptotic
cells
Increase in
apoptotic PC3
and DU145 cells
- -
pRb Expression - Dose-dependent
decrease in PC3 - -
Cdk4 Expression - Dose-dependent
decrease in PC3 - -
102
p27 Expression - Dose-dependent
increase in PC3
Mitogenicity -
Decrease in
proliferation at
24, 48 hours in
PC3
- -
Table 4: Summary of In Vivo Results
WIN 55,212-2
Animal Body Weight No changes relative to control
Tumor Size Decrease
Pathology No differences between groups (Data not
shown)
103
Chapter 5 Discussion
Discussion
In addition to the well-known palliative effects of cannabinoids on cancer-associated symptoms,
a large body of evidence suggests these molecules may inhibit the growth of tumor cells in
culture and animal models by modulating key signalling pathways. The first evidence of these
anticancer effects was reported in lung cancer by Munson et al (Munson, Harris, Friedman,
Dewey, & Carchman, 1975) and since then, numerous studies have been carried out,
investigating the anti-tumor effects of cannabinoids in a variety of cancers.
Several cannabinoids, including plant-derived, endogenous, and synthetic cannabinoids, are
known to exert antiproliferative actions in a variety of cancer cells, such as breast, brain, skin,
thyroid and colorectal cancers (Guzmán, Sánchez, & Galve-Roperh, 2002). Additionally,
administration of cannabinoids to nude mice slows tumor growth rate in lung, glioma, thyroid
epithelioma, skin carcinoma and lymphoma xenograft models (Bifulco et al., 2001; Casanova et
al., 2003; McKallip et al., 2002). Despite evidence indicating the anti-cancer effect of
cannabinoids in other tumor types, there is very limited evidence for the use of cannabinoids,
anandamide and WIN 55,212-2 as a therapeutic agent in prostate cancer, and our study was
designed to address this gap in knowledge.
In this thesis, we have provided evidence supporting the hypothesis that cannabinoids,
anandamide and WIN 55,212-2, reduce proliferation, migration and invasion, and induce
apoptosis and cell cycle arrest in preclinical models of prostate cancer possibly through a CB2
receptor mediated pathway.
Initially, we demonstrated that treating various prostate cancer cell lines with anandamide or
WIN 55,212-2 caused a reduction in proliferation. A dose-dependent effect was seen upon
administration of AEA or WIN to prostate cancer cells, however, these effects were not seen in
PC3 cells treated with AEA. Furthermore, analysis by flow cytometry showed a decrease in
proliferation and an increase in apoptosis of LNCaP cells treated with AEA, however no
increases in apoptosis of DU145 cells. This may be due to inherent differences between the two
cells lines. It is possible that the apoptotic effects in LNCaP cells are dependent on AR or p53
104
status, both of which are nonfunctional in DU145 cells. Our studies revealed an increase in
apoptosis in PC3 and DU145 cells treated with WIN, confirming results obtained through the
MTS cell proliferation assay. We were unable to demonstrate an induction of apoptosis in
LNCaP cells despite reports by Sarfaraz et al. This may be due to differences in experimental
approaches; we conducted our apoptosis studies using Annexin V, however Sarfaraz et al
completed a more detailed study exploring the expression of several apoptotic markers, including
Bax, Bcl-2, caspase 3,9, and PARP. It is possible that the assay used in our study is not sensitive
enough to detect the pro-apoptotic effect of WIN on LNCaP cells. Hence, conducting further
mechanistic studies may support the work published by Sarfaraz et al. Cell cycle analysis
revealed that WIN 55,212-2 caused cells to arrest in G1 and G2/M phase, while the percentage of
cells progressing to S phase was decreased. Studies combining AEA or WIN with the CB2
antagonist AM630 demonstrated a reversal in the cannabinoids’ anti-proliferative effects,
suggesting that alterations in cell proliferation may be occurring through a CB2 dependent
pathway. In PC3 cells, AM630 partially restored proliferation in the presence of WIN, relative to
control. These effects are possibly due to activation of the CB1 receptor, which may be counter-
acting the increase in proliferation observed through inhibition of CB2. Our migration and
invasion studies indicate a decrease in cell migration and invasion after treatment with AEA or
WIN for 24 hours in DU145 and PC3 cells. Mechanistic studies performed on key cell cycle
regulator proteins reveal that WIN may be exerting its effects through upregulation of the cyclin
dependent kinase inhibitor p27, downregulation of the tumor suppressor protein phosphorylated
retinoblastoma, and downregulation of the cell division protein kinase Cdk4.
Based on these in vitro findings, we went on to investigate the effects of WIN in a xenograft
mouse model. We found that animals treated with WIN over a period of three weeks
demonstrated significantly reduced tumor sizes. Moreover, administration was safe and well
tolerated, supporting its role as a safe and effective anti-cancer agent. Mice treated with WIN
demonstrated small, but non-significant reductions in weight loss. We did not observe
differences in food intake between the two groups, however the small, yet insignificant
reductions in body weight may be due to the influence of cannabinoid-induced changes in
metabolic activity. Future studies could explore changes in glycolysis through AMPK activation.
Subsequent mitogenicity studies using serum collected from mice showed significant reductions
in human prostate cancer cell proliferation.
105
In this thesis, we have focused solely on the cannabinoid receptor 2, mainly due to its ability to
generate limited psychoactive side effects when stimulated. However, for the purpose of
gathering a better understanding of the mechanism through which cannabinoids exert their anti-
cancer effects in prostate cancer, it would be beneficial to explore a variety of the known
cannabinoid receptors discussed in literature, including the cannabinoid receptor 1, GRP55 and
TRPV1. A deeper understanding of role of these receptors could be achieved through the use of
antagonists or genetic silencing. This has been partially completed in prostate cancer, where
silencing of GPR55 was associated with reduced PC3 proliferative rates and was crucial for
anchorage-dependent and anchorage-independent cell growth though a mechanism involving
ERK (Pĩeiro, Maffucci, & Falasca, 2011). In glioblastoma xenograft mice models, silencing the
expression of GPR55 lead to significantly slower tumor growth rates compared to control
siRNA-treated mice (Andradas et al., 2011). With regard to TRPV1, studies have shown a
cannabidiol driven impairment in the invasion of human lung and cervical cancer cells through
activation of the MAPks p38 and p42/44, which was reversed by the TRPV1 antagonist
capsazepine (Ramer, Merkord, Rohde, & Hinz, 2010). These studies shed light on the unclear
aspects of the TRPV1 and GRP55 receptors, including their physio-pathological role and the
signalling pathways they are coupled with, and may help to understand their relevance in human
cancer.
Several studies could be conducted on a variety of promising cannabinoids, including
phytocannabinoids such as THC and CBD, synthetic cannabinoids and the endogenous
cannabinoid 2-AG. Majority of these compounds have shown promise in other areas of cancer;
however, a very limited number have been explored for therapeutic use in PCa. Synthetic
cannabinoids that bind to the CB2 receptor with higher affinity would be of greater efficacy for
its transition to clinical trials, as psychoactive effects would be mitigated or completely avoided.
Several of these cannabinoids alter varying pathways, thus their individual mechanisms of action
offer great knowledge into future pathways to target for the prevention and treatment of PCa. A
few of the pathways commonly discussed in the literature were mentioned in the molecular
targets section of this thesis (see section 1.7). Conducting more detailed mechanistic studies on
key proteins in each of the pathways related to endoplasmic reticulum stress, oxidative stress, as
well as RhoA GTPase, will allow for a more solid foundation into how these compounds are
involved in the anti-proliferative, anti-migratory, anti-invasive, and pro-apoptotic processes
106
explored in this thesis. Additional experiments, including oxidative stress assays, calcium level
assays, ceramide kinase assays, immunostaining to examine structural changes in cytoskeleton,
in addition to the expression of autophagy-related markers would allow for a more in-depth
analysis of the processes involved in cannabinoid administration.
Targeting the cell cycle pathway is crucial in cancer therapy, as cancer development has been
associated with dysregulation of cell cycle machinery. Cell cycle arrest represents a survival
mechanism that provides the tumor cell with the opportunity to repair its damaged DNA or can
activate the apoptotic cascade, leading to cell death (Schwartz & Shah, 2005). Several
cannabinoids have been shown to target the cell cycle through inducing cell cycle arrest in G1
phase, allowing for a reduction in the percentage of cells in S phase (See Table 1). In this study
we have demonstrated that treatment with WIN 55,212-2 induces cell cycle arrest in PC3 cells
through a mechanism involving p27, Cdk4, and pRb. Our results indicate that WIN may be
regulated via this pathway and may provide a potential target for future therapy in the field of
prostate cancer. One approach to further validating these mechanistic studies are to inhibit the
various aspects of the signalling pathway using commercially available inhibitors. This would
validate my Western blot findings and add further credence to the mechanism of action.
With regard to the in vivo portion of this thesis, the bioavailability of WIN 55,212-2 in mice is
not well documented and very limited clinical studies have been conducted on the metabolism of
WIN in humans. In the first study discussing the metabolism of WIN 55,212-2 in humans, it was
reported that WIN is extensively metabolized in the liver, with a predicted human clearance rate
of 16mL/min/kg, suggesting a fast and nearly complete metabolism in vivo, as well as a short
half-life of the drug (Mardal, Gracia-Lor, Leibnitz, Castiglioni, & Meyer, 2016). In our study,
there was no indication of toxicity after intraperitoneal administration of 5mg/kg of WIN,
demonstrating that WIN is well tolerated.
Serum concentrations and metabolism of WIN were not detailed in this study. Experiments
analyzing the precursors or metabolites of WIN in media or mice serum would help us to better
understand concentrations and time points for optimal treatment. Thus, more in-depth analysis on
the bioavailability and pharmacokinetics of WIN 55,212-2 in vivo is warranted through serum
analysis of xenograft mice models.
107
In summary, we have revealed that cannabinoids WIN and anandamide reduce the growth,
migration and invasion of prostate cancer cells. Furthermore, WIN induces cell cycle arrest in G1
and G2/M phase and alters expression levels of key proteins in the cell cycle pathway. The in
vivo studies revealed that intraperitoneal administration of 5mg/kg WIN 55,212-2 thrice weekly
in mice is well tolerated and significantly reduces the tumor growth rate, without significant
impact on body weight. Based on these findings, it would be important to conduct further studies
to assess the anti-cancer effects of WIN and anandamide and to determine their potential as a
treatment option for prostate cancer through future clinical trials.
108
Chapter 6 Future Directions and Overall Conclusion
Future Directions and Overall Conclusion
Throughout the course of my studies, several questions arose. However, due to time constraints,
we were unable to address these questions or conduct the necessary experiments to investigate
them in greater detail. In the following section, some of the emerging areas of interest from this
thesis will be outlined.
6.1 Potential In Vivo Studies
Despite the promising anti-cancer properties of cannabinoids observed in vitro, there is quite a
gap in our understanding of the roles cannabinoids and the endocannabinoid system play in the
development of prostate cancer. To further explore this aspect, the use of transgenic model
systems that very closely resemble human prostate cancer development are necessary. Utilizing a
transgenic model will allow for the ability to commence treatment at different stages of disease
progression, including prior to cancer development, after PIN lesion development, or after the
development of metastasis. This will provide better insight as to optimal time point for maximum
treatment effectiveness and will allow us to determine the effect of cannabinoid treatment on
metastasis.
Another potential extension of this thesis is the completion of in vivo studies using cannabinoid
receptor knockout mice models. The development of CB1 and CB2 knockouts in prostate cancer
mouse models will provide a clearer insight into the role of CB1/2 in prostate cancer. This could
lead to the discovery of potential alterations in tumor microenvironment through changes in
immune cell populations, downstream signalling, or the promotion of inflammation. Thus far,
there are three lines of cannabinoid receptor 1 knockout mice and two lines of cannabinoid
receptor 2 knockout mice (N. E. Buckley, 2008). These knockouts have allowed researchers to
discover the role of the endocannabinoid system in vivo (Vincenzo Di Marzo et al., 2000), how
immunomodulation is affected (Nancy E. Buckley et al., 2000), and the involvement of the CB
receptors in immune cell function and development, autoimmune inflammation, apoptosis,
chemotaxis, and infection (Buckley et al 2008). Thus far, no prostate cancer mouse models
lacking either of these receptors have been developed. In colorectal cancer (CRC), azoxymethane
109
(AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models have been developed in
which the CB1 and GPR55 receptors are knocked out. Using these models, Hasenoehrl et al
(2017) were able to demonstrate that GPR55 and CB1 play differential roles in colon
carcinogenesis where the former acts as an oncogene and the latter as a tumor suppressor. These
results help us understand the pathway in which cannabinoid receptor activation results in an
inhibitory effect on carcinogenesis and if translated to a prostate cancer model would provide
convincing evidence as to its relationship to prostate cancer.
6.2 Current and Potential Clinical Trials
Thus far, there are no clinical trials testing the use of cannabinoids as treatment for prostate
cancer. However, a few clinical trials have been launched testing the safety and efficacy of
cannabinoids in the treatment of cancers. The ongoing clinical trials involve the use of
dexanabinol, a synthetic cannabinoid which does not produce cannabis-like psychoactive effects,
and Sativex, an oromucosal spray containing a 1:1 ratio of CBD and THC.
In 2016, Phase I and II clinical trials were completed to assess the tolerability, safety and
pharmacodynamics of Sativex in combination with dose-intense temozolomide in patients with
recurrent glioblastoma. In part one of this study, 6 patients with recurrent glioblastoma
multiforme were treated with Sativex adjunct to dose-intense temozolomide to assess safety of
the combination. Part two involved 20 patients receiving either their individualized dose of
Sativex or placebo plus temozolomide. Results of this two-part safety and exploratory study was
recently published in the Journal of Clinical Oncology (Twelves et al 2017). There were no
Grade 3 or 4 toxicities associated with use of the drug, and patients in the Sativex treatment
group had a higher median survival and higher one-year survival rate compared to the
chemotherapy group.
In another clinical trial completed in 2016, varying doses of dexanabinol were used on patients
with solid tumors in order to determine the maximum safe dose and to further understand the
safety of the drug and measure any reductions in tumor size. In this Phase I clinical trial, 40
patients were assigned to 9 treatment arms where dosages of dexanabinol ranged from 2mg/kg to
36mg/kg. Results indicate the drug is well tolerated up to 22mg/kg, with minimal adverse events
reported. The patient’s progression free survival was increased to a maximum of 293 days in the
110
group treated with 22mg/kg dexanabinol, and tumor development was delayed by the treatment
(ClinicalTrials.gov Identifier: NCT01489826).
Both of these studies have shown that cannabinoid treatment offers some efficacy in cancer
patients and confirms the safety and feasibility of individualized dosing. They set the basis for
future trials aimed at evaluating the antitumoral activity of cannabinoids.
Within the field of prostate cancer, little information is available on the pharmacokinetics,
metabolism and route of administration of cannabinoids both in animals and human, making the
transition towards clinical studies challenging. Future studies should explore the
pharmacokinetics of drug administration and investigate optimal drug dosage by administering
cannabinoids to patients through different routes and/or at a variety of dosages.
Additionally, one simple study that could be done to provide a proof of principle for a potential
pathway of action is to give patients the drug over a short period of time before surgery and look
for apoptosis or any changes in the expression of key proteins in the pathways of interest (i.e.
pRb, p27, Cdk4). This would confirm our in vitro findings and help us to determine whether or
not this proposed pathway is targeted by cannabinoids in humans, allowing for the potential to
examine this pathway as a target for future therapy of prostate cancer.
6.3 Overall Conclusion
In conclusion, this work provides novel evidence for the use of cannabinoids anandamide and
WIN 55,212-2 as a therapeutic for the treatment of prostate cancer. We initially reported that
treating various prostate cancer cells with anandamide and WIN 55,212-2 causes a reduction in
proliferation, migration, and invasion, and an increase in apoptosis. Cell cycle analysis revealed
that WIN 55,212-2 caused alterations in cell cycle distribution, whereby the proportion of cells in
G1 phase increased and the proportion of cells in S phase decreased. Mechanistic studies
revealed that WIN 55,212-2 exerts its anti-cancer effects through upregulation of the cell cycle
inhibitor protein, p27, downregulation of the tumor suppressor protein, phosphorylated
retinoblastoma protein, and downregulation of the cyclin dependent kinase protein, Cdk4.
Furthermore, blocking the activation of the CB2 receptor caused cell proliferation to increase,
suggesting anandamide and WIN 55,212-2’s anti-proliferative effects may be occurring through
a CB2-dependent manner.
111
Based on our in vitro findings, we went on to investigate the effects of WIN 55,212-2 in a
xenograft model of prostate cancer. We found that administration of WIN 55,212-2 was well
tolerated by animals and tumor growth rate was significantly reduced. Mitogenicity studies
revealed decreases in cell growth following treatment with cannabinoid-containing mouse serum.
We strongly believe that the novel work presented in this thesis will further the understanding of
the therapeutic benefits of cannabinoids in prostate cancer. This information will provide a
framework for future studies and clinical trials that will help us to better understand the potential
benefit of cannabinoid use in prostate cancer and improve the way in which this disease is
managed and treated.
112
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Appendix
Contributions
With the support and guidance of my supervisor Dr. Vasundara Venkateswaran and my co-
supervisor Dr. Laurence Klotz, I have gained the knowledge and experience to design and
perform experiments, and to analyze and interpret my results. They have contributed to my
successful publications and conference presentations and have ensured that my project remained
scientifically sound.
Dr. Geneve Awong and Courtney McIntosh provided me with the necessary training to complete
flow cytometry experiments for cell cycle analysis and apoptosis, and microscopy training for
wound healing experiments. Dr. Katerina Molnarova provided me with animal care training
needed to complete my in vivo studies. Dr. Roman Bass assisted with the in vivo component of
the project. Dr. Linda Sugar, pathologist at Sunnybrook Hospital, reviewed xenograft tumours
and liver samples obtained from the in vivo component of the project and contributed to
histopathological review of all animal tissues. Results were interpreted with the assistance of my
supervisor Dr. Vasundara Venkateswaran, and my co-supervisor Dr. Laurence Klotz.