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Theses and Dissertations

August 2015

Antioxidant Function of Pigment EpitheliumDerived Factor in Adipose Tissue, the Prostate, andProstate CancerLyndsey Sandra CrumpUniversity of Wisconsin-Milwaukee

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Recommended CitationCrump, Lyndsey Sandra, "Antioxidant Function of Pigment Epithelium Derived Factor in Adipose Tissue, the Prostate, and ProstateCancer" (2015). Theses and Dissertations. 948.https://dc.uwm.edu/etd/948

ANTIOXIDANT FUNCTION OF PIGMENT EPITHELIUM

DERIVED FACTOR IN ADIPOSE TISSUE, THE PROSTATE,

AND PROSTATE CANCER

by

Lyndsey Crump

A Thesis Submitted in

Partial Fulfillment of the

Requirements for the Degree of

Master of Science

in Biomedical Sciences

at

The University of Wisconsin- Milwaukee

August 2015

ii

ABSTRACT

ANTIOXIDANT FUNCTION OF PIGMENT EPITHELIUM DERIVED FACTOR IN ADIPOSE TISSUE, THE PROSTATE,

AND PROSTATE CANCER

by

Lyndsey Crump

The University of Wisconsin- Milwaukee, 2015 Under the Supervision of Jennifer A. Doll, PhD

Aggressive prostate cancer (PCa) is positively correlated with obesity and

a high fat diet, suggesting that dysregulated lipid metabolism promotes

PCa progression. Pigment epithelium-derived factor (PEDF) regulates

angiogenesis, lipid metabolism, and has antioxidant function in other cell

types. In the prostate, PEDF inhibits angiogenesis, and its expression is

decreased in PCa. However, PEDF’s role in regulating lipid metabolism

and oxidative stress levels has not been investigated, and, as such, was

the goal of the present study. Oxidative stress levels were evaluated in

vivo and ex vivo in prostate and adipose tissues from wildtype (C56Bl/6J)

and PEDF knockout (KO) mice. In vitro, human normal prostate or PCa

cell lines were treated with PEDF with or without oleic acid (lipid overload

model). Reactive oxygen and reactive nitrogen species (ROS/RNS) and

antioxidant enzyme levels (GPx-3 and mSOD) were quantified using an

ROS/RNS detection assay and Western blot, respectively. Loss of PEDF

in adipose tissue increased ROS/RNS, indicating elevated oxidative

stress. PEDF loss also decreased GPx-3 and mSOD levels in the adipose

tissue. However, these results were not seen in the prostate or in PCa

cells. While these data do support the hypothesis that PEDF has some

iii

antioxidant function in adipose tissue, further studies are needed to

elucidate this role.

iv

TABLE OF CONTENTS

List of Figures..............................................................................................v

List of Tables..............................................................................................vi

List of Abbreviations..................................................................................vii

Acknowledgments .....................................................................................ix

INTRODUCTION.........................................................................................1

Prostate Cancer……...................................................................................1

PCa Diagnosis and Treatment…….............................................................2

PCa Risk Factors………………………………………………………………..5

Obesity and PCa…………………………………………….………………..…7

Pigment Epithelium Derived Factor………………………………..…...……11

PEDF’s Role in Tumor Progression……...................................................14

PEDF and Metabolism .............................................................................16

PEDF and Oxidative Stress……..………..................................................18

Summary and Gap in Knowledge.............................................................20

Hypothesis and Specific Aims...................................................................21

MATERIALS AND METHODS .................................................................22

RESULTS .................................................................................................30

DISCUSSION ...........................................................................................46

CONCLUSIONS AND FUTURE DIRECTIONS........................................61

REFERENCES .........................................................................................63

v

LIST OF FIGURES

Figure 1. Loss of PEDF increases oxidative stress in adipose but not

prostate tissue...........................................................................................31

Figure 2. PEDF loss exhibits no significant change on GPX-3 and mSOD

levels in adipose tissue.............................................................................32

Figure 3. PEDF loss exhibits no significant change on GPX-3 and mSOD

levels in prostate tissue.............................................................................32

Figure 4. Loss of PEDF exhibits no significant change on oxidative stress

in response to H2O2 stimulus in adipose tissue.........................................33

Figure 5. Loss of PEDF exhibits no significant change on oxidative stress

in response to H2O2 stimulus in prostate tissue........................................34

Figure 6. PEDF treatment has no effect RWPE-1 cell viability or

ROS/RNS..................................................................................................36

Figure 7. PEDF treatment does not significantly affect mSOD or GPX-3

expression in RWPE-1 cells in an in vitro obesity setting.........................37

Figure 8. PEDF treatment has no effect LNCaP cell viability or

ROS/RNS..................................................................................................39

Figure 9. PEDF treatment does not significantly affect mSOD or GPX-3

expression in LNCaP cells in an in vitro obesity setting............................40

Figure 10. PEDF treatment has no effect PC-3 cell viability or

ROS/RNS..................................................................................................42

Figure 11. PEDF treatment does not significantly affect mSOD or GPX-3

expression in PC-3 cells in an in vitro obesity setting...............................43

Figure 12. PEDF treatment has no effect DU145 cell viability or

ROS/RNS..................................................................................................45

Figure 13. PEDF treatment does not significantly affect mSOD or GPX-3

expression in DU145 cells in an in vitro obesity setting............................46

vi

LIST OF TABLES

Table 1. In vitro treatment groups…..........................................................24

vii

LIST OF ABBREVIATIONS

ADT androgen deprivation therapy

AHB hydroxybutyrate

AKT protein kinase B

AP anterior prostate

ATGL adipose triglyceride lipase

BMI body mass index

BPH benign prostatic hyperplasia

BRCA breast cancer

CGS cysteine-glutathione disulfide

CRPC castration-refractory PCa cells

DCFH-DiOxyQ DiOxyQ fluorogenic probe.

DLP dorsolateral prostate

DMEM Dulbecco's modified eagle medium

DRE digital rectal exam

FGF fibroblast growth factor

GPx glutathione peroxidase

GSSG oxidized glutathione

IL interleukin

KO knockout

MAPK mitogen-activated protein kinase

MITF microphthalmia-associated transcription factor

OA oleic acid

PBS phosphate buffered saline

PCa prostate cancer

PDGF platelet derived growth factor

PEDF pigment epithelium derived factor

PI3-K phosphatidylinositol 3-kinase

PMSF phenylmethanesulfonyl fluoride solution

PPA periprostatic adipose

viii

PSA prostate specific antigen

RNS reactive nitrogen species

ROS reactive oxygen species

RPMI Roswell Park Memorial Institute medium

SDS sodium dodecyl sulfate

SOD superoxide dismutase

SQA subcutaneous adipose

TBS tris-buffered saline

TBS-T tris-buffered saline + tween

TNF-α tumor necrosis factor α

TNM tumor node metastasis

TRAMP transgenic adenocarcinoma of mouse prostate

TSP thrombospondin

VEGF vascular endothelial growth factor

VFP visceral fat pad

VP ventral prostate

WT wildtype

ix

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my thesis advisor, Dr.

Jennifer Doll, for her continued guidance, comments, and feedback during

my time in this program. I would like to thank my thesis committee

members, Dr. Janis Eells and Dr. Dean Nardelli, for all of their support and

instruction, and I would also like to thank my fellow laboratory members,

Neil Madisen, Nizar Khamjan, and Allison Werner, for their assistance

throughout the completion of this project. Finally, I would like to thank my

family for their endless support and encouragement, and I would like to

dedicate this work to my mother and friend. Thank you for your never-

ending love and support.

x

“Somewhere, something incredible is waiting to be known.”

-Carl Sagan

1

Introduction:

Prostate Cancer

Prostate cancer (PCa) is the second most prevalent form of cancer

in men in the United States. In 2013, there were over 238,000 new cases

of PCa in American males (1). It is also a leading cause of cancer-related

death, second only to lung cancer. A male in the United States has a

16.2% chance of developing PCa over his lifetime and a 12.5% chance of

dying from it (2). Although frequently asymptomatic, the primary symptoms

experienced as a result of PCa include difficult, painful, or frequent

urination, a struggle to completely empty the bladder, any blood in the

seminal fluid or urine, pain while ejaculating, or persistent pain in the back,

waist, or pelvis (1).

The prostate is an important gland in the male reproductive system.

It is located inferior to the urinary bladder, directly adjacent to the rectum,

and it is responsible for the production and secretion of seminal fluid,

which functions in the protection and motility of the sperm (3). The

prostate is comprised of four functioning zones: the peripheral zone, the

central zone, the transitional zone, and the anterior fibromuscuar zone (4).

All zones, with the exception of the fibromuscuar zone, are glandular in

structure (4). Over 95% of PCa cases are adenocarcinomas, which are

derived from the epithelial cells within these glandular structures (5). Of

these adenocarcinomas, 68% originate in the peripheral zone, 24% in the

2

transition zone, and 8% in the central zone (6). Prostates typically weigh

14-26 grams and do not usually continue to grow once a male reaches

adulthood. Adenocarcinomas typically occur in a region of the prostate

that is also susceptible to benign prostatic hyperplasia (BPH) (6). BPH is a

post-adolescent enlargement of the prostate, which may cause the

prostate to reach up to 49 grams (7). Although BPH has many similarities

to PCa, including androgen-dependency and age-associated risk, its

presence does not increase the risk of PCa in humans (8).

PCa Diagnosis and Treatment

Diagnosing PCa can be challenging for several reasons. One of the

primary obstacles is that diagnosis involves a digital rectal exam (DRE)

and a blood test for a prostate specific antigen (PSA) value, both of which

may lead to unwillingness to participate by the patient, contributing to

greater difficulty generating an accurate diagnosis (9). Additionally, the

DRE is only 52-61% accurate (10), and the PSA antigen blood test lacks

the high specificity desired in a diagnostic exam. Catalona et al. showed

that of men with a serum PSA value ≥4.0 μg per liter, as values of <4.0 μg

per liter are considered normal, only 22% actually had PCa (11). If

elevated PSA is detected and if the DRE findings are abnormal, a

transrectal ultrasound imaging technique is used to guide the collection of

needle biopsy samples for histological evaluation (9). It is only with this

histological evaluation that a firm diagnosis of PCa can be made.

3

A common scale for assessing prostate tumors at the time of

biopsy is the Gleason score tumor grading system, which is based solely

on the histologic appearance of the tumor cells and glandular structures.

The tumor cells and glandular structures are graded on a scale of 1 to 5,

based on their histological pattern, with 1 being benign and 5 being a

highly aggressive cancer. As cancer in the prostate is typically

heterogeneous, the two most common grades are summed to produce a

final score. If the final score is less than 6, the tumor is considered to be

well-differentiated and clinically non-cancerous. If a score is equal to 6, the

tumor is considered to be moderately-differentiated and cancerous,

although it is not considered an aggressive tumor. If the score is 7-10, the

tumor is considered poorly-differentiated, cancerous, and aggressive (12).

Once the biopsy is identified as positive for PCa, the tumor can then be

staged by its histological characteristics, tumor size, and primary or

metastatic locations using the tumor, node, metastasis (TNM) system (13).

When staging cancer, it is especially important to note whether the tumor

has reached the regional lymph nodes, as this indicates that the tumor is

able to spread to other locations throughout the body (14).

A conservative management approach is generally used for men

with low grade tumors (a Gleason score of 6). To minimize the side effects

from other treatment options, this approach is generally taken when a

patient already has a life expectancy of less than 10 years (15).

Conservative management is comprised of androgen deprivation therapy

4

(ADT) (16). Androgens are sex hormones that are normally responsible for

the general maintenance and function of the prostate in healthy

individuals. However, in high amounts, they have been shown to

contribute to the pathogenesis of cancerous prostate cells (17). ADT has

been shown to increase patient survival rates by blocking the androgen

activity that promotes tumor growth (16). ADT can cause severe adverse

effects, such as hot flashes, gynecomatia, and the flare phenomenon,

which is a dangerous rise in serum testosterone levels (18). Due to these

risks, an active surveillance approach has recently gained favor over other

treatment options for low-grade cancers, particularly for men older than 70

(19). During this approach, no treatment is administered, and monitoring

tests are periodically administered to assess the growth rate of the cancer

(19).

If the tumor has remained localized to the prostate, surgery is also

an option. A radical prostatectomy is the complete removal of the prostate

gland and usually some of the surrounding tissue. It has been shown to

significantly decrease PCa related mortality rates (20). However, the side

effects of surgery may be more severe, including erectile dysfunction and

urinary leakage (21). As with most cancers, chemotherapy and radiation

therapy are also treatment options available to PCa patients.

5

PCa Risk Factors

The main risk factors for PCa are age, diet, and obesity, although

race and family history are also important contributors (22). As men grow

older, particularly over 50 years of age, they become far more susceptible

to PCa due to the higher risk of accumulating enough sporadic genetic

mutations in the prostatic epithelial cells to advance the cells to a

cancerous phenotype (9). Some of these mutations may lead to the

activation of oncogenes, which stimulate tumor growth, or to the

inactivation or down-regulation of tumor suppressor genes, which prevent

tumor growth (23). Hanahan and Weinberg have previously identified the

six definitive characteristics tumor cells must acquire through genetic

mutations to support their malignancy: 1) the ability to evade apoptosis, 2)

the capability to metastasize and invade surrounding tissues, 3) the

capacity to replicate limitlessly, 4) the capacity to sustain angiogenesis,

the growth of new blood vessels from the existing vasculature, 5) the

ability to maintain self-sufficiency in growth signal production, and 6) the

ability to evade anti-growth signals (24). After their initial publication, they

expanded these hallmarks to include 7) the deregulation of cellular

energetics, 8) the avoidance of destruction by the immune system, 9)

tumor-promoting inflammation, and 10) increased genomic instability and,

thus, increased mutation (25). In addition to sporadic mutations, some

individuals are carriers of tumor suppressor gene loss of function alleles

that predispose them to developing PCa. Such loss of function alleles

6

occur in tumor suppressor genes such as breast cancer 1 (BRCA1),

breast cancer 2, ataxia telangiectasia, checkpoint kinase 2, and BRCA1-

interacting protein 1 (26). These mutations can serve as cancer

biomarkers and aid in screening and tumor detection (9).

The clinical incidence rate of PCa is highest in African American

men, due, in part, to several polymorphisms and androgen receptor

mutations found in the African American population (27). African American

men also have higher rates of obesity, which may contribute to their

elevated PCa incidence rates (28). PCa incidence is significantly lower in

men in certain Asian countries, including China and Japan (29). However,

the incidence rate of PCa increases once a Chinese or Japanese man

immigrates to the United States (30). These data collectively suggest that

the differing clinical incidence rates between men from these regions and

men who immigrated to the United States are due to factors other than

genetic differences. Instead, environmental factors, such as altered diets

or decreased exercise regimens, may explain these differences.

The transition to a Western diet, which generally includes an

increased consumption of animal products high in fat and cholesterol and

a decreased consumption of fruits and vegetables, is suspected to play a

role. Such a change in diet would lead to increased lipid levels and weight

gain, which could contribute to the increased clinical PCa incidence rates

(31). This type of diet may also lead to a decreased intake of vitamins and

minerals (32). Increased serum levels of certain vitamins, such as vitamin

7

D, are associated with decreased clinical incidence rates of PCa (33),

indicating a role in the prevention of tumor cell proliferation.

Cholesterol, which is a steroid found in the cell membrane of animal

cells, is also consumed in high amounts in an animal-based Western diet.

Moreover, it has been shown to accumulate in the prostate during tumor

formation, and the ability of prostate cells to maintain an appropriate

balance of cholesterol is altered with increased age (34). Conversely, a

study of New York men found that diets high in phytochemicals, which are

naturally occurring chemicals in plants such as β-sitosterol, campesterol,

stigmasterol, and phytosterols, are correlated with lower PCa incidence

rates (35). Diets high in these phytochemicals are also generally low in fat

and cholesterol (36). In addition, diets with high vegetable consumption,

especially cruciferous vegetables, have been correlated with decreased

PCa incidence rates (37).

Obesity and PCa

While there are conflicting studies as to whether or not obesity

increases PCa incidence, the majority of studies show a clear association

with more aggressive prostate tumors (38). The U.S. Centers for Disease

Control and Prevention defines an obese individual as someone with a

body mass index (BMI) of 30 or higher (39). The BMI measurement is

calculated by a formula using a person’s weight and height (kg/m2) (39).

Obese individuals are more likely to die once a prostate tumor has formed

8

(40), and obesity has been associated with higher recurrence rates of PCa

after remission (41). Studies using a murine PCa model support these

observations. Llaverias et al. conducted a dietary study using transgenic

adenocarcinoma of mouse prostate (TRAMP) mice, which have a high

PCa occurrence rate due to the insertion of the SV40 T antigen gene.

After 20 weeks on a Western diet (21.2% fat and 0.2% cholesterol by

weight), they found that prostatic tumors had increased microvascular

density compared to TRAMP mice fed a control diet (0.5% fat and 0.002%

cholesterol by weight) (31), indicating that the high fat diet was promoting

angiogenesis, a well-established hallmark of cancer (44). The TRAMP

mice fed a Western diet also had increased tumor incidence, enlarged

prostates, and higher numbers of lung metastases compared to the

controls (31).

While the above studies strongly support a role for diet in PCa

progression, the mechanism by which consumption of a high fat diet and

obesity specifically contributes to PCa progression is unclear. Increased

adipose depots may promote the progression of PCa through a variety of

molecular mechanisms, including the increased secretion of pro-

angiogenic factors, growth factors or cytokines, sex hormones, and fatty

acids. The enlarged adipose tissue may also lead to a chronic

inflammatory state that could promote tumor growth. Obesity-stimulated

PCa progression is likely due to a combination of these contributing

factors (42).

9

More specifically, obesity may contribute to metabolic syndrome, a

group of metabolic abnormalities such as glucose intolerance and

dyslipidemia, the presence of which positively correlates with the presence

of PCa (43). The altered levels of certain adipose-derived cytokines, also

known as adipokines, may also be a link between obesity and aggressive

PCa. When treated with obese human periprostatic adipose tissue

secretions taken from lean and obese PCa patients, both PCa and normal

prostate epithelial cells demonstrated an increase in cellular proliferation

(44). Additionally, the unsaturated to saturated fat ratio from these tissues

was negatively correlated with the histological grade of the tumor (44).

Certain adipokines, such as leptin, are thought to promote PCa

progression. In an in vitro study, Hoda and Popken showed that leptin

treatment promoted PCa cellular proliferation and decreased the number

of apoptotic PCa cells via the mitogen-activated protein kinase (MAPK)

and phosphatidylinositol 3-kinase (PI3-K) signaling pathways (45). In

addition to an increase in cellular proliferation in vitro, Moreira et al. also

showed that treatment of RM1 PCa cells with 3T3-L1 mature adipocyte

cell conditioned media led to an increase in invasion and migration (46).

Furthermore, Noda et al. were able to obtain similar results after treating

PCa cells with leptin for 28 days, and they also found that leptin treatment

increased the expression of the leptin receptor, ObR, in the PCa cells (47).

Conversely, decreased plasma levels of a different adipokine, adiponectin,

have been linked to increased PCa incidence, tumor grade, and disease

10

stage (48). Tan et al. showed that adiponectin is down-regulated in PCa

tissues compared to those taken from BPH patients (49). They also

showed that silencing exogenous adiponectin in 22RV1 human PCa cells

further promoted tumor cell proliferation and initiated the epithelial to

mesenchymal transition process, contributing to their invasive potential

(49).

Another theory regarding the link between obesity and aggressive

PCa is the contribution of a chronic inflammatory state caused by

increased adipose tissue. Obesity leads to an increase in the release of

fatty acids and pro-inflammatory molecules. Weisberg et al. found that

pro-inflammatory macrophages accumulate in obese adipose tissue and

contribute to the secretion of tumor necrosis factor-α (TNF-α) and

interleukin-6 (IL-6), further promoting a pro-inflammatory state (50).

Subsequently, Park et al. demonstrated that obesity-fueled hepatocellular

carcinomas are dependent on the increased production of TNF-α and IL-6,

which lead to the activation of an oncogenic transcription factor, STAT3,

further supporting the role of an inflammatory state in obesity-induced PCa

progression (51).

One final theory regarding the mechanism linking obesity and

aggressive PCa is that lipid overload in both adipose and non-adipose

cells causes altered cellular metabolism. This may be an effect of a high

fat diet, obesity, and/or increased age and accumulated genetic mutations,

which are characteristics in cancer cells. Cancer cells are known to have

11

altered metabolism, with one example being the increased amount of

glucose uptake and lactate production of cancer cells, a metabolic change

known as aerobic glycolysis or the Warburg effect (52). These alterations

in cellular metabolism may lead to the increased generation of by-products

in the mitochondria that contribute to increased oxidative stress within the

tissue (53), further promoting PCa progression.

Pigment Epithelium Derived Factor

First identified as a secreted protein in retinal cells by Joyce

Tombran-Tink and Lincoln Johnson in the 1980s, pigment epithelium-

derived factor (PEDF), a 50 kDa glycoprotein, is a non-inhibitory member

of the serpin family proteins. In the eye, PEDF is present in

photoreceptors and inner retinal cells, although it may be secreted into the

interphotoreceptor matrix (54). Tombran-Tink et al. have identified PEDF

in a variety of tissues, including the liver, testis, ovaries, pancreas, and

brain (55). The full crystal structure of glycosylated PEDF is 2.85

angstroms and has an asymmetric charge distribution (56). PEDF has a

variety of functions, including the regulation of cell migration, proliferation,

the inhibition of vasculature development, the regulation of cancer cell

apoptosis, and the promotion of retinal neuron differentiation. PEDF has

two functional epitope regions, a 34-mer peptide and a 44-mer peptide

(57). The 44-mer peptide can interact with receptors to induce a

neurotrophic function, protecting and aiding in the development of neurons

12

(58), and the 34-mer peptide induces apoptosis and blocks the migration

of endothelial cells (59).

Compared to benign prostate tissue, PEDF expression is

decreased or absent in human PCa tissues (60). In Dunning rats, more

aggressive metastatic PCa tumors also have decreased PEDF expression

compared to benign tissues, suggesting that loss of PEDF may promote a

metastatic phenotype (61). Additionally, PEDF is a potent anti-angiogenic

factor; it inhibits angiogenesis, the growth of new blood vessels from the

existing vasculature, via the binding of multiple integrins on endothelial

cells, although the exact mechanisms remain unclear (62). This

angiogenic process is regulated by a number of factors that either inhibit

or promote angiogenesis. Pro-angiogenic factors include interleukin-8,

fibroblast growth factor (FGF-2), vascular endothelial growth factor

(VEGF), and platelet derived growth factor (PDGF) (63), while

angiogenesis inhibitors include thrombospondin-1 (TSP-1) (64) and

interleukin-12 (IL-12) (65). Famulla et al. have shown that during

adipogenesis in human primary adipocytes and smooth muscle cells, the

expression of PEDF significantly increases, while TNF-α and hypoxic

conditions both contribute to the down-regulation of PEDF (66). However,

in contrast, Yang et al. found that hypoxic conditions cause an increase in

the expression of PEDF and VEGF in retinal glial Müller cells (67). In a

mouse model, lack of PEDF promoted the development of stromal

vascularity and epithelial cell hyperplasia in the prostate (60). These data

13

cumulatively suggest that PEDF may potentially have therapeutic use for

cancer patients by inhibiting blood vessel growth (68).

In murine studies, in vivo PEDF has demonstrated the ability to

suppress neovascularization in the eye (69). Regulation of blood vessel

growth in the eye is necessary for proper visual function. This regulatory

function was also shown in an in vitro setting, along with the ability to

inhibit VEGF-promoted retinal endothelial proliferation, suggesting PEDF

treatment as a potentially effective treatment for retinal neovascular

diseases (70). PEDF has also demonstrated the ability to prevent light-

induced photoreceptor cell death via the activation of the Akt signaling

pathway when introduced in vitro (71). Additionally, Spranger et al.

analyzed the expression of PEDF in patients with and without hypoxic eye

diseases, including proliferative diabetic retinopathy and extensive

nondiabetic retinal neovascularization, and found that PEDF is under-

expressed in individuals with these diseases (72). They also proposed that

these patients may benefit from treatment with angiogenesis inhibitors,

including PEDF (72). PEDF has been described as having a high

therapeutic potential due to its ability to target new blood vessel growth,

easy administration as a soluble protein or via viral-mediated gene

transfer, and due to its high stability and efficacy (73). In a phase I clinical

trial, patients with advanced macular degeneration were given intravenous

injections of an adenoviral vector expressing PEDF. PEDF treatment

produced an anti-angiogenic effect in these patients, with 94% and 71%

14

experiencing a decrease or no increase, respectively, in lesion size at 3

and 6 months (74).

PEDF’s Role in Tumor Progression

PEDF expression is decreased in a variety of tumor types. In

human breast cancer tissue, PEDF expression is significantly decreased

compared to benign breast tissue (75). In melanoma cells, PEDF

expression positively correlates with microphthalmia-associated

transcription factor (MITF) expression, which is characteristic of less-

aggressive tumors (76). The silencing of MITF also down-regulates PEDF

expression in these cells, implicating a causal relationship (76). In breast

cancer, PEDF inhibited cancer cell migration and invasion via the down-

regulation of fibronectin and MMP2/MMP9 by utilizing the p-ERK and p-

AKT signaling pathways (77). An in vivo study with mouse

neuroblastomas showed that Schwann and ganglionic cells produce

PEDF, but more poorly-differentiated tumor cells do not; additionally,

recombinant PEDF was shown to have antitumor function in

neuroblastomas both in vitro and in vivo (78).

The expression of PEDF is also down-regulated in human PCa

tissues and in PCa cell lines (DU145, LNCaP, and PC-3) compared to

benign human tissues and normal prostate epithelial cells (60). Treatment

of xenograft PCa tumors from the PC-3 and DU145 cell lines with PEDF

targets angiogenesis and initiates apoptosis, supporting PEDF as a

15

treatment to suppress tumor growth (60). In vitro, increased expression of

PEDF inhibits the migratory ability of castration-refractory PCa (CRPC)

PC-3 cells. CRPC is an aggressive form of PCa currently without viable

treatment options. In vivo, PEDF decreased the growth of subcutaneous

LNCaP, PC-3, and CL1 xenograft tumors (79). Nelius et al. showed that

PEDF treatment, in conjunction with low-dose chemotherapy, significantly

increased the survival time of mice with LNCaP-derived CRPC xenograft

tumors (79).

In the PC-3 prostate cancer cell line, the overexpression of PEDF

via an adenoviral vector caused a reduction in cell proliferation (80).

Microarray analysis showed that PEDF regulates genes responsible for

angiogenesis, signal transduction, cell growth, and apoptosis (80).

Administration of PEDF to DU145 and PC-3 PCa cells has also been

shown to alter expression of genes controlling catalytic activity (17β-

hydroxysteroid dehydrogenases), cell proliferation (fibroblast growth factor

3), angiogenesis (brain-specific angiogenesis inhibitor 2), and apoptosis

(growth arrest and DNA-damage-inducible protein alpha) (81). When co-

cultured with PEDF and adipose-derived mesenchymal stromal cell

conditioned media, PC-3 cells exhibited down-regulation of a metastatic

gene (pescadillo homolog), androgen-encoding genes (androgen receptor,

sex hormone-binding globulin), genes regulating the phosphoinositide 3-

kinase- protein kinase B (AKT) signaling pathway (AKT1, B-cell lymphoma

2, S-specific cyclin-D2), an inflammatory gene (cytochrome C oxidase

16

subunit 3), and a survival gene (insulin-like growth factor 1), reducing the

invasiveness and growth potential of the PC-3 cells (82).

PEDF knockout (KO) C57BL6 mice, which do not express the

PEDF protein, develop prostate hyperplasia, a PCa precursor in mice, by

three months of age (60). Doll et al. characterized this development via

increased cellular proliferation and microvessel density (60). PEDF KO

mice also have elevated intracellular lipid accumulation in both adipose

and non-adipose tissues, including the prostate (83). Additionally, PEDF

KO mice exhibit increased visceral adipose tissue surrounding the

prostate (Doll et al., unpublished observations) and have 50% increased

total body fat compared to controls (84). PEDF deficient mice also develop

hepatic steatosis, also referred to as fatty liver disease (83).

PEDF and Metabolism

PEDF is also believed to play an important role in lipid metabolism.

Elevated lipid levels, like those occurring with a high fat diet and ectopic

lipid accumulation, cause cellular dysfunction and death in many normal

tissue types (85). In murine skeletal muscles, PEDF has been shown to

induce the metabolism of fatty acids via the promotion of lipolysis (86), a

mechanism dependent on adipose triglyceride lipase (ATGL) (87). Db/db

obese mice, which are null for the leptin receptor (88), are known to

develop hepatic steatosis (89). In one study, the authors observed

decreased PEDF levels in the liver of db/db mice compared to wildtype

17

C57BL/6J liver tissues (89). Together, these data suggest that PEDF

regulates lipid deposits in the liver. Chung et al. also showed that PEDF

regulates lipid metabolism in hepatocytes treated with recombinant PEDF

(83). However, cancer cells have altered metabolisms, and how PCa cells

react to lipid overload and the role of PEDF in this process remains

unknown.

In one study using C57BL/6J mice fed a high fat diet (60% calories

from fat) for 16 weeks, the average plasma PEDF concentration, 4.9 ± 1.6

ng/ml, was increased 3.2-fold as compared to mice fed a control diet (86).

In a given patient population, elevated serum PEDF levels positively

correlate with the presence of metabolic syndrome (90). In human

adipocytes, secretion of PEDF is also increased by insulin, a hormone

secreted to aid in the regulation of lipid metabolism (66). Additionally, in

Japanese patients, elevated serum levels of PEDF have been positively

associated with the metabolic features of type 2 diabetes, including

elevated levels of triglycerides, TNF-α, and creatinine (91). While these

data implicate PEDF in disease processes, it is still unclear if PEDF plays

a causative role or if it is functioning as a counter-balance system in

glucose and/or lipid metabolism, as proposed by Gattu et al (92).

PEDF and Oxidative Stress

Ectopic lipid accumulation, such as occurs in an obese setting, can

cause increased oxidative stress in cells. Cellular damage due to oxidative

18

stress results from increased levels of reactive oxygen species (ROS) and

reactive nitrogen species (RNS). These molecules are highly reactive

oxygen-containing and nitrogen-containing molecules, respectively, and

they are the by-products of normal cellular reactions, primarily in the

mitochondria. Low levels of ROS/RNS molecules are crucial for multiple

biochemical processes, including those for cellular differentiation (93),

apoptosis (94), and immunity (95). Some examples of ROS include the

hydroxyl radical, nitric oxide, and the superoxide anion, while examples of

RNS include nitrogen dioxide and peroxynitrite. The levels of ROS and

RNS are tightly regulated by antioxidants, such as glutathione peroxidase

(GPx) and superoxide dismutase (mSOD), due to the potential cellular

damage that these molecules can inflict (96). GPx functions to prevent the

lipid peroxidation of cellular membranes by reducing free hydrogen

peroxide to water, while mSOD catalyzes the conversion of superoxide to

hydrogen peroxide, which is then reduced to water by GPx or other

antioxidants (97). When ROS/RNS levels rise above the antioxidant

capacity, cellular damage occurs. Decreasing lipid levels can reduce

oxidative stress. For example, decreasing dietary lipid consumption has

been shown to reduce ROS and other oxidative stress markers in the

endothelial cells of rabbits (98). Damaged molecules, such as proteins

and lipids, must then be degraded and replaced. Damage to cellular

nucleic acids, such as DNA and RNA, can result in mutations that, in turn,

may promote tumor formation and progression.

19

In PCa cells, ROS levels are increased as compared to normal

WPMY1 and RWPE1 prostate epithelial cell lines (99). Higher ROS levels

have also been found in more aggressive PCa cell lines, such as PC-3

(99). In humans, PCa and prostatic intraepithelial neoplasia tissues also

have decreased levels of certain antioxidant enzymes, including SOD1,

mSOD, and catalase, further implicating changes in oxidative stress in

tumor development and progression (100). Additionally, sera metabolic

data reveal changes in circulating markers of oxidative stress in PEDF KO

mice as compared to wildtype (WT) C57BL/6J mice. WT mice fed a high

fat diet (32.2% kcal from fat) had elevated levels of oxidized glutathione

(GSSG) and cysteine-glutathione disulfide (CGS) and decreased levels of

2-hydroxybutyrate (AHB) (Doll et al., unpublished observations), reflective

of a normal response to the oxidative stress induced by a high fat diet

(101). In PEDF KO mice fed the same high fat diet, AHB levels

significantly increased, but GSSG and CGS remained unchanged

compared to PEDF KO fed a control diet. On the control diet, PEDF KO

mice had significantly decreased levels of AHB compared to the WT on a

control diet (Doll et al. unpublished observations). As discussed above,

PEDF KO mice develop a prostatic intraepithelial neoplasia-like

hyperplasia phenotype, a precursor lesion to cancer. Then the PEDF KO

mice were fed a high fat diet, the precursor phenotype was pushed to

cancerous phenotype (Doll et al. unpublished observations).

20

Additionally, PEDF has demonstrated antioxidant activity by

decreasing H2O2-induced apoptosis in granuloma cells (102), although

another study on granuloma cells suggested that PEDF treatment induced

ROS production in a concentration-dependent manner (10-100nM) (103).

PEDF treatment also blocked angiotensin II-induced ROS generation in

human lymphoblastic leukemia MOLT-3 T cells (104). These data

cumulatively suggest that PEDF loss interferes with the normal cellular

response to oxidative stress.

Summary and Gap in Knowledge

The presence of aggressive PCa is positively correlated with

obesity and consumption of a high fat diet (38). PEDF, an angiogenesis

inhibitor, has been shown to regulate lipid metabolism and have

antioxidant properties in normal cell types (89) and may play a key role in

lipid regulation in the prostate. Additionally, PEDF has demonstrated

antioxidant activity in some other cell types (102). While PEDF function as

an angiogenesis inhibitor in the prostate has been well established (60),

its function in lipid regulation and as an antioxidant have not been

established in the prostate. Although ROS has been linked to increased

tumor progression (99), PEDF’s antioxidant ability has not been explored

in the prostate, adipose tissue, or in PCa cells. Here, studies were

conducted to begin to elucidate the molecular pathway of PEDF-mediated

regulation of lipid metabolism and to establish a potential antioxidative role

21

of PEDF in prostate tissue, adipose tissue, and PCa cells. Toward these

studies, two specific aims were addressed.

Hypothesis and Specific Aims:

Central Hypothesis: PEDF demonstrates antioxidant activity in the

prostate and adipose tissue by reducing ROS/RNS and increasing

antioxidant enzyme levels.

Specific Aim 1: Quantify oxidative stress levels in prostate and

adipose tissues taken from wildtype C57BL/6J and PEDF KO mice.

Working hypothesis: Loss of PEDF will lead to increased ROS/RNS levels

and decreased antioxidant enzyme levels in both prostate and adipose

tissues.

Specific Aim 2: Determine the effect of PEDF on lipid accumulation-

induced changes to oxidative stress levels in prostate and PCa cells.

Working hypothesis: Lipid accumulation will increase ROS/RNS and

decrease antioxidant enzyme levels in prostate and PCa cells, while the

addition of PEDF will decrease ROS/RNS and increase antioxidant

enzyme levels.

22

Materials and Methods:

Prostate and Adipose Tissue Collection and Explant Culture

The ventral (VP), anterior (AP), and dorsolateral (DLP) lobes of the

prostate and the subcutaneous (SQA), visceral fat pad (VFP), and

periprostatic (PPA) adipose tissues were dissected from 4-month-old wild

type and PEDF KO mice. All mice were of C57BL/6J background. The

mice were anesthetized with isoflurane and euthanized by cervical

dislocation. The tissues were exposed via a midline incision through the

abdominal wall. The SQA tissue was excised, and an incision was made

through the peritoneum. The VFP was harvested, and then the PPA tissue

and VP, DLP, and AP prostate lobes were excised under a dissecting

microscope. All tissues were immediately frozen in liquid nitrogen for later

use in oxidative stress assays or placed in a tube on ice for explant

culture.

For the explant culture, the weight of each tissue was recorded,

and the tissue was washed twice with PBS in a culture dish. The tissues

were cut and weighed, then plated with serum-free (basal) Dulbecco's

Modified Eagle Medium (DMEM) a concentration of 25 μg/mL. The explant

cultures were treated with 10 μM H2O2 or an equal volume of PBS and

incubated at 37°C for 3 hours. After incubation, the tissues were collected,

washed with PBS, and stored at -80°C until use in the oxidative stress

assay.

23

Cell lines and culture conditions

RWPE-1, DU145, PC-3, and LNCaP cell lines were purchased from

American Type Culture Collection Company (Manassas, Virginia). RWPE-

1 cells were originally isolated from normal human prostatic epithelial cells

and transfected with human papillomavirus 18 to immortalize them (105).

DU145 cells were originally isolated from a brain metastasis of a human

prostate adenocarcinoma (106). PC-3 cells were isolated from a human

prostate adenocarcinoma that metastasized to bone (107). LNCaP cells

were isolated from a human prostatic carcinoma that metastasized to a

regional lymph node (108). Cells were maintained in growth media

[Keratinocyte growth media for RWPE-1; DMEM with 10% FBS and 1%

P/S for DU145 and PC-3, and Roswell Park Memorial Institute (RPMI)

media for LNCaP] at 37°C with 5% CO2.

In vitro treatment with PEDF, Oleic Acid, and H2O2

Cells were plated at 25,000 cells/cm2 on 10 cm tissue culture

dishes and incubated at 37°C, 5% CO2 overnight. After incubation, the

growth medium was gently aspirated, and the cells were washed with

sterile PBS. Basal media was added to each plate, and the cells were

returned to the incubator for 4 hours. The basal media was gently

aspirated, and new basal media was added with the treatments outlined in

Table 1. Group 1, with only basal media, served as the negative control.

24

The cells were incubated with oleic acid (Sigma-Aldrich, Saint Louis, MO)

and PEDF (Sigma-Aldrich, Saint Louis, MO) only for 45 hours at 37°C, 5%

CO2, then H2O2 was added for the final 3 hours. OA was chosen to mimic

an obesity setting in vitro, as it has been observed to promote the

proliferation of PCa cells and inhibit PEDF expression (Doll lab,

unpublished observations). After incubation, the conditioned media and

the cell lysates were collected as described below.

Table 1: In vitro treatment groups

Treatment

Group PEDF

(50 nm) OA

(1 mM) H2O2

(10 μM)

1 - - -

2 + - -

3 + + -

4 + + +

5 - + -

6 - + +

7 + - +

8 - - +

Cell lysate and conditioned media collection

The conditioned media (CM) was collected and placed on ice. The

CM was then centrifuged at 2500 x g for 10 minutes to pellet any cellular

debris and transferred to a new tube. 1X protease inhibitor cocktail

(Sigma-Aldrich, Saint Louis MO) and 100 nM phenylmethanesulfonyl

fluoride solution (PMSF, Sigma-Aldrich, Saint Louis MO) was added to a

final dilution of 1:100. The cells remaining on the plate were washed with

PBS and trypsinized. 4 mL of growth media was added to stop

25

trypsinization, and the cells and media were collected into a conical vial. A

50 μL aliquot of the cell solution was mixed with 50 μL of 0.4% trypan blue

solution (Sigma-Aldrich, Saint Louis, MO) to provide a cell count and

permit the assessment of cell viability using a Cellometer (Nexcelom,

Lawrence, MA). 20 μL of the cell mixture was placed in each chamber of a

Cellometer counting slide. The cell aliquots were counted in duplicate on

the Cellometer, per manufacturer’s instructions. The total cell number, live

cell number, and cell viability were recorded. The remaining cells were

pelleted by centrifugation at 800 x g for 8 minutes, and the supernatant

was gently aspirated. The cell pellets and conditioned media were stored

at -80°C until needed.

Oxidative Stress Assay

Oxidative stress levels were assessed using the Oxiselect STA-347

ROS/RNS Assay Kit from Cell Biolabs, Inc. (San Diego, CA), which

quantifies the total amount of ROS/RNS in a tissue or cell lysate sample,

using a dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ) fluorogenic

probe. The primary tissues, explant culture tissues, cell lysates, and

conditioned media were assayed. Cell pellets were resuspended in sterile

PBS at a concentration of 1 x 106 cells/mL. Cells were sonicated at 40%

amplitude for 30 seconds in duplicate. Tissues were homogenized in

sterile PBS at a concentration of 25 mg/mL using a Kontes motor and

disposable pestles (Fisher Scientific, Waltham, MA). A standard curve was

26

created using H2O2. Samples and standards were loaded onto a 96-well

plate. A catalyst was added to each well as the DiOxyQ probe was primed

and stabilized in solution per the manufacturer’s instructions. The probe

was added to each well and incubated, protected from light, for 30

minutes. The plate was then read at the excitation and emission

wavelengths of 480 nm and 530 nm, respectively, using a Synergy HT

plate reader (Biotek, Winooski, VT) and Gen5 software.

Assessment of GPx-3 and mSOD Levels

The total amount of protein in each cell lysate and tissue

homogenate sample was quantified using a Coomassie dye-binding

assay. 490 μL of Coomassie dye was aliquoted into microfuge tubes, and

5 μL of each sample was added. Samples were incubated for 10 minutes,

and then transferred to a 96 well plate, along with albumin standards in

PBS. The standards had final concentrations of 0 μg/mL, 25 μg/mL, 125

μg/mL, 250 μg/mL, 500 μg/mL, 750 μg/mL, 1000 μg/mL, and 1500 μg/mL.

The plate was read at 595 nm using the Synergy HT plate reader. Cell

pellets were resuspended in 1 mL sterile PBS and sonicated at 40%

amplitude for 30 seconds in duplicate. Tissues were homogenized in

tissue extraction reagent (Invitrogen, Carlsbad, CA) at a concentration of

10 mg/mL using a Kontes motor and disposable pestles (Fisher Scientific,

Waltham, MA). The volume of cell lysate and tissue homogenate needed

27

for 20 μg of protein was transferred to a new tube and lyophilized in 3-4 20

minute cycles each, with 15 minutes of medium-high heat.

A Western blot was then used to compare the amount of GPx3 and

mSOD in the cell lysate of each treatment group, as the levels of these

enzymes are indicators of oxidative stress within the cells. The proteins

were separated using a sodium dodecyl sulfate (SDS)-polyacrylamide gel

comprised of a 5% stacking gel and a 7.5% resolving gel in a 0.1% SDS

running buffer. 20 μg of the total protein was placed in a microfuge tube

with an equal volume of 2X Laemmeli sample loading buffer. The lysate

samples were incubated at 100°C for 10 minutes to ensure denaturation of

the proteins. 30 μL of each sample and 10 μL of a Precision Plus protein

molecular weight standard (BioRad, Hercules, CA) was loaded onto the

gel. The gel was run at 110 volts for 1.5 hours to separate the proteins

based on size, with GPx-3 measuring 92 kDa, mSOD measuring 22 kDa,

and GAPDH measuring 37 kDa.

Next, the proteins in the gel were transferred to a nitrocellulose

membrane by electroblotting. Prior to setup, the membrane was soaked in

methanol for 30 seconds, then rinsed 10 times with diH2O. The gel and

membrane were positioned between 4 pieces of Whatman filter paper and

2 sponges soaked in 1X Tris-glycine transfer buffer for 10 minutes prior to

blotting. The transfer setup was placed in a transfer cassette, and the

transfer was conducted at 70 volts for 2 hours on ice. The membrane was

28

washed with 1X tris-buffered saline (TBS) + 0.1% tween (TBS-T) for 5

minutes to remove any residual SDS.

To prepare for antibody hybridization, the membrane was blocked

using TBS with 5% powdered milk to prevent any nonspecific binding by

the antibody to the membrane. The membrane was then washed with

TBS-T and incubated with the primary antibody at a 1:1000 dilution in

TBS-T with 5% milk overnight at 4°C. Anti-GPx3, anti-mSOD, and anti-

GAPDH (Cell Signaling, Danvers, MA) antibodies were the primary

antibodies used for protein detection. GAPDH served as a control for total

protein loading between samples. After washing 3 times for 10 minutes

with TBS-T, a horseradish peroxidase-linked secondary antibody was

used to bind to the primary antibody for 1 hour at room temperature. 3

final TBS-T washes were performed, and chemiluminescence was used to

visualize the protein. Super Signal West Pico ELC substrates (Thermo

Fisher Scientific, Milwaukee, WI) were mixed in a 1:1 ratio. The membrane

was placed, protein-face down, into the solution for 1 minute. The

membrane was then saran-wrapped and placed in a film cassette. In a

dark room, several film exposures at various times were performed, and

the film was developed with an automated TI-200 developer (Kodak,

Rochester, NY). The exposure time was adjusted as needed to obtain an

appropriate image for comparison between lanes. If no bands were visible

after development, the membrane was re-washed with TBS-T and a more

sensitive chemiluminescence detection system was used. Super Signal

29

West Femto ELC substrates (Thermo Fisher Scientific, Milwaukee, WI)

were mixed in a 1:1 ratio, and the membrane was placed, protein-face

down, into the solution for 1 minute. The membrane was placed in the

cassette and exposed as described above.

To probe with a second primary antibody, the first antibody was

stripped from the membrane. The membrane was washed with TBS-T for

5 minutes, then submerged in a boiling 0.1% SDS stripping buffer and

allowed to cool to room temperature. It was washed thrice with TBS-T.

The second primary antibody was hybridized, beginning with the blocking

step as described above. The amount of GPx3 and mSOD was

normalized to GAPDH as fold over values. Protein levels were compared

between sample lanes, relative to untreated wild type tissues, by

densitometry using ImageJ software.

Statistics

All in vitro experiments were repeated in duplicate. A Student’s t-

test was used to analyze the results. For analyses where normality or

equal variance was not reached, a Mann Whitney U test was used. For

each tissue type, the ROS/RNS assay levels were compared between WT

and PEDF KO groups. For each cell type, the levels of oxidative stress

markers were compared between each treatment group. Oxidative stress

markers were compared between the PCa cell lines and the normal

prostate cell line for each treatment group. All analyses were performed by

30

statistical analysis software within the SigmaPlot program (v12.0, Systat

Software, Inc, San Jose California). A result was considered statistically

significant when P≤0.05.

Results:

Part I. In vivo and ex vivo studies

The effect of PEDF loss on oxidative stress in prostate and adipose

tissues

The oxidative stress levels in prostate and adipose tissues were

quantified by both an ROS/RNS assay (Oxiselect) and by comparison of

GPx3 and mSOD levels. Of the three adipose tissue depots tested, the

PPA and the VFP tissues from PEDF knockout mice had significantly

increased ROS/RNS levels compared to wildtype tissues as a control

(Figure 1A; n=4/group; p=0.0380 and p=0.0342, respectively). The

ROS/RNS levels in the SQA tissue from PEDF KO mice were also

increased; however, this did not reach statistical significance (Figure 1A;

n=4/group; p=0.057). In the prostate tissues, while a trend toward increase

in ROS/RNS levels was observed in the VP and AP tissues in the PEDF

KO mice, this was not statistically significant (Figure 1B).

31

Loss of PEDF exhibits no significant change on GPX-3 and mSOD

levels in adipose and prostate tissue

Homogenized adipose (SQA, PPA, and VFP) and prostate (VP,

DLP, and AP) mouse tissues were assessed by Western blot for GPx-3

and mSOD levels. For both the adipose and prostate tissues, there were

no statistically significant changes in mSOD or GPx-3 expression levels

between PEDF KO and control wildtype tissues (Figure 2 and Figure 3,

respectively). Within the adipose tissue depots, PEDF KO mice showed a

trend toward decrease in mSOD expression in both VFP and PPA,

although none of these differences reached statistical significance (Figure

2A). Similarly to mSOD, GPx-3 expression in the SQA, VFP, and PPA was

also decreased in PEDF KO mouse tissues (Figure 2B). In the prostate

Figure 1. Loss of PEDF increases oxidative stress in adipose but not prostate tissues. Prostate and adipose tissues were collected from wildtype C57Bl/6J and PEDF KO mice (n=4/group). Tissues were homogenized, and RNS/RNS levels were quantified using an Oxiselect ROS/RNS assay in the (A) subcutaneous adipose (SQA), periprostatic adipose (PPA), visceral fat pad (VFP), and (B) ventral prostate (VP), dorsolateral prostate (DLP), and anterior prostate (AP) tissue homogenates. Levels from PEDF KO mice were normalized to wildtype. * p<0.05 compared to WT, # p = 0.057 compared to WT

32

tissues, no noticeable differences were seen between the PEDF KO and

WT mSOD expression levels (Figure 3A), and only the DLP tissues

demonstrated a change in enzyme expression with an increase in GPx-3

expression (Figure 3B).

Figure 2. PEDF loss exhibits no significant change on GPX-3 and mSOD levels in adipose tissue. Adipose tissues were collected from wildtype C57Bl/6J and PEDF KO mice (n=2/group). Tissues were homogenized, and GPx-3 and mSOD levels were assessed via Western blotting from subcutaneous adipose (SQA), periprostatic adipose (PPA), and visceral fat pad (VFP) tissue homogenates. Levels were normalized to GAPDH, and Image J software was used to assess relative (A) mSOD

and (B) GPx-3 expression levels.

Figure 3. PEDF loss exhibits no significant change on GPX-3 and mSOD levels in prostate tissue. Prostate tissues were collected from wildtype C57Bl/6J and PEDF KO mice (n=2/group). Tissues were homogenized, and GPx-3 and mSOD levels were assessed via Western blotting from ventral prostate (VP), dorsolateral prostate (DLP), and anterior prostate (AP) tissue homogenates. Levels were normalized to GAPDH, and Image J software was used for densitometry, and relative (A) mSOD and (B) GPx-3 expression levels are presented. No values reached statistical significance.

33

Absence of PEDF does not alter response to H2O2 ex vivo in adipose

or prostate tissues

To assess if absence of PEDF impairs the tissue response to an

oxidative stress stimulus, in a pilot study, SQA, PPA, and VFP adipose

tissues were treated with or without H2O2 for 3 hours ex vivo (n=2/group).

An Oxiselect assay was performed on the tissues to measure the total

ROS/RNS concentration in each tissue sample. No statistically significant

differences were observed between the wildtype and PEDF KO adipose

tissues (Figure 4). Surprisingly, the H2O2-treated tissues also exhibited no

significant changes compared to the respective untreated tissues.

Likewise, no statistically significant differences were observed between

the wildtype and PEDF KO prostate tissues (Figure 5), nor did the tissues

cultured with H2O2 exhibit any statistically significant change compared to

the untreated tissues.

Figure 4. Loss of PEDF exhibits no significant change on oxidative stress in response to H2O2 stimulus in adipose tissue. Adipose tissues were collected from wildtype C57Bl/6J and PEDF KO mice (n=2/group). Tissues were treated with H

2O

2 or PBS, and then homogenized. RNS/RNS levels were assessed for

the (A) subcutaneous adipose tissue, (B) periprostatic adipose tissue, and (C) visceral fat pad using an Oxiselect ROS/RNS assay. No values reached statistical significance.

34

Part II. In vitro obesity studies

Section A. RWPE-1

PEDF treatment has no effect on RWPE-1 cell viability

Normal prostatic epithelial cells, RWPE-1, were treated with PEDF

in the presence and absence of OA. H2O2 was added to specified

treatment groups to induce the production of ROS and as a positive

control for the ROS/RNS assay. The effect of PEDF on cell viability was

being measured with and without OA present to simulate an obese

environment. A negative control group received only basal media in lieu of

additional treatment. In the presence and absence of OA, PEDF had no

significant effect on cell viability of RWPE-1 cells (Figure 6A). OA

treatment alone also produced no change in cell viability, nor did H2O2

Figure 5. Loss of PEDF exhibits no significant change on oxidative stress in response to H2O2 stimulus in the prostate. Prostate tissues were collected from wildtype C57Bl/6J and PEDF KO mice (n=2/group). Tissues were treated with H

2O

2

or PBS and homogenized. ROS/RNS levels were assessed from (A) ventral, (B) dorsolateral, and (C) anterior prostate tissue homogenates using the Oxiselect assay. No values reached statistical significance.

35

treatment alone. Only the dual OA and H2O2 treated cells exhibited a

significant decrease in cell viability compared to untreated (Figure 6A,

p=0.00851).

PEDF treatment does not affect oxidative stress in RWPE-1 cells

An Oxiselect assay was performed on PEDF, OA, and H2O2 treated

RWPE-1 cell lysate and conditioned media to measure the total ROS/RNS

concentration in each sample. No statistically significant changes in

ROS/RNS levels were observed in RWPE-1 cell lysate (Figure 6B) or

conditioned media (Figure 6C). Interestingly, cells treated with H2O2 alone

did not exhibit an increase in ROS/RNS levels. The expression levels of

mSOD, GPx-3, and GAPDH were also assessed in the cell lysates of the

treated RWPE-1 cells via Western blotting and antibody hybridization. For

densitometry, mSOD (Figure 7A) and GPx-3 (Figure 7B) expression levels

were normalized to GAPDH expression within each sample. These results

show that treatment with PEDF, in the presence and absence of OA, did

not significantly affect the expression of mSOD or GPx-3 compared to the

untreated control group or the OA-treated group.

36

Figure 6. PEDF treatment has no effect on prostate or PCa cell viability or ROS/RNS. Normal prostate epithelial cells, RWPE-1, were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and OA treatments were for

48 hours, with the H2O

2 treatment for the last 3 hours of the treatment. Conditioned

media was collected, then cells were trypsinized, an aliquot was taken for cell count, and cell lysate was collected. (A) Cell viability was assessed on a Cellometer, and ROS/RNS levels were quantified in the cell lysate (B) and conditioned media (C) using an Oxiselect assay. ** p<0.00002 compared to untreated cells. Results shown are the combined data of two independent experiments.

37

Section B. LNCaP

PEDF treatment has no effect on LNCaP cell viability

Androgen-dependent PCa cells, LNCaP, were treated with PEDF in

the presence and absence of OA and H2O2. PEDF treatment alone did not

affect the viability of LNCaP cells (Figure 8A). OA treatment alone

significantly decreased cell viability compared to untreated (Figure 8A,

p=0.0135). In the presence of both PEDF and H2O2, OA further decreased

cell viability compared to the PEDF and H2O2-treated (Figure 8A,

p=0.0195). Additionally, dual treatment with OA and H2O2 significantly

decreased viability compared to untreated (Figure 8A, p=0.00308).

Figure 7. PEDF treatment does not significantly affect mSOD or GPX-3 expression in RWPE-1 cells in an in vitro obesity setting. RWPE-1 cells were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H2O2 (10 µM). PEDF and OA treatments were 48 hours, while the H2O2 treatment was 3 hours. The cells were trypsinized, and the cell lysate was collected. mSOD and GPx-3 expression was assessed via Western blotting of the cell lysates. Protein levels were normalized to GAPDH, and Image J software was used to assess relative mSOD (A) and GPx-3 (B) expression. No statistically significant changes were observed with PEDF treatment in the presence and absence of OA. Each experiment was performed in duplicate.

38

PEDF treatment does not affect oxidative stress pathways in LNCaP

cells

An Oxiselect assay was performed on PEDF, OA, and H2O2 treated

LNCaP cell lysate and conditioned media to measure the total amount of

ROS/RNS in each sample. For the cell lysate, only the cells treated with

PEDF, OA, and H2O2 demonstrated an increase in fluorescence, relative

to untreated cells, indicative of elevated ROS/RNS (Figure 8B, p=0.0110).

In the conditioned media samples, surprisingly, the presence of OA alone,

and when combined with PEDF and H2O2, decreased ROS/RNS levels

compared to the untreated control (Figure 8C, p=0.0261). However, when

OA and H2O2 were added together, the ROS/RNS significantly increased

compared to the control group. Similarly to RWPE-1 cells, H2O2 treatment

did not produce the expected increase in ROS/RNS in either the

intracellular or secreted environment, with the exception of the conditioned

media treated with PEDF and H2O2.

The expression levels of mSOD, GPx-3, and GAPDH were

assessed in PEDF, OA, and H2O2 treated LNCaP cell lysates via Western

blotting and antibody hybridization. After performing densitometry, mSOD

(Figure 9A) and GPx-3 (Figure 9B) expression levels were normalized to

GAPDH expression within each sample. In both the presence and

absence of OA, treatment with PEDF did not significantly affect the

expression of mSOD or GPx-3 compared to the untreated control group or

the OA-treated group.

39

Figure 8. PEDF treatment has no effect on LNCaP cell viability or ROS/RNS. LNCaP PCa cells were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and OA treatments were 48 hours, while the H

2O

2 treatment

was for the final 3 hours of treatment. Conditioned media was collected, then cells were trypsinized, an aliquot was taken for cell count, and the cell lysate was collected. (A) Cell viability was assessed on a Cellometer, and ROS/RNS levels were quantified in the cell lysate (B) and conditioned media (C) using an Oxiselect assay. * p<0.05, ** p<0.009 compared to untreated cells. Results shown are the combined data of two independent experiments.

40

Section C. PC-3

PEDF treatment has no effect on PC-3 cell viability

Androgen-independent PCa cells, PC-3, were treated with PEDF

both with and without OA and H2O2. In the presence and absence of OA,

the addition of PEDF had no effect on PC-3 cell viability compared to OA-

alone treated and untreated, respectively (Figure 10A). PEDF also had no

effect on cell viability in the presence of an additional ROS inducer, H2O2,

compared to cells that only received the H2O2 treatment. Treatment with

both OA and H2O2 did not decrease cell viability compared to untreated.

However, OA treatment alone and PEDF and OA dual treatment

Figure 9. PEDF treatment does not significantly affect mSOD or GPX-3 expression in LNCaP cells in an in vitro obesity setting. LNCaP cells were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and OA

treatments were 48 hours, while the H2O

2 treatment was 3 hours. The cells were

trypsinized, and the cell lysate was collected. mSOD and GPx-3 expression was assessed via Western blotting of the cell lysates. Protein levels were normalized to GAPDH, and Image J software was used to assess relative mSOD (A) and GPx-3 (B) expression. No statistically significant changes were observed with PEDF treatment in the presence and absence of OA. Each experiment was performed in duplicate.

41

decreased viability compared to the untreated control (Figure 10A,

p=0.041and p=0.029, respectively).

PEDF treatment does not affect oxidative stress pathways in PC-3

cells

To measure the total ROS/RNS in PEDF, OA, and H2O2 treated

PC-3 cells, an Oxiselect assay was performed on cell lysate and

conditioned media from each sample. No statistically significant changes

in ROS/RNS levels were observed in PC-3 cell lysate (Figure 10B) or

conditioned media (Figure 10C). Additionally, neither cell lysate nor

conditioned media treated with H2O2 exhibited an increase in ROS/RNS

levels. The expression levels of mSOD, GPx-3, and GAPDH were

assessed in the treated PC-3 cell lysates via Western blotting and

antibody hybridization (Figure 11). In both the presence and absence of

OA, treatment with PEDF did not significantly affect the expression of

mSOD or GPx-3 compared to the untreated control group or the OA-

treated group.

42

Figure 10. PEDF treatment has no effect on PC-3 cell viability or ROS/RNS. PCa cells, PC-3, were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF

and OA treatments were 48 hours, while the H2O

2 treatment was for the final 3 hours of

treatment. Conditioned media was collected, then cells were trypsinized, an aliquot was taken for cell count, and the cell lysate was collected. (A) Cell viability was assessed on a Cellometer, and ROS/RNS levels were quantified in the cell lysate (B) and conditioned media (C) using an Oxiselect assay. * p<0.05, ** p<0.009 compared to untreated cells. Results shown are the combined data of two independent experiments.

43

Section D. DU145

PEDF treatment has no effect on DU145 cell viability

Androgen-independent PCa cells, DU145, were treated with PEDF

both with and without OA and H2O2. PEDF had no effect on DU145 cell

viability compared to untreated (Figure 12A). PEDF had no effect on cell

viability in the presence OA alone compared to only OA-treated cells

(Figure 12A). PEDF also had no effect on cell viability in the presence of

H2O2 compared to only H2O2-treated cells (Figure 12A). Dual OA and

H2O2 treatment decreased cell viability compared to the untreated control,

Figure 11. PEDF treatment does not significantly affect mSOD or GPX-3 expression in PC-3 cells in an in vitro obesity setting. DU145 cells were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and OA treatments

were 48 hours, while the H2O

2 treatment was 3 hours. The cells were trypsinized, and

the cell lysate was collected. mSOD and GPx-3 expression was assessed via Western blotting of the cell lysates. Protein levels were normalized to GAPDH, and Image J software was used to assess relative mSOD (A) and GPx-3 (B) expression. No statistically significant changes were observed with PEDF treatment in the presence and absence of OA. Each experiment was performed in duplicate.

44

as did OA treatment alone (Figure 12A, p=0.0245 and p=0.00137,

respectively).

PEDF treatment does not affect oxidative stress pathways in DU145

cells

To measure the total ROS/RNS in PEDF, OA, and H2O2 treated

PC-3 cells, an Oxiselect assay was performed on cell lysate and

conditioned media from each sample. DU145 cells treated with only H2O2

or with H2O2 and OA had a significant increase in intracellular ROS/RNS

levels (Figure 12B, p=0.00237 and p=0.0329, respectively). OA treatment

and PEDF treatment had no significant effect on ROS/RNS, although in

the presence of OA, the addition of PEDF did decrease ROS/RNS

compared to the OA-treated group (Figure 12B). In the conditioned media,

no statistically significant changes in ROS/RNS levels were observed in

DU145 samples as compared to untreated cells (Figure 12C).

The expression levels of mSOD, GPx-3, and GAPDH were

assessed in PEDF, OA, and H2O2 treated DU145 cell lysates by Western

blotting and antibody hybridization. As measured by densitometry, PEDF

alone, or with OA treatment, did not significantly affect the expression of

mSOD (Figure 13A) or GPx-3 (Figure 13B) compared to the untreated

control group or the OA-treated group. H2O2 treatment also had no

significant effect on mSOD or GPx-3 expression.

45

Figure 12. PEDF treatment has no effect on DU145 cell viability or ROS/RNS. PCa cells, DU145, were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and OA treatments were 48 hours, while the H

2O

2 treatment

was for the final 3 hours of treatment. Conditioned media was collected, then cells were trypsinized, an aliquot was taken for cell count, and the cell lysate was collected. (A) Cell viability was assessed on a Cellometer, and ROS/RNS levels were quantified in the cell lysate (B) and conditioned media (C) using an Oxiselect assay. * p<0.05, ** p<0.009 compared to untreated cells. Results shown are the combined data of two independent experiments.

46

Discussion

The presence of aggressive PCa is positively correlated with

obesity and a high fat diet (38), suggesting that dysregulated lipid

metabolism may promote PCa progression (89). One potential contributing

mechanism resulting from dysregulated lipid metabolism is increased

production of ROS/RNS. PEDF regulates lipid metabolism and exhibits

antioxidant function in other cell types (102), and its expression is

decreased in PCa (60). However, whether PEDF loss contributes to

dysregulated lipid metabolism and increased oxidative stress levels has

Figure 13. PEDF treatment does not significantly affect mSOD or GPX-3 expression in DU145 cells in an in vitro obesity setting. DU145 cells were treated with PEDF (50 nM), oleic acid (OA; 1 mM), and/or H

2O

2 (10 µM). PEDF and

OA treatments were 48 hours, while the H2O

2 treatment was 3 hours. The cells were

trypsinized, and the cell lysate was collected. mSOD and GPx-3 expression was assessed via Western blotting of the cell lysates. Protein levels were normalized to GAPDH, and Image J software was used to assess relative mSOD (A) and GPx-3 (B) expression. No statistically significant changes were observed with PEDF treatment in the presence and absence of OA. Each experiment was performed in duplicate.

47

not previously been investigated. The objective of this study was to

determine if PEDF regulates oxidative stress levels in the prostate and in

adipose tissues, operating under the hypothesis that PEDF demonstrates

antioxidant activity by reducing ROS/RNS and increasing antioxidant

enzyme levels.

In vivo effects of PEDF on oxidative stress

To test the effect that loss of PEDF has on oxidative stress

identifiers, ROS/RNS levels were assessed in WT and PEDF KO mouse

adipose and prostate tissues and in PEDF-treated prostate and PCa cells.

In the adipose tissue, PEDF KO mice exhibited higher levels of ROS/RNS

compared to their wildtype counterparts, with PPA and VFP tissue levels

reaching statistical significance and SQA levels approaching significance

(Figure 1A). These data support the central hypothesis of this study,

suggesting that loss of PEDF directly increases oxidative stress in these

tissues. This result is consistent with previous in vitro studies in granuloma

cells (102), leukemia cells (104), and retinal pericytes (109), which have

documented decreased ROS generation with exogenous PEDF treatment.

The increased ROS/RNS levels in PEDF KO adipose tissues may also be

due to the increased visceral adipose tissue surrounding the prostate (Doll

et al., unpublished observations) or overall increased fat mass of PEDF

deficient mice (84), as it is known that obese adipose tissues have

increased oxidative stress levels (110). This is a likely cause of this

48

increase, as C57Bl/6J mice fed a high fat diet have experienced a two-fold

increase in the generation of ROS in adipocytes and visceral adipose

tissues (111). Additionally, C57Bl/6J mice on a high fat diet have

increased fatty acid oxidation and GPx-1 expression in the liver and

adipose tissues (112). In humans, higher BMI is associated with elevated

lipid peroxidation and lower plasma adiponectin levels (110). Pou et al.

also showed that increased SQA tissue and visceral adipose tissue is

correlated with higher inflammatory and oxidative stress biomarkers (113).

In comparison to other cell types, human primary adipocytes also have

increased expression of PEDF (66).This increase in PEDF expression

may plays a role in the regulation of oxidative stress in adipose tissues.

In PPA and VFP tissues, loss of PEDF decreased the expression of

mSOD (Figure 2A); however, as tissues from only 2 mice were assessed,

this did not reach statistical significance. Similar results were seen in the

expression of GPx-3 in the PPA, VFP, and SQA (Figure 2B). These trends

are consistent with the in vitro data presented by Amano et al., who

showed that the administration of PEDF to pericytes, which are the

contractile cells that wrap around endothelial cells, increased the mRNA

expression of GPx-3 (109), and Zhang et al., who showed that PEDF

treatment increased the expression of SOD1 protein in pericytes (114).

The trend of decreased mSOD and GPx-3 expression, coupled with the

increased ROS/RNS levels in the PEDF KO mice, support an antioxidant

function for PEDF in these adipose tissue depots. These data support the

49

proposed mechanism that PEDF regulates antioxidant enzyme levels.

These results are also consistent with a more recent study by

Sheikpranbabu et al., who showed that PEDF inhibits the generation of

ROS via increased expression of SOD and GPx in porcine retinal

pericytes (115). The relative changes in both the SQA and visceral

adipose tissues may be important as increased visceral adipose tissue

and higher visceral adipose to SQA ratio has demonstrated stronger

correlations with negative health impacts, including a higher risk for more

aggressive PCa (116). Interestingly, the SQA, which exhibited the smallest

change in mSOD and GPx-3 expression, also had the highest relative

ROS/RNS levels (Figure 1A). Overall, these data support the hypothesis

that PEDF has antioxidant function in the visceral adipose tissue depots.

While the VP, DLP, and AP tissues from PEDF KO mice showed a

similar trend toward increased ROS/RNS as compared to wildtype tissues,

none of these differences neared statistical significance likely due to the

variance in the samples (Figure 1B). These trends are consistent with

reported in vitro studies describing the decreased generation of ROS with

PEDF treatment in other cell types (102, 104, 109). Interestingly, although

the ROS/RNS trends in the prostate tissues are similar the results seen in

the adipose tissues, the antioxidant enzyme expression levels are quite

different. In the PEDF-deficient mice, only the DLP showed a notable

decrease in GPx-3 expression compared to wildtype; no other tissue

demonstrated a change (Figure 3B). In the VP, mSOD expression is

50

decreased in the PEDF KO mice, although no other tissue demonstrated a

change (Figure 3A). While the trend toward an increase in ROS/RNS

levels in the adipose tissues may support an antioxidant role of PED,

these data suggest that the antioxidant function is not via the regulation of

mSOD and GPx-3 expression levels in prostate tissue lobes. The trend

toward increased ROS/RNS in the PEDF deficient VP may be due to

decreased mSOD expression. In the human prostate, mSOD has

increased expression in mid-grade tumors compared to noncancerous

prostate (117). Future studies should expand the number of animals per

group to confirm if the noted trends are statistically significant, for both the

prostate and adipose tissues.

Ex vivo oxidative stress challenge

H2O2 is routinely used as a positive control for the induction of

oxidative stress (118). In an effort to determine if absence of PEDF

affected a tissue’s response to an oxidative stress inducer, a pilot study

was performed comparing wildtype and PEDF KO adipose and prostate

tissues responses to H2O2 treatment ex vivo. In the explant-cultured

adipose tissues, H2O2 did not significantly increase ROS/RNS values. This

unexpected result could be due to technical difficulties with the Oxiselect

assay, such as the inability to consistently detect H2O2. However, as

ROS/RNS levels were detected in primary untreated tissues (uncultured),

this is unlikely. No previous publications describe the use of the Oxiselect

51

kit on H2O2-treated tissue, nor were any listed on the company’s website

(119). Therefore, this assay may not be suitable for the detection of H2O2

in these tissue types. Other factors that may have affected the assay

results include the concentration of H2O2 and the length of the treatment.

While the H2O2 treatment was optimized on cell culture treatments, it is

possible that higher concentrations are necessary for tissue treatments. It

is also possible that the treatment time was not long enough to produce a

measurable response. Additionally, there were no significant differences

between WT and PEDF KO adipose and prostate tissues. However, the

ROS/RNS levels in the PEDF KO SQA (Figure 4A) and PPA (Figure 4B)

tissues were elevated compared to wildtype, although these trends did not

reach statistical significance. The PPA and SQA tissue trends are

consistent with the ROS/RNS data from the primary tissue assay (Figure

1A). While the trend is similar to the fresh tissue analysis, the lack of

statistical significance in the ex vivo experiment is likely due to only

tissues from two animals per group being tested for these two adipose

tissues. Increasing the number of animals may allow for a clearer picture

of the differences between these groups.

In contrast, in the VFP tissue, a small decrease in oxidative stress

was observed in the PEDF KO tissue compared to the wildtype without

H2O2 treatment (Figure 4C). This data is not consistent with the primary

tissue data (Figure 1A). The discrepancy may be due to the fact that, with

the ROS/RNS assay, samples were run as a single sample (i.e. not in

52

duplicate as with other samples) due to the small amount of tissue

obtained. The PPA culture samples could not be completed in duplicate

due to the extremely low weight of the harvested tissues.

Similar results were observed in the prostate tissue samples

(Figure 5). The elevated ROS/RNS values for the PEDF KO tissues

observed in the fresh tissue assays were not observed in the PEDF KO

tissues in the ex vivo assay. However, the addition of H2O2 did increase

ROS/RNS values in the VP, although not to a statistically significant level.

This trend was evident in both the WT and PEDF KO tissues (Figure 5A).

The difference seen in the VP compared to the DLP and AP may be due

to the structural differences between these prostate lobes. Want et al.

showed that, in TRAMP mice, over 66% of prostate tumors are found in

the VP, whereas only 11.1% are in the DLP and 5.6% in the AP (120),

suggesting that the VP may be more susceptible to tumor-promoting

stimuli.

In vitro effects of PEDF on cell viability

The in vitro experiments in this study assessed the effect of PEDF

on OA treated cells. OA stimulates triglyceride accumulation (121), which

serves as an in vitro mimic of obesity. As with the ex vivo studies, H2O2

was added as a positive control for induction of ROS/RNS. Despite careful

optimization for each cell line for optimal cell numbers for the Oxiselect

assay, low levels of fluorescence were obtained and large variances within

53

samples were observed. Due to this high variance, few statistically

significant differences were observed. However, there were discernable

trends. In the absence of OA, PEDF treatment did not affect RWPE-1 cell

viability (Figure 6A). However, in the presence of OA, the addition of

PEDF increased cell viability compared to OA-alone treated groups,

although not to a significant level. PEDF treatment alone had no effect on

the viability of the PCa cell lines, LNCaP (Figure 8A), PC-3 (Figure 10A),

and DU145 (Figure 12A). This result was interesting, as PEDF has shown

the ability to inhibit the formation of tumors when over-expressed in PC-3

cells (57). Therefore, it is possible that PEDF exhibited very low activity in

this setup. Based on the data presented here, PEDF does not have a

significant effect on the viability of normal or PCa cells.

The PCa cell lines all exhibited a decrease in viability with OA

treatment alone and dual OA and PEDF treatment compared to the

untreated control groups, while the normal RWPE-1 cells did not. The only

RWPE-1 group that experienced a significant decrease in viability was the

group treated with OA and H2O2 (Figure 6A). In contrast, every treatment

group that included OA, including the OA-only group, produced a

decrease in viability for LNCaP (Figure 8A) and DU145 (Figure 12A). In

PC-3 cells, all treatment groups with OA produced a decrease in viability

except for OA and H2O2 dual-treated cells (Figure 10A). These data

support that lipid accumulation actually contributes to cell death in PCa

cells, while normal prostate epithelial cells are more resilient to an obesity-

54

challenge. These findings are consistent with a study by Hagen et al., in

which they showed that a 48 hour 100 μM OA treatment significantly

decreased the viability in DU145 cells and showed a trend toward

decreased viability in PC-3 and LNCaP cells, although this did not reach

significance (122). However, these findings are inconsistent with a study

presented by Gasmi et al., in which they treated RWPE-1, LNCaP, and

PC-3 cells with two isomers of oleic acid, trans-vaccenic and cis-vaccenic,

at a concentration of 100 μM and found no change in viability in any of the

cell lines (123). These different results could be due to different viability

tests being used. In the Hagen study, a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) assay was used, while in the Gasmi

study, the WST-1 cell viability assay used. The difference between the

studies could also be due to the difference in culture times, with Gasmi et

al. assessing 24-hour treatments (123), whereas Hagan et al treated cells

for 48 hours (122). However, Gasmi et al. did find that five other fatty

acids, jacaric acid, punicic acid, alpha-calendic acid, beta-calendic acid,

and catalpic acid decreased the viability of LNCaP and PC-3, but not

RWPE-1 (123). These fatty acids, which are also found in dietary sources

like plant seed oils, likely induced a change in viability due to their

octadecatrienoic structure; whereas, in contrast, trans-vaccenic and cis-

vaccenic are both octadecaenoic (123).

Treatment with H2O2 at a concentration of 10 μM did not decrease

cell viability for RWPE-1 (Figure 6A), LNCaP (Figure 8A), PC-3 (Figure

55

10A), or DU145 (Figure 12A) cells compared to untreated controls. These

results are partially supported in a previous study by Freitas et al., which

showed that treatment with 500 μM H2O2 did not affect the viability of PC-3

cells, although it did lead to a significant decrease in the viability of

RWPE-1 cells (124). This difference is likely due to the higher

concentration of H2O2 used by Freitas et al. Additionally, Bao et al.

showed that treatment with H2O2 did not affect the viability of DU145 cells,

although it did lead to a decrease in the viability of RWPE-1 cells in a

dose-dependent manner (125).

In vitro effects of PEDF on oxidative stress

For normal prostate RWPE-1 cells, no statistically significant

changes in intracellular (Figure 6B) or secreted (Figure 6C) ROS/RNS

levels were detected due to the large amount of variance with the assay.

However, in the presence of OA, the addition of PEDF did decrease the

amount of intracellular ROS/RNS, although not to a significant level.

Neither OA nor PEDF appears to influence mSOD (Figure 7A) or GPx-3

(Figure 7B), suggesting that obesity may not affect antioxidant regulation

via the upregulation of these particular enzymes. These data do not

support a role for PEDF in the regulation of this antioxidant pathway for

normal RWPE-1 prostate cells.

For LNCaP cells, only cells treated with PEDF, OA, and H2O2 had a

significant increase in intracellular ROS/RNS compared to the untreated

56

control group (Figure 8B). In the presence of OA, the addition of PEDF

decreased the amount of intracellular ROS/RNS, although not to a

significant level, and the addition of H2O2 to the OA and PEDF-treated

group significantly increased intracellular ROS/RNS (Figure 8B). In

LNCaP conditioned media, ROS/RNS values showed a significant

decrease with PEDF and OA treatment, OA treatment alone, and OA and

H2O2 treatment (Figure 8C). With the addition of H2O2 alone, secreted

ROS/RNS increased compared to the untreated control (Figure 8C). OA

and PEDF have no effect on mSOD (Figure 9A) or GPx-3 (Figure 9B)

expression, suggesting that neither lipid accumulation nor PEDF

influences antioxidant regulation via the upregulation of these particular

enzymes. Combined with the ROS/RNS data, an antioxidant function for

PEDF in LNCaP cells is not supported.

PC-3 cells showed no change in intracellular (Figure 10B) or

secreted (Figure 10C) ROS/RNS values. OA and PEDF exhibited no

effect on mSOD (Figure 11A) or GPx-3 (Figure 11B) expression. These

results suggest that neither lipid-accumulation nor PEDF influence

antioxidant regulation via the upregulation of these antioxidant enzymes.

DU145 cells only showed an increase in intracellular ROS/RNS

with OA and H2O2 dual treatment and H2O2 treatment alone (Figure 12B).

No significant changes in secreted ROS/RNS levels were observed

(Figure 12C). As with RWPE-1 and LNCaP, in the presence of OA, the

addition of PEDF decreased the amount of intracellular ROS/RNS,

57

although not to a significant level. This is, however, an interesting trend to

note. The lack of effect seen with OA treatment was an unexpected result,

as OA treatment was being used to simulate a mock-obesity environment

in vitro. OA treatment was expected to increase oxidative stress, as

obesity contributes to oxidative stress in PCa cells (126). PCa cells treated

with serum from obese mice experience an increase in markers of aerobic

glycolysis and ROS (127). OA was chosen for this model system due to its

ability to stimulate the accumulation of triglycerides in PC-3 cells (Doll et

al. unpublished observations). However, based on these findings, other

fatty acids, such as punicic acid or catalpic acid may be considered for

future studies. Additionally, these results are inconsistent with data

presented by Bao et al., who found that treatment with 1 mM H2O2

increased ROS in RWPE-1 cells, but not DU145 cells (125). This

discrepancy is likely due to the higher concentration of H2O2 used by Bao

et al, which led to the elevated ROS seen in RWPE-1 cells.

While OA treatment alone increased GPx-3 expression in DU145

cells (Figure 13B), PEDF appeared to cause an increase in mSOD

expression (Figure 13A). In a prospective clinical study, Li et al. showed

that the risk of aggressive PCa is increased in the presence of a somatic

mSOD polymorphism that decreases enzymatic function, supporting a role

for mSOD in PCa tumor progression (128). Similarly, Yu et al. reported

that GPx-3 is inactivated in PCa, and overexpression of GPx-3 in PCa

cells led to their reduced invasive potential (129).

58

The overall ROS/RNS values and mSOD/GPx-3 expression data

from the prostate and PCa cells presented here do not seem to support an

antioxidant effect of PEDF in the prostate. There were no changes in the

antioxidant enzyme expression levels, except for in the DU145 cell line, in

which PEDF increased mSOD expression. PEDF treatment of prostate

and PCa cells induced no changes in ROS/RNS levels, further supporting

this lack of anti-oxidant function in the prostate. Additionally, the mock

obesity treatment of OA did not induce a change in mSOD and GPX-3

expression, with the exception of in DU145 (Figure 13), where OA

increased GPx-3 expression. However, this overall lack of effect seen with

OA treatment was surprising, as OA treatment was expected to increase

oxidative stress due to the previously reported contribution to oxidative

stress by obesity in PCa cells (126). While our studies do not necessarily

support a link between PEDF and antioxidant enzyme levels in the

prostate, some studies have shown a link between a high fat diet and

decreased antioxidant enzyme expression. In TRAMP mice, a transgenic

prostate cancer model, a high fat diet led to a decrease in GPx-3

expression in the DLP and AP as compared to animals on a control diet

(130).

It has been noted that there are several evident differences in the

results between the three PCa cell lines used in this study. Despite the

fact that LNCaP, PC-3, and DU145 are all cells derived from human PCa

tumors, there are distinct genetic differences between these cell lines,

59

most notably with the well-characterized tumor suppressor genes, PTEN,

P53, and Rb. LNCaP produces non-functional PTEN (131) protein, but

normal p53 (132) and Rb protein (133). In contrast, PC-3 produces no

PTEN (131) or P53 (132) protein, but normal Rb protein (133), and DU145

produces functional PTEN protein (131), but non-functional P53 (132) and

Rb protein (133). The presence, absence, or loss of function of these

prominent anti-tumor proteins may affect the cellular responses to the

given treatments and lead to the observed varying results between the cell

lines.

Overall, the high variability in the data of the cell treatments is likely

due, in part, to the Oxiselect assay. This assay was first introduced in

2010 (119) and was also a novel assay to our lab. While the

manufacturer’s instructions appear straightforward, several technical

difficulties were experienced in optimizing and completing the assays for

these studies. Most notably, despite several optimization trials to

determine the most appropriate concentration of cells needed to detect

ROS/RNS, concentration levels were often on the low end of the

spectrum, below the quantification values of the standard curve, but well

above background levels. This is why the values for the in vitro studies are

reported as fluorescence values, relative to the untreated groups.

However, as fluorescence is a direct by-product of ROS/RNS in this

assay, these values are still appropriate representations of oxidative

stress levels within the cell lysate and conditioned media groups. In

60

contrast, homogenates from fresh tissue produced calculable ROS/RNS

concentration values. No published studies reporting similar problems with

low ROS/RNS readings with this assay were found with a literature

search. Additionally, no reports on the use of this assay on PCa cells were

identified.

Another unexpected result observed in the Oxiselect assay data is

that the addition of H2O2 did not significantly increase the ROS/RNS

values of RWPE-1, LNCaP, and PC-3 cells, despite the fact that H2O2 is

an inducer of oxidative stress. It is possible that the concentration of H2O2

was not high enough to cause a significant change in the cells. Despite

the fact that in several optimization trials run prior to these experiments,

ROS/RNS levels with the concentration of H2O2 used were detectable.

The difference between the optimization trials and the experimental trials

were the number of cells per plate when the 3-hour H2O2 treatment was

added. For the optimization trials, the treatment was added when plates

were near confluent, although no cell counts were conducted prior to

treatment. For the experimental trials, cells were plated at 25,000

cells/cm2 and left overnight before adding the treatments the next

morning. Therefore, the difference in cell density on the plate may be

responsible for these varying results. Nonetheless, future studies of this

nature should assess the effect of cell number on ROS/RNS levels as well

as test higher concentrations of H2O2.

61

Conclusions and Future directions

The purpose of this study was to determine if PEDF has an

antioxidant function in adipose tissue and prostate, including in PCa. The

data presented in this thesis partially support the central hypothesis, in

that loss of PEDF in vivo led to a significant increase in total ROS/RNS

levels in murine adipose tissue. These adipose tissues also had trends of

lowered antioxidant enzymes (mSOD and GPx-3) further supporting an

antioxidative role for PEDF. PEDF loss in normal prostate tissues led to an

increase in total ROS/RNS in the prostate, although not to a statistically

significant level. However, similar results were not observed in the normal

prostate or in PCa cells in vitro. In these studies, PEDF did not appear to

influence total ROS/RNS or antioxidant expression levels, except for the

increase of mSOD in DU145 treated cells. Further studies are needed to

validate PEDF’s antioxidant function in the adipose tissue and to

determine if the trends seen in the prostate and PCa cells are significant.

In particular, the in vivo studies should be repeated with larger group

numbers. Increasing the number of mice may help to determine if the

trends seen in the expression levels of mSOD and GPx-3 were statistically

significant. In addition, as these data do not support a strong role for GPx3

or mSOD in the prostate, other antioxidant enzymes, such as catalases or

peroxiredoxins, should be investigated in future studies to attempt to

62

better understand the underlying molecular mechanisms responsible for

the increased oxidative stress in PEDF deficient mice.

Several adjustments should also be made in the in vitro

experiments for future studies; a higher concentration of H2O2 should be

used, and other fatty acids should be used to simulate an oxidative stress-

inducing obese environment. A well-established antioxidant, such as

vitamin C or vitamin E, could also be introduced into the treatment system

to determine if there is the expected decrease in oxidative stress. These

future studies would help to expand upon this work and further establish a

full antioxidant role for PEDF. Overall, the work presented in this study

does support that PEDF has an antioxidant function in adipose tissue,

although its role in the prostate remains unclear. It also supports the

justification of the outlined future experiments towards better establishing

PEDF’s function in obesity-mediated PCa progression.

63

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