antioxidant function of pigment epithelium derived factor
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University of Wisconsin MilwaukeeUWM Digital Commons
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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