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Monoamine oxidase A inhibitors and androgen receptor antagonists regulate mitochondrial function in prostate cancer cells
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Monoamine oxidase A inhibitors and androgen receptor antagonists regulate mitochondrial function in prostate cancer cells
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Content
Monoamine Oxidase A Inhibitors and Androgen
Receptor Antagonists
Regulate Mitochondrial Function in
Prostate Cancer Cells
by
Jinghua Cai
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(Pharmaceutical Science)
May 2019
2
Acknowledgements
First, I would like to express the deepest appreciation to my thesis professor, Dr.
Jean C. Shih. Her ideas and knowledge were the basis of my research. Without her guidance
and persistent help this thesis would not have been completed.
I would like to thank Dr. Yong Zhang and Dr. Enrique Cadenas for serving on my
thesis committee and providing me with valuable insights. My deep gratitude also goes to
Ronald W. Irwin, Bin Qian, Jami Pei-Chuan Li, Zeyang Li. I would like to especially thank
Dr. Irwin for his guidance and assistance in my thesis writing.
Finally, I would like to thank my family for the continuous support they have given
me throughout the years. Without their support, I would not have been able to study and
work in such a great environment and reach my academic goals.
3
Table of Contents
Acknowledgements ………………………………………………………………….….…2
Abbreviations ……………………………………………………………………….……...5
Abstract ……………………………………………………………………………….……6
List of Figures ……………………………………………………………………………...8
List of Tables ………………………………………………………………………………9
1. Introduction ……………………………………………………………………………10
1.1 Cancer
1.2 Prostate cancer
1.3 Androgen receptor
1.4 Monoamine oxidase
1.5 Mitochondrial function
1.6 Objective of the project
2. Materials and methods ………………………………………………………………..19
2.1 Cell culture and supplies
2.2 Western blot analysis
2.3 Seahorse assay
2.4 Mitochondrial oxidation of fatty acid
2.5 Statistical analysis
3. Results ………………………………………………………………………………….23
3.1 MAOA expression and androgen sensitivity in human prostate cancer cells
3.2 Clorgyline produces additive effect in combination with enzalutamide in
inhibiting the cell proliferation in LNCaP cells
3.3 Clorgyline, enzalutamide and their combination treatment decrease oxygen
consumption rate (OCR) and mitochondrial function in LNCaP cells
3.4 Dose response for clorgyline, enzalutamide and their combination treatment in
oxygen consumption rate in LNCaP cells
3.5 Clorgyline, enzalutamide and their combination treatment decrease glycolytic
function in LNCaP cells
4
3.6 Clorgyline decrease fatty acid dependency in LNCaP cells
3.7 Clorgyline, enzalutamide and their combination treatment decrease oxygen
consumption rate and mitochondrial function in 22RV1 cells
3.8 Clorgyline, enzalutamide and their combination treatment have no effect on
oxygen consumption rate (OCR) and mitochondrial function in PC3 cells
3.9 Clorgyline, enzalutamide and their combination treatment decrease the expression
of androgen receptor in LNCaP cells
4. Discussion and conclusion …………………………………………………….……….41
Reference ………………………………………………………………………………….45
5
Abbreviations
2DG 2-deoxy-glucose
ADP Adenosine diphosphate
ADT Androgen deprivation therapy
ATP Adenosine triphosphate
CNS Central nervous system
CRPC Castrate-resistant prostate cancer
DHT Dihydrotestosterone
DRE Digital rectal exam
ECAR Extracellular acidification rate
ETC Electron transport chain
FBS Fetal bovine serum
MAOA Monoamine Oxidase A
MAPK Mitogen-activated protein kinase
mtDNA Mitochondria DNA
OCR Oxygen consumption rate
OXPHOS Oxidation phosphorylation
PCa Prostate cancer
PSA Prostate specific antigen
PVDF Polyvinylidene fluoride
ROS Reactive oxygen species
SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis
RPMI Roswell Park Memorial Institute
6
Abstract
Prostate cancer is the most common diagnosed non-cutaneous cancer and second
leading cause of death in men in the United States. One of the biggest problems in the
prostate cancer field is that patients become resistant to therapy and the prostate cancer will
continue to proliferate and metastasize to other organs most often the lymph nodes or bone
(Sardana et al., 2008).
Monoamine oxidase A (MAOA), a mitochondrial enzyme, which can catalyze the
oxidative deamination of monoamine neurotransmitters and generate hydrogen peroxide. It
has been found that MAOA is highly expressed in high grade prostate cancer and can
mediate prostate cancer progression and metastasis (Wu et al., 2014). MAOA inhibitors can
effectively reduce cancer growth (Wu et al., 2014). Androgen receptor antagonists such as
enzalutamide is used to treat prostate cancer, however prostate cancer patients often become
treatment resistant leading to aggressive androgen independent cancer. Here, we investigate
the effect of MAOA inhibitors alone or in combination with enzalutamide to understand
how MAOA and AR inhibition regulate metabolic mechanisms in prostate cancer cells.
In this study, we analyzed pharmacologic responses in three prostate cancer cell
lines: LNCaP-androgen sensitive with high MAOA activity, CWR22RV1-androgen
independent with high MAOA activity, and PC3-androgen independent with low MAOA
activity. Our results showed that treatment of prostate cancer cells with MAOA inhibitor
clorgyline decrease the mitochondrial function of LNCaP cells and 22RV1 cells.
Enzalutamide alone was moderately effective in decreasing mitochondrial respiratory
function and the effect was enhanced when combined with clorgyline, further decreasing
oxygen consumption rate. Glycolytic function was likewise reduced by both treatments
indicating that overall metabolic function and cancer cell growth were suppressed. Cell
7
viability data confirmed these results. Expression of MAOA was not altered by clorgyline
or enzalutamide. Expression of androgen receptor was decreased by both enzalutamide and
clorgyline. Furthermore, we found that clorgyline reduced fatty acid dependency in LNCaP
cells.
Collectively, these results indicate that MAOA inhibitors suppress prostate cancer
cell metabolism and further suppress metabolism and cell growth in combination with
antiandrogen therapy. MAOA inhibitors provide a novel therapeutic approach to regulate
mitochondrial function and fatty acid dependency in androgen resistant advanced prostate
cancer.
8
List of Figures
Figure 1. Summary of the major androgen receptor signaling pathways and MAOA
signaling pathway in prostate cancer.
Figure 2. Images of LNCaP, 22RV1 and PC3 cell lines.
Figure 3. The effect of clorgyline when combined with enzalutamide on the cell
proliferation in LNCaP cells.
Figure 4. Clorgyline, enzalutamide and combination treatment reduced prostate
mitochondrial aerobic metabolism.
Figure 5. Dose response for clorgyline, enzalutamide and their combination treatment in
oxygen consumption rate of LNCaP cells.
Figure 6. Analysis of clorgyline, enzalutamide and their combination treatment regulation
of extracellular acidification rate (ECAR).
Figure 7. XF analysis of fuel flex shows reduced dependency on fatty acids after 1uM
clorgyline exposure in LNCaP cells.
Figure 8. Clorgyline, enzalutamide and combination treatment reduced prostate
mitochondrial aerobic metabolism in androgen independent 22RV1 cells.
Figure 9. Clorgyline, enzalutamide and combination treatment have no effect on prostate
mitochondrial aerobic metabolism in androgen independent and MAOA independent
PC3 cells.
Figure 10. Clorgyline and enzalutamide reduced the expression of AR.
9
List of Tables
Table 1. Characteristics of three human prostate cancer cell lines: LNCaP, 22RV1, and
PC3.
10
1 Introduction
1.1 Cancer
Normally, human cells grow and divide to form new cells when the body needs them.
In the normal cell, complex signaling mechanisms control cell division and apoptosis.
Cancer is the rapid production of abnormal cells that divide without stopping and can
metastasize into surrounding tissues. Cancer cells are able to influence the normal cells,
molecules and blood vessels. This makes it difficult for the body to work the way it should.
Cells in any part of body can become cancerous. Metastasis is the major cause of death from
cancer. The most common type of cancer refers to a malignant tumor originating from
epithelial tissue in a process known as epithelial mesenchymal transition (Kalluri and
Weinberg, 2009).
1.2 Prostate cancer
Prostate cancer (PCa) is the development of cancer in the prostate, which is a gland
in the male reproductive system. Almost all prostate cancers are adenocarcinomas which
develop from the gland cells. The primary function of the gland is to secrete fluid that is
part of semen. The one important component of this fluid is prostate specific antigen (PSA)
which is secreted by the epithelial cells of the prostate gland. PSA levels are typically less
than 4 ng/mL in men with healthy prostate but the exact number may vary and is often
elevated in prostate cancer or other prostate disorders (Catalona et al., 2017). The main
function of PSA is to stop sperm cells from sticking together. PSA can leave the prostate
gland and travel through the blood in the body, thus why we can measure the PSA level by
taking the blood test. For healthy people, only a small amount of PSA leaks out of the cells
of the glands into prostate and get into the bloodstream.
11
However, as the prostate cancer continues to grow and eventually metastasize, the cells in
the glands of the prostate become disorganized and the layer between the prostate and blood
vessel becomes damaged. Therefore, as more PSA leaks into the blood stream the stage of
prostate cancer advances. Besides PSA test, digital rectal exam (DRE), Xrays, or cystoscopy
can also be used to diagnose prostate cancer.
Prostate cancer is the most common diagnosed non-cutaneous cancer and second
leading cause of death in men in the United States (Shafi et al., 2015). According to
statistics, 1 in 9 men will have prostate cancer during their lifetime, and about 1 man in 41
will die of it. Prostate cancer is mainly developed in older men and African-American men.
The American Cancer Society’s estimates that there will be 174,650 new cases and 31,620
prostate cancer–related deaths are expected in the United States in 2019
(Cancer.org, 2019).
1.3 Androgen receptor
Androgens, natural (such as testosterone or dihydrotestosterone (DHT)) or synthetic
steroid hormones (such as the anabolic steroids nandrolone or metribolone (R1881)) that
activate the androgen receptor, regulate the rate of cell proliferation and metabolism and
regulate male characteristics. Androgens are mainly produced by the male testes and adrenal
gland. Androgen receptors are primarily located in the cytoplasm as a classical ligand
(androgen)-activated transcription factor that belongs to the nuclear receptor superfamily.
Androgen receptor also plays a critical role both in the growth and development of benign
and malignant prostate tissue. AR protein has four main domains, including N-terminal
regulatory domain, DNA-binding domain, hinge region and ligand-binding domain. One of
12
the cytochromes P450 enzyme called 5αreductase is highly expressed within the prostate
and it can convert testosterone to DHT
(Lonergan and Tindall, 2011). After binding to dihydrotestosterone, androgen receptors are
activated and inducing conformational change. Then the AR-dimers translocate to the
nucleus, where they bind to their target genes and regulates their expression (Figure 1).
Furthermore, androgen receptor can also be activated in the absence or very low
dihydrotestosterone levels. The activation signals come from several non-repulsive
mechanism including extracellular peptides such as interleukin-6 (Lonergan and Tindall,
2011).
Since prostate cancer is primarily an androgen-dependent disease, AR become the
main therapeutic target for the PCa. Androgen deprivation therapy (ADT) is the firstline
treatment for locally advanced or metastatic disease (Shafi et al., 2015) and it has long been
used as treatment of choice for PCa. Although androgen ablation therapy initially decreases
the tumor volume and expression of the androgen-dependent gene, most patients within 5
years become resistant to ADT and progress to a state which is known as castrate-resistant
prostate cancer (CRPC), because despite the low level of hormone, the proliferation of
CRPC cells continue depend on androgen receptor activation. The reactivation of androgen
receptors within these cells shows an adaptive stress response after exposure to ADT
(Adeniji et al., 2017). Moreover, the role of AR mediated gene activation in CRPC is
supported by the expression of androgen-regulated gene (Gregory et al., 2001). Possible
mechanisms for AR reactivation in CRPC include
AR gene amplification or AR protein overexpression, stimulation of ligand-independent AR
activity by mitogen-activated protein kinase (MAPK) cascade, and the expression of
constitutively active AR splice variants (Leung and Sadar, 2017). In the absence or presence
13
of low androgen levels, AR interacts with Src activates MAPK pathways therefore enhance
cell proliferation and survival in a non-genomic fashion (Figure 1). The
immunohistochemical study by Lonergan showed that 63% of CRPC samples have
increased Src1 and TIF2 expression (Lonergan and Tindall, 2011).
Figure 1. Summary of the major androgen receptor signaling pathways and MAOA signaling pathway in
prostate cancer. 1. Phosphorylation (P) happens before and after androgen binding. As binding to
dihydrotestosterone (DHT), AR translocates to the nucleus, forms dimerization, binds to its target genes
and regulates their expression. Androgen receptor can also be transactivated in the absence, or in very
low levels of dihydrotestosterone. Enzalutamide blocks the binding of androgen (Lonergan and Tindall,
2011). 2. Monoamine oxidases catalyze the oxidative deamination of monoamines and generate ammonia
and hydrogen peroxide (Shih et al., 1999). MAO inhibitors restrain the activity of MAOA.
Enzalutamide is a second-generation androgen receptor antagonist, and it was
approved by FDA for the treatment of CRPC. Enzalutamide was derived from a nonsteroidal
14
androgen receptor agonist, RU59063. The way enzalutamide inhibits the androgen receptor
signaling is by competitively inhibiting the binding of androgens, therefore, preventing the
nuclear translocation of the receptor and tumor growth.
However, the patients who initially responded eventually developed resistant to
enzalutamide, and the survival benefits were only achieved in up to 50% patients (Schalken
and Fitzpatrick, 2016). New approaches for overcoming the mechanism of enzalutamide
resistance are needed.
1.4 Monoamine oxidase
Monoamine oxidases (MAOs) catalyze the oxidative deamination of biogenic
monoamines neurotransmitters and during this process they generate aldehydes, ammonia
and hydrogen peroxide (Shih et al., 1999). In humans, there are two forms of MAOs located
on the outer mitochondrial membranes: MAOA and MAOB. Both forms can inactivate
monoamine neurotransmitters in the central nervous system (CNS) such as dopamine and
tryptamine. They share 70% amino acid identity, but have difference tissue distribution,
substrate and inhibitor specificities (Bach et al., 1988; Grimsby et al., 1991). Particularly,
MAOA is responsible for breaking down serotonin, norepinephrine and epinephrine while
phenylethylamine is mainly oxidized by MAOB. MAO inhibitors are a class of drug that
inhibit the activity of one or both MAO isoenzymes and they have shown therapeutic value
in psychiatric and neurologic disorders including depression and Parkinson’s disease. The
early MAO inhibitors bound covalently to the MAO enzymes and irreversibly inhibit them,
thereby blocking the enzyme function until the cells synthesize and replace the enzyme pool.
On the other hand, the newly developed MAOIs such as moclobemide are reversible.
15
Although we normally consider MAOA as a neurotransmitter regulator for brain
disorders, recent studies have revealed that there is a higher expression of MAOA in both
prostatic epithelium and high-grade (Gleason grades 4 and 5) prostate cancer, low
expression in low grade prostate cancer but still higher than normal (Flamand et al.,
2010). As well as, the patients had higher levels of MAOA protein had higher level of serum
prostate specific antigen also showed the contribution of MAOA to the growth of high-
grade cancer. Clorgyline, a selective irreversible MAOA inhibitor, has antioncogenic and
pro-differentiation effect in a model of high risk PCa. According to the research from
Flamand, clorgyline suppresses oncogenic pathways and clinically decreases the
proliferation of PCa (Flamand et al., 2010). Furthermore, our laboratory has shown that
clorgyline actively inhibits cell viability in LNCaP cells (Gaur et al., 2019). Therefore, we
hypothesize that clorgyline could be a potential therapy for advanced stages of PCa.
1.5 Mitochondria Respiration and Mitochondria Function
The mitochondrion is the powerhouse organelle, and it undertakes many key functions
in the cell. In addition to distinguishing the metabolic pathways and physiological states of
the cell, mitochondria perform cellular respiration and supply biological energy source
adenosine triphosphate (ATP), generate and detoxify reactive oxygen species (ROS),
initiate cellular apoptosis, regulate cytoplasmic and mitochondrial matrix Ca
2+
, as well as
synthesis and catabolize metabolites (Brand and Nicholls, 2011). All eukaryotes generate
ATP via glycolysis and oxidation phosphorylation (OXPHOS). During the oxidation
phosphorylation, electrons are transferred from electron donor to electron acceptor in redox
reaction which releases energy to form ATP. The redox reaction is carried out through a
series of protein complexes within the inner membrane of the mitochondria of the cell, and
16
those linked proteins are called electron transport chain (ETC). The electron released
through the ETC, in which a proton gradient established across the boundary of inner
membrane by oxidizing the NADH produced from the Krebs cycle. There is a large enzyme
located on the inner membrane of mitochondrion called ATP synthase which use the energy
from the proton flow to transform adenosine diphosphate (ADP) to ATP. Glycolysis can
function with or without the presence of oxygen. Within this metabolic pathway, glucose is
converted to pyruvate and generate energy used to form ATP.
Mitochondria were originally proposed to be associated with cancer by German physiologist
Otto Heinrich Warburg who reported that cancer cells produce excessive amounts of lactate
in the presence of oxygen and he named this state “aerobic glycolysis” (Warburg et al., 1927).
This view helped a lot for the development of powerful imaging tools which are still in
clinical use. As the research continued to a deeper level, mitochondria attracted more
attention from a metabolic perspective. Some studies have approved that the Warburg effect
promotes tumor growth and metastasis. Increased glycolysis produces more metabolic
intermediates to fuel several anabolic processes and results in providing more substance for
the cells to proliferate rapidly (Shafi et al., 2015).
Moreover, activated AR alters cell metabolism by stimulating aerobic glycolysis. It
becomes more and more clear that mitochondria play a key role in oncogenesis and confers
considerable metabolic plasticity to malignant cells (Fendt et al., 2013; Wise et al., 2011).
Because the mitochondrial function is essential for cancer cells, any changes on it can be
identified as the factors in diseases. The mutation in mitochondria DNA (mtDNA) may
cause impairment on ETC and result in the increased production of ROS (Porporato et al.,
2014). The high production of ROS by mitochondria facilitate the accumulation of potential
17
oncogenic DNA defects and alteration cellular redox regulation, thereby contributing to the
progression of cancer (Sabharwal and
Schumacker, 2014).
1.6 Lipid metabolism
Lipid metabolism is the synthesis and degradation of lipids in cells, and it involves
the decomposition or storage of lipids to obtain energy. Changes in metabolic activities have
been shown to support the malignant properties of cancer cells. In addition, extracellular
fatty acids are the major source (~83%) of carbons to the total lipid pool in all cell lines,
compared with glucose (~13%) and glutamine (~4%) and the released fatty acid are taken
up and stored by prostate tumor cells (Balaban et al., 2019). Prostate cancer is characterized
by a low glycolytic rate and increased dependency on ATP generation from fatty acid
oxidation to promote cell growth and proliferation. Thus, blocking fatty oxidation becomes
an important target to reduce prostate cancer cell viability (Liu et al., 2010). Watt also shows
that increased fatty acid uptake was found in prostate cancer cells (Watt et al., 2019).
Furthermore, uptake of free fatty acids stimulates the expression of the isoform of NADPH
oxidase which can increase intracellular ROS, activated HIF1, then increase tumor cell
invasion (Laurent et al., 2019).
Cancer cells typically have higher oxidative metabolism and they are more dependent
on mitochondrial functions to meet energy demands than normal cells (Ertel et al., 2012).
In accordance with those findings, altering mitochondrial function has become an important
target in developing a prostate cancer treatment.
18
1.7 Objectives of the project
The main aim of this study was to find out if MAOA inhibitors may enhance the effect of
enzalutamide on prostate cancer cells. The effect of clorgyline, enzalutamide and their
combination treatment on mitochondrial function in LNCaP, 22RV1 and PC3 cell lines was
investigated. We also tried to examine how those treatments affected the expression of
MAOA and AR.
19
2. Materials and methods
2.1 Cell culture and supplies
Human prostate cancer cell lines LNCaP, CWR22RV-1 (RV1) and PC3 were purchased
from American Type Culture Collection (Manassas, Va). The tumor cells were maintained
in culture in humidified atmosphere at 37 °C and 5% CO2 in Roswell Park Memorial
Institute (RPMI) 1640 media (ThermoFisher, USA) supplemented with 10% heat
inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 0.01 mg/ml streptomycin.
The cells were passage twice a week and discarded after 10 passages. Enzalutamide (MDV-
3100) were purchased from Selleckchem (Houston, TX) and clorgyline were obtained from
Sigma-Aldrich (St. Louis, MO). Antibodies against different proteins were obtained
from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA).
Figure 2. Images of LNCaP, 22RV1 and PC3 cell lines.
2.2 Western blot analysis
The cells were plated in RPMI and treated for 24 hours. After 24h, medium was removed,
and the cells were washed with PBS. Cells were lysed by using cell culture lysis buffer from
Promega (Madison, WI) and protease inhibitor Sigma-Aldrich (St. Louis, MO) and lysates
transferred into tubes. After centrifugation, the supernatants were saved and pellet
LNCaP 22 RV 1 PC3
20
discarded. The protein concentrations were determined from supernatants using the
Bradford assay (Sigma-Aldrich). Protein was measured by using Bio-Rad protein assay kit
(Bio-Rad, Hercules, CA) and 10% sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was used to resolve 25 µg of protein. The proteins were
transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham, Arlington
Heights, IL) and then detected using MAOA antibody at 1:1000 dilution (G-10, Santa Cruz,
CA) and AR (C-19, Santa Cruz, CA) antibody at 1:1000 dilution. Blots were then incubated
with HRP-conjugated secondary antibody (Amersham) followed by ECL western blotting
substrate detection from Thermo Fisher Scientific (Waltham, MA).
GAPDH was measured as the protein for loading control. The blots were imaged with
BioRad ChemiDoc imaging system (Hercules, CA). Image lab software 6.0.1 was used to
adjust for brightness and contrast for minimum backgrounds and also quantify protein
expression (Bio-Rad).
2.3 Seahorse assay
Seahorse Bioscience XF96 analyzer (Seahorse Bioscience, North Billerica, MA, USA) was
used to detect the extracellular acidification rate (ECAR), which is a measure of the rate of
glycolysis and oxygen consumption rate (OCR), which is a measure of the rate of oxidative
phosphorylation (OXPHOS). In the Seahorse mito stress test, the mitochondrial inhibitors
were added serially: oligomycin (1 µM) to inhibit ATP synthase, FCCP (1 µM) to uncouple
oxygen consumption from ATP production, and a mix of rotenone and antimycin A to
inhibit complex I and complex III of the electron transport chain. ATP production, maximal
respiration, and nonmitochondrial respiration were measured respectively. Proton leak and
spare respiratory capacity were then calculated using these parameters and basal respiration.
21
In the Seahorse glycolysis stress test, glucose (10 mM), oligomycin (1 µM) and 2-DG (50
µM) were consecutively injected to measure glycolysis, glycolytic capacity and non-
glycolytic acidification. Glycolytic reserve is calculated using these parameters. LNCaP
cells were plated at 20,000 cells/well in Seahorse XF96 plates and incubated overnight.
Cells were then treated with vehicle, clorgyline, enzalutamide and combination at difference
concentration for 24 hours. The medium used for the Seahorse mito stress test was XF assay
medium: 10 mM glucose, 2 mM glutamine and 1 µM pyruvate, pH 7.4 ± 0.05 (37°C).
Oligomycin (63 nmol), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)
(72 nmol), and a mix of rotenone/ antimycin A (27 nmol of both) were from XF Agilent
Seahorse XF Cell Mito Stress Test Kit. Glucose (300 µmol) and 2-DG (1500 µmol) were
from Seahorse XF Glycolysis Stress Test Kit.
2.4 Mitochondrial oxidation of fatty acid
Oxidation rate of fatty acid was measured using a Seahorse XF96 analyzer (Seahorse
Bioscience, North Billerica, MA, USA). LNCaP cells were treated with vehicle and 1 µM
clorgyline for 24 hours prior to the XF Fuel Flex Test Kit performed in accordance with
manufacturer’s instructions. Each plotted value is the mean of at least 3 replicates and is
normalized to protein concentration in each well.
2.7 Statistical analysis
Each data set is presented as mean ± standard error mean of at least three
independent cell populations. Two-tailed student’s t-test was used for statistical analysis
and p-value was determined using Microsoft Excel Program 2011 and Prism 6 (GraphPad,
22
Inc, USA). Differences were considered statistically significant at * p < 0.05, ** p < 0.01
and *** p < 0.001.
23
3. Results
3.1 MAOA expression and androgen sensitivity in human prostate cancer cells
MAO A is known to be expressed in localized prostate cancer especially associated
with the high-grade disease. However, the activity level of MAOA is different in the
different prostate cancer cell lines. Three human prostate cancer cell lines: LNCaP, 22RV1
and PC3 are involved in this research. Characteristics of these cell lines were listed in Table
1. According to the data from Dr. Shih’s laboratory, LNCaP cells have the highest MAOA
activity and 22RV1 also has relatively high MAOA activity, but PC3 has extremely low
MAOA activity. 22RV1 and PC3 are androgen independent cell lines and LNCaP is
androgen sensitive cell line. The original tumor sites of LNCaP and PC3 are metastatic
lymph node and bone, respectively.
24
MAOA activity Androgen Metastatic
Prostate cancer cell Phenotype
(nmol/20min/mg protein) Sensitivity Site
LNCaP 139.48±15.23* + Lymph node
adherent, single
cells and loosely
attached cluster
22RV1 86.89±10.74* - NA Adherent
PC3 0.05±0.019* - Bone Adherent
NA: not available
*Data from Dr. Jean Shih’s Lab
Table 1. Characteristics of three human prostate cancer cell lines: LNCaP, 22RV1 and PC3 (ATCC.org, 2019)
25
3.2 Clorgyline produces additive effect in combination with enzalutamide in inhibiting
the cell proliferation in LNCaP cells
As we know that both clorgyline and enzalutamide can decrease the proliferation of
the LNCaP cells. We would like to see how clorgyline effect cell proliferation when
combining with enzalutamide. Gaur Shika, who from Dr. Shih’s lab, performed MTS assay
to test the number of LNCaP cell after 24 hours treatment of different concentration of
clorgyline and enzalutamide. Based on the result she obtained (Figure 3), enzalutamide at
10 µM, 25 µM and 50 µM concentration inhibited cell proliferation by 28.3%, 33% and
32.5%, respectively. Clorgyline, when combined with enzalutamide, decrease cell
proliferation by more than 10% as compared to enzalutamide alone.
26
LNCaP
0
Enzalutamide (μM)
0 1 2.5 5.0 10.0 25.0 50.0 0.0
Clorgyline (μM)
0 0 0 0 10.0 10.0 10.0 10.0
Figure 3. The effect of clorgyline when combined with enzalutamide on the cell proliferation in LNCaP
cells (Gaur et al., 2019).
27
3.3 Clorgyline, enzalutamide and their combination treatment decrease oxygen
consumption rate (OCR) and mitochondrial function in LNCaP cells
To investigate the effect of clorgyline, enzalutamide and their combination treatment on
prostate cancer cells, we carried out Seahorse mito stress test on LNCaP cells to measure
OCR, a measure of oxidative phosphorylation. Seahorse XF provides a method to assess the
key parameters of mitochondrial function: Basal respiration, ATP-linked respiration, H+
(Proton) Leak, Maximal Respiration. Mitochondrial respiration demonstrates significant
differences between each group. For the basal respiration, clorgyline has 52% decrease
(p<0.02), enzalutamide has 29% decrease and combination treatment group has 48%
decrease. For the proton leak, clorgyline has 45% decrease (p<0.04), enzalutamide has 17%
decrease and combination treatment group has 31% decrease. For the ATP production,
clorgyline has 59% decrease (p<0.02), enzalutamide has 40% decrease and combination
treatment group has 66% decrease (p<0.02) (Figure 4B). The result shows that all
treatments reduced prostate mitochondrial aerobic metabolism.
28
Figure 4. Clorgyline, enzalutamide and combination treatment reduced prostate mitochondrial aerobic
metabolism. LNCaP cells were treated with vehicle, 1 µM clorgyline, 1 µM enzalutamide and 1 µM
clorgyline + 1 µM enzalutamide for 24 hours prior to Seahorse assay. A. Oxygen consumption rate
(OCR) were measured using the Seahorse Bioscience-XF96 Analyzer. Values were normalized to protein
concentration. Cells were treated with different mitochondrial inhibitors (Oligomycin, FCCP, Rotenone
& AA) over the course of the experiment. B. Significant difference in respiratory parameters from
groups. Data are presented as the average 4-8 replicate wells ± SEM.
** p< 0.01, * p<0.05 compared to respective vehicle.
B asa l R es p ir ati o n A T P
P r o d u c ti o n
Ma xim al Re sp ira tio n P r o to n
L e a k
Oligomycin
FCCP
Rotenone & AA
A
29
3.4 Dose response for clorgyline, enzalutamide and their combination treatment in oxygen
consumption rate in LNCaP cells.
Exposure of LNCaP cells to clorgyline (0.5 µM, 1 µM and 5 µM), enzalutamide
(0.2 µM, 1 µM and 2 µM) and the combination of clorgyline and enzalutamide (0.5 µM
+0.2 µM, 1 µM +1 µM and 5 µM +2 µM) resulted in decrease in OCR, demonstrated using
a mito stress test (Figure 5). For all three treatment groups, we found a dosedependent OCR
reduction
30
Figure 5. Dose response for clorgyline, enzalutamide and their combination treatment in oxygen
consumption rate of LNCaP cells. (A) LNCaP cells were treated in vitro with clorgyline (0.5 µM, 1 µM
and 5 µM) and basal OCR was assessed. A mito stress test was then performed in order to assess the
effect of clorgyline on mitochondrial function. Graph represent OCR outputs from the XF96 analyzer of
control (blue line) and clorgyline treated (various colors, 0.5 µM, 1 µM and 5 µM) and the response to
oligomycin, FCCP and rotenone/antimycin A. (B) LNCaP cells were treated in vitro with enzalutamide
(0.2 µM, 1 µM and 2 µM) and basal OCR was assessed. (C) LNCaP cells were treated in vitro with
combination of clorgyline and enzalutamide (0.5 µM +0.2 µM, 1 µM +1 µM and 5 µM +2 µM) and basal
OCR was assessed.
Clorgyline 5uM
Clorgyline 0.5uM
Vehicle
Clorgyline 1uM
Vehicle
Enzalutamide 0.2uM
Enzalutamide 1uM
Enzalutamide 2uM
A
C
B
Vehicle
Clg 0.5uM + ENZ 0.2uM
Clg 1uM + ENZ 1uM
Clg 5uM + ENZ 2uM
Oligomycin
FCCP
Rotenone/AA
Rotenone/AA
Oligomycin
FCCP
Rotenone/AA
Oligomycin
FCCP
31
3.5 Clorgyline, enzalutamide and their combination treatment decrease glycolytic function
in LNCaP cells
Some studies have shown that activation of AR with androgen receptor agonist
(R1881) or induced androgen receptor splice variant increased glycolysis (Shafi et al.,
2015). To determine whether androgen receptor inhibitor have reverse effect, a Seahorse
assay was done to measure ECAR, a measure of glycolysis. Contrary to R1881 treatment
and induction of AR-V7, enzalutamide decreases ECAR (Figure 6A and 6B). Clorgyline
and combination treatment groups resulted in a reduction on the glycolysis.
32
Figure 6. Analysis of clorgyline, enzalutamide and their combination treatment regulation of extracellular
acidification rate (ECAR). LNCaP cells were treated with vehicle (RPMI), 1 µM clorgyline, 1 µM
enzalutamide and 1 µM clorgyline +1 µM enzalutamide. A. Extracellular acidification rate (ECAR). B.
Glycolytic capacity calculated for ECAR. Values were normalized to protein concentration. Cells were
given difference substance (Glucose, Oligomycin, 2DG) over the course of the experiment. The cells use
the glucose injection and catabolize it through the glycolytic pathway to pyruvate. Oligomycin is an ATP
synthase inhibitor which inhibit mitochondrial ATP production and shifts the energy production to
glycolysis. 2-deoxy-glucose (2DG) is a glucose analog that inhibits glycolysis through competitively
binding to glucose hexokinase. Data are presented as the average of 3-4 replicate wells ± SEM. *** p<
0.001, ** p<0.01, * p< 0.05.
Vehicle
Clorgyline
Enzalutamide
Combination
A .
B.
33
3.6 Clorgyline decreases fatty acid dependency in LNCaP cells
Although many studies have shown reduced LNCaP’s cell viability in clorgyline
exposure, the effect on mitochondrial fuel utilization has not been defined. The seahorse XF
Fuel Flex Test kit was used to test parameters of fatty acid utilization in exposure of medium
and 1 µM clorgyline (Figure 7). This process involves using specific inhibitor to compare
decrease in oxygen consumption after inhibition. Etomoxir, an inhibitor of long chain fatty
acid oxidation, inhibit carnitine palmitoyl-transferase 1A. Targeting of the fatty acid
oxidation pathway resulted in a 21% decrease in fatty acid utilization after clorgyline
exposure.
34
Figure 7. XF analysis of fuel flex shows reduced dependency in fatty acid after 1 µM clorgyline exposure
in LNCaP cells. Seahorse XF analysis using the fuel flex test kits was performed with pretreatment (24
hrs) with medium or 1uM clorgyline. Dependency and flexibility were calculated with Wave 2.0 software
for fatty acid fuel capacity, dependency, and flexibility.
35
3.7 Clorgyline, enzalutamide and their combination treatment decrease oxygen
consumption rate (OCR) and mitochondrial function in 22RV1 cells
To investigate the effect of clorgyline, enzalutamide and their combination treatment
on MAOA sensitive and androgen receptor insensitive prostate cancer cells, we carried out
Seahorse mito stress test on 22RV1 cells to measure OCR, a measure of oxidative
phosphorylation. Mitochondrial respiration demonstrates significant differences between
each group. For the basal respiration, clorgyline has 42.9% decrease, enzalutamide has 22%
decrease and combination treatment group has 61% decrease. For the ATP production,
clorgyline has 54% decrease, enzalutamide has 27% decrease and combination treatment
group has 63% decrease (p<0.02) (Figure 8).
Results show that all treatments reduced prostate mitochondrial aerobic metabolism.
36
Figure 8. Clorgyline, enzalutamide and combination treatment reduced prostate mitochondrial aerobic
metabolism in androgen independent 22RV1 cells. 22RV1 cells were treated with vehicle, 5 µM
clorgyline, 2 µM enzalutamide and 5 µM clorgyline + 2 µM enzalutamide for 24 hours prior to Seahorse
assay. Oxygen consumption rate (OCR) were measured using the Seahorse BioscienceXF96 Analyzer.
Values were normalized to protein concentration. Significant difference in respiratory parameters from
groups. Data are presented as the average 4-5 replicate wells ± SEM. * p<0.05, ** p<0.01 compared to
respective vehicle.
37
3.8 Clorgyline, enzalutamide and their combination treatment have no effect on oxygen
consumption rate (OCR) and mitochondrial function in PC3 cells
PC3 cell is androgen independent with extremely low MAOA activity. To determine
whether clorgyline, enzalutamide and their combination treatment effect the mitochondrial
function, a Seahorse assay was done to measure OCR (Figure 9). As expected, those
treatment did not change the OCR levels.
38
Figure 9. Clorgyline, enzalutamide and combination treatment have no effect on prostate mitochondrial
aerobic metabolism in androgen independent and MAOA independent PC3 cells. PC3 cells were treated
with vehicle, 5 µM clorgyline, 2 µM enzalutamide and 5 µM clorgyline + 2 µM enzalutamide for 24
hours prior to Seahorse assay. Oxygen consumption rate (OCR) were measured using the Seahorse
Bioscience-The XF96 Analyzer. Values were normalized to protein concentration. Data are presented as
the average 3-5 replicate wells ± SEM.
39
3.9 Clorgyline, enzalutamide and their combination treatment decrease the expression of
androgen receptor in LNCaP cells
Next, we investigated the effects of clorgyline, enzalutamide and their combination
treatment on AR expression. LNCaP cells are androgen-sensitive human prostate
adenocarcinoma cells, and AR gene is itself regulated by androgenic activity, so we
examined effects of those treatment on AR expression levels. Clorgyline, enzalutamide and
combination treatment decrease AR expression by 10%, 41% and
39%, respectively (Figure 10).
40
Figure 10. Clorgyline and enzalutamide reduced the expression of AR. LNCaP cells were treated with
vehicle, 1 µM clorgyline, 1 µM enzalutamide or both (clg+ENZ) for 24 hours. The cells were collected
and processed for western blot analysis. The experiment was repeated at least three times and shows the
representative blot. The bar diagram (mean±SEM, n=4) showing the fold change in the intensity of the
band with respect to GAPDH. * p<0.05 compared to respective vehicle.
41
4. Discussion and conclusion
Enzalutamide is one of the main line chemotherapy treatments for prostate cancer patients,
however, the response to enzalutamide is temporary and people become resistant to this
drug. It has been shown that androgen deprivation therapy can induce EMT in metastatic
prostate cancer (Nouri et al., 2014). Inhibition of the androgen axis promotes tumor cells to
adapt and survive to chemotherapy and can cause cancer recurrence and progression. MAO
inhibitors have been shown to prevent EMT (Wu et al., 2014) thus adding MAO inhibitors
to prostate cancer therapies may prevent the EMT escape from anti-androgens treatment
making the combined treatment more effective.
In this study, I reported that combination of MAO inhibitor, clorgyline and androgen
receptor antagonist, enzalutamide provides potential strategy for the treatment of prostate
cancer in cell culture models. My goal was to figure out possible molecular mechanisms of
action for the compounds contributing to the reduction of mitochondrial function of human
prostate cancer cells.
Two human prostate cell lines, LNCaP and 22RV1, both have MAOA catalytic
activities. The difference between these two cell lines is androgen receptor sensitivity.
LNCaP depends on androgen receptors but 22RV1 does not. To determine the effect of
clorgyline when combined with enzalutamide, LNCaP cells were treated by increasing
doses of the two treatments and then MTS cell viability assay was performed. According to
the graph of clorgyline and enzalutamide (Figure 3), clorgyline has an additive effect on the
inhibition of cell proliferation in LNCaP when combined with enzalutamide.
To determine the effect of MAOA inhibitor and androgen receptor antagonist on
mitochondrial function, cells were treated at the same doses of treatments for 24 hours and
then Seahorse mito stress test was performed. We found that clorgyline effectively
42
decreased the oxygen consumption rate of prostate cancer cell lines regardless of their
sensitivity to androgen receptor by using the Seahorse mitochondrial stress test (Figure 4).
Shafi reported that LNCaP cells treated with androgen receptor agonist (R1881) have higher
oxygen consumption rate compare to the vehicle control group (Shafi et al., 2015).
Furthermore, MAOA inhibitor revealed additive effects in combination with androgen
receptor antagonist in androgen receptor sensitive prostate cancer cell line, LNCaP. 22RV1
is androgen independent and exhibited a minimal decrease in oxygen consumption rate with
enzalutamide. However, clorgyline enhanced the effect of enzalutamide in these cells
(Figure 8) indicating that a combination of MAOA inhibitor and antiandrogen may be a
more effective treatment strategy for androgen resistant prostate cancer.
I repeated the Seahorse protocol shown in Figure 4 on LNCaP treated with different
concentration of clorgyline, enzalutamide and their combination treatment. After 24 hours,
I observed a dose dependent decrease in oxygen concentration rate which is an indicator of
mitochondrial respiratory function (Figure 5).
Previous studies have shown that activated AR affect cellular metabolism by
stimulating aerobic glycolysis and anabolic metabolism in prostate cancer (Massie et al.,
2011; Shafi et al., 2015). Thus, we tried to compare the actions of androgen receptor
inhibitor and androgen receptor agonist in altering metabolism. The AR inhibitor elicited an
opposite effect on glycolysis compared to R1881 when measured by ECAR (Figure 6). Our
results show that clorgyline, enzalutamide and combination treatment decreased glycolysis
of androgen sensitive cells.
As free fatty acids have been shown to contribute to tumor progression mainly
through modulation of tumor cell metabolism toward fatty acid oxidation (Laurent et al.,
2019), it is important to investigate and acknowledge the possibility of disruption of fatty
43
acid utilization. It has been found that fatty acid oxidation rates are greater in prostate cancer
cells compared with a control human prostate epithelial cell line
(Balaban et al., 2019). To determine the effect of 1uM clorgyline on fatty acid utilization,
cells were treated for 24 hours and Seahorse fuel flex test was performed (Figure 7).
Clorgyline effectively decreased the fatty acid dependency.
Since both clorgyline and enzalutamide decreased the mitochondrial function of
LNCaP cells, we sought to find if the reduced function is related to the change of expression
of MAOA and androgen receptor by those treatments. Thus, we used the same concentration
(1 µM) for all the treatments to detect expression of protein by using western blot.
Consistent with the result from Shikha (Gaur et al., 2019), there is no change in the
expression of MAOA when treated with clorgyline, enzalutamide or the combination
treatment. On the other hand, those three treatments inhibited the expression of androgen
receptor. Previous studies have shown that clorgyline decrease the MAOA activity in
LNCaP cells. The unchanged MAOA expression reveals that the mechanism of clorgyline
is only on the enzyme activity but not protein expression.
Both clorgyline and enzalutamide decreased mitochondrial respiration, glycolytic
function and AR expression in androgen dependent cell line and clorgyline itself reduced
fatty acid utilization. Together, these results suggest that MAOI inhibits mitogenic pathway
downstream and add to effects of antiandrogen-treatment in both androgen-dependent and
androgen-independent prostate cancer cell. However, the exact mechanisms that associate
MAOA and androgen receptor and their combination
treatment reduce the mitochondrial function of the prostate cancer cells are not yet clear.
44
In conclusion, we found that MAOA inhibitor clorgyline and androgen receptor
antagonist enzalutamide regulated the mitochondrial function of prostate cancer cell lines.
Clorgyline exhibited additive effects when combined with enzalutamide as a novel
combination therapy to potentially test in other androgen-sensitive prostate cancer models.
Our findings suggest that MAOA inhibitors and androgen antagonists may serve as an
effective combination treatment strategy to treat advanced prostate cancer.
45
References
ATCC.org (2019).
Bach, A.W., Lan, N.C., Johnson, D.L., Abell, C.W., Bembenek, M.E., Kwan, S.W., Seeburg,
P.H., and Shih, J.C. (1988). cDNA cloning of human liver monoamine oxidase A and B:
molecular basis of differences in enzymatic properties. Proc Natl Acad Sci U S A 85,
4934-4938.
Balaban, S., Nassar, Z.D., Zhang, A.Y., Hosseini-Beheshti, E., Centenera, M.M.,
Schreuder, M., Lin, H.M., Aishah, A., Varney, B., Liu-Fu, F., et al. (2019).
Extracellular Fatty Acids Are the Major Contributor to Lipid Synthesis in Prostate
Cancer. Mol Cancer Res.
Brand, M.D., and Nicholls, D.G. (2011). Assessing mitochondrial dysfunction in cells.
Biochem J 435, 297-312.
Cancer.org (2019). Global Cancer Facts and Figures, Key Statistics for Prostate
Cancer. American Cancer Society.
Catalona, W.J., Richie, J.P., Ahmann, F.R., Hudson, M.A., Scardino, P.T., Flanigan, R.C.,
DeKernion, J.B., Ratliff, T.L., Kavoussi, L.R., Dalkin, B.L., et al. (2017).
Comparison of Digital Rectal Examination and Serum Prostate Specific Antigen in
the Early Detection of Prostate Cancer: Results of a Multicenter Clinical Trial of
6,630 Men. J Urol 197, S200-S207.
Ertel, A., Tsirigos, A., Whitaker-Menezes, D., Birbe, R.C., Pavlides, S.,
MartinezOutschoorn, U.E., Pestell, R.G., Howell, A., Sotgia, F., and Lisanti, M.P. (2012).
Is cancer a metabolic rebellion against host aging? In the quest for immortality,
tumor cells try to save themselves by boosting mitochondrial metabolism. Cell Cycle
11, 253-263.
Fendt, S.M., Bell, E.L., Keibler, M.A., Olenchock, B.A., Mayers, J.R., Wasylenko, T.M.,
Vokes, N.I., Guarente, L., Vander Heiden, M.G., and Stephanopoulos, G. (2013).
Reductive glutamine metabolism is a function of the α-ketoglutarate to citrate ratio
in cells. Nat Commun 4, 2236.
Flamand, V., Zhao, H., and Peehl, D.M. (2010). Targeting monoamine oxidase A in
advanced prostate cancer. J Cancer Res Clin Oncol 136, 1761-1771.
Gaur, S., Gross, M.E., Liao, C.P., Qian, B., and Shih, J.C. (2019). Effect of Monoamine
oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant
prostate cancer cells. Prostate.
Gregory, C.W., He, B., Johnson, R.T., Ford, O.H., Mohler, J.L., French, F.S., and Wilson,
E.M. (2001). A mechanism for androgen receptor-mediated prostate cancer
recurrence after androgen deprivation therapy. Cancer Res 61, 43154319.
Grimsby, J., Chen, K., Wang, L.J., Lan, N.C., and Shih, J.C. (1991). Human monoamine
oxidase A and B genes exhibit identical exon-intron organization.
Proc Natl Acad Sci U S A 88, 3637-3641.
Kalluri, R., and Weinberg, R.A. (2009). The basics of epithelial-mesenchymal
transition. J Clin Invest 119, 1420-1428.
Laurent, V., Toulet, A., Attané, C., Milhas, D., Dauvillier, S., Zaidi, F., Clement, E.,
46
Cinato, M., Le Gonidec, S., Guérard, A., et al. (2019). Periprostatic Adipose Tissue
Favors Prostate Cancer Cell Invasion in an Obesity-Dependent Manner: Role of
Oxidative Stress. Mol Cancer Res 17, 821-835.
Leung, J.K., and Sadar, M.D. (2017). Non-Genomic Actions of the Androgen Receptor
in Prostate Cancer. Front Endocrinol (Lausanne) 8, 2.
Liu, Y., Zuckier, L.S., and Ghesani, N.V. (2010). Dominant uptake of fatty acid over
glucose by prostate cells: a potential new diagnostic and therapeutic approach.
Anticancer Res 30, 369-374.
Lonergan, P.E., and Tindall, D.J. (2011). Androgen receptor signaling in prostate
cancer development and progression. J Carcinog 10, 20.
Massie, C.E., Lynch, A., Ramos-Montoya, A., Boren, J., Stark, R., Fazli, L., Warren, A.,
Scott, H., Madhu, B., Sharma, N., et al. (2011). The androgen receptor fuels prostate
cancer by regulating central metabolism and biosynthesis. EMBO J 30, 2719-2733.
Nouri, M., Ratther, E., Stylianou, N., Nelson, C.C., Hollier, B.G., and Williams, E.D.
(2014). Androgen-targeted therapy-induced epithelial mesenchymal plasticity and
neuroendocrine transdifferentiation in prostate cancer: an opportunity for
intervention. Front Oncol 4, 370.
Porporato, P.E., Payen, V.L., Pérez-Escuredo, J., De Saedeleer, C.J., Danhier, P.,
Copetti, T., Dhup, S., Tardy, M., Vazeille, T., Bouzin, C., et al. (2014). A mitochondrial
switch promotes tumor metastasis. Cell Rep 8, 754-766. Sabharwal, S.S., and
Schumacker, P.T. (2014). Mitochondrial ROS in cancer: initiators, amplifiers or an
Achilles' heel? Nat Rev Cancer 14, 709-721. Sardana, G., Jung, K., Stephan, C., and
Diamandis, E.P. (2008). Proteomic analysis of conditioned media from the PC3,
LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate
prostate cancer biomarkers. J Proteome Res 7, 3329-3338.
Schalken, J., and Fitzpatrick, J.M. (2016). Enzalutamide: targeting the androgen
signalling pathway in metastatic castration-resistant prostate cancer. BJU Int 117,
215-225.
Shafi, A.A., Putluri, V., Arnold, J.M., Tsouko, E., Maity, S., Roberts, J.M., Coarfa, C.,
Frigo, D.E., Putluri, N., Sreekumar, A., et al. (2015). Differential regulation of
metabolic pathways by androgen receptor (AR) and its constitutively active splice
variant, AR-V7, in prostate cancer cells. Oncotarget 6, 31997-32012. Shih, J.C.,
Chen, K., and Ridd, M.J. (1999). Monoamine oxidase: from genes to behavior. Annu
Rev Neurosci 22, 197-217.
Warburg, O., Wind, F., and Negelein, E. (1927). THE METABOLISM OF TUMORS IN
THE BODY. J Gen Physiol 8, 519-530.
Watt, M.J., Clark, A.K., Selth, L.A., Haynes, V.R., Lister, N., Rebello, R., Porter, L.H.,
Niranjan, B., Whitby, S.T., Lo, J., et al. (2019). Suppressing fatty acid uptake has
therapeutic effects in preclinical models of prostate cancer. Sci Transl Med 11. Wise,
D.R., Ward, P.S., Shay, J.E., Cross, J.R., Gruber, J.J., Sachdeva, U.M., Platt, J.M., DeMatteo,
R.G., Simon, M.C., and Thompson, C.B. (2011). Hypoxia promotes isocitrate
dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell
growth and viability. Proc Natl Acad Sci U S A 108, 19611-19616.
Wu, J.B., Shao, C., Li, X., Li, Q., Hu, P., Shi, C., Li, Y., Chen, Y.T., Yin, F., Liao, C.P., et al.
47
(2014). Monoamine oxidase A mediates prostate tumorigenesis and cancer
metastasis. J Clin Invest 124, 2891-2908.
Abstract (if available)
Abstract
Prostate cancer is the most common diagnosed non-cutaneous cancer and second leading cause of death in men in the United States. One of the biggest problems in the prostate cancer field is that patients become resistant to therapy and the prostate cancer will continue to proliferate and metastasize to other organs most often the lymph nodes or bone. ❧ Monoamine oxidase A (MAOA), a mitochondrial enzyme, which can catalyze the oxidative deamination of monoamine neurotransmitters and generate hydrogen peroxide. It has been found that MAOA is highly expressed in high grade prostate cancer and can mediate prostate cancer progression and metastasis. MAOA inhibitors can effectively reduce cancer growth (Wu et al., 2014). Androgen receptor antagonists such as enzalutamide is used to treat prostate cancer, however prostate cancer patients often become treatment resistant leading to aggressive androgen independent cancer. Here, we investigate the effect of MAOA inhibitors alone or in combination with enzalutamide to understand how MAOA and AR inhibition regulate metabolic mechanisms in prostate cancer cells. ❧ In this study, we analyzed pharmacologic responses in three prostate cancer cell lines: LNCaP-androgen sensitive with high MAOA activity, CWR22RV1-androgen independent with high MAOA activity, and PC3-androgen independent with low MAOA activity. Our results showed that treatment of prostate cancer cells with MAOA inhibitor clorgyline decrease the mitochondrial function of LNCaP cells and 22RV1 cells. Enzalutamide alone was moderately effective in decreasing mitochondrial respiratory function and the effect was enhanced when combined with clorgyline, further decreasing oxygen consumption rate. Glycolytic function was likewise reduced by both treatments indicating that overall metabolic function and cancer cell growth were suppressed. Cell viability data confirmed these results. Expression of MAOA was not altered by clorgyline or enzalutamide. Expression of androgen receptor was decreased by both enzalutamide and clorgyline. Furthermore, we found that clorgyline reduced fatty acid dependency in LNCaP cells. ❧ Collectively, these results indicate that MAOA inhibitors suppress prostate cancer cell metabolism and further suppress metabolism and cell growth in combination with antiandrogen therapy. MAOA inhibitors provide a novel therapeutic approach to regulate mitochondrial function and fatty acid dependency in androgen resistant advanced prostate cancer.
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Cai, Jinghua
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Monoamine oxidase A inhibitors and androgen receptor antagonists regulate mitochondrial function in prostate cancer cells
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04/29/2019
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