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Monoamine oxidase inhibitors regulate tumorigenesis and mitochondrial function in a prostate cancer mouse model

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Monoamine oxidase inhibitors regulate tumorigenesis and mitochondrial function in a prostate cancer mouse model

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Monoamine Oxidase Inhibitors Regulate
Tumorigenesis and Mitochondrial Function in a
Prostate Cancer Mouse Model

by

Jiachun Li

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 Sciences)

May
2019
i

Table of Contents
Acknowledgement………………………………………………………….………………...ii
List of Figures…………………………………………………………………………...…….iii
Abbreviations……………………………………………………………….………………...iv
Abstract………………………………………………………………………………………....v
Chapter 1. Introduction………………………………………………………………...…...1
1.1 Cancer and prostate cancer
1.2 Monoamine oxidase and prostate cancer
1.3 Monoamine oxidase inhibitor and cancer
1.4 Mitochondrial function in cancer and mitochondrial respirometric assay
1.5 Aim of study
Chapter 2. Materials and Methods……………………………….…………………...…12
2.1 Animals and chemicals
2.2 Bioluminescence imaging (BLI)
2.3 MAO A catalytic activity/inhibition assay
2.4 Mitochondrial isolation
2.5 Respiratory measurement
2.6 Western blot analysis
2.7 Statistical analysis
Chapter 3. Results……………………………………………………………..…………….18
3.1 Phenelzine reduces prostate weight in Pten KO mice
3.2 Phenelzine reduces the MAO A activity of prostate from Pten KO mice
3.3 Phenelzine inhibits prostate tumorigenesis
3.4 Phenelzine reduces the mitochondrial aerobic metabolism of prostate from Pten KO
mice
3.5 Phenelzine decreases the expression of mitochondrial OXPHOS complexes in Pten KO
prostate
Chapter 4. Conclusion and Discussion.............................................................................32
References...……………………………………………………………………………...…....34
ii

Acknowledgement
First of all, I am very grateful for Dr. Jean Chen Shih for not only giving me
the opportunity to perform this study but also for mentoring me to be a scientist. I
am very thankful for her guidance and support at the School of Pharmacy, University
of Southern California.
Moreover, I am sincerely thankful to senior research associate, Ronald Irwin,
PhD, for teaching me during this project and for giving me valuable scientific advice.
I would also like to thank Jami Pei-Chuan Li and Bin Qian for their help and
giving me important advice during the experiments.
Overall, I want to thank all of the group members in Prof. Shih’s lab, it was
really nice to work with you!
Finally, I owe my warmest and personal gratitude to my family for their
everlasting support and encouragement in my life!

iii

List of Figures
Figure 1. Representative prostate anatomy of human and mouse.
Figure 2. Modeling prostate cancer in the Pten conditional knockout mouse model.
Figure 3. Schematic representation of the electron transfer, ATP synthesis, and proton leak in
mitochondria
Figure 4. Purified mitochondria at the interface of premade percoll gradient.
Figure 5. Prostate mitochondria under microscope
Figure 6. Phenelzine reduces prostate weight without changing body weight
Figure 7. Phenelzine reduces the MAO A activity of prostate
Figure 8. BLI imaging shows abrogated cancer development by phenelzine
Figure 9. Phenelzine attenuates the cancer morphology in Pten KO prostate
Figure 10. Phenelzine reduces the mitochondrial aerobic metabolism of prostate
Figure 11. Phenelzine increases Complex II-driven respiration
Figure 12. Phenelzine decreases the expression of mitochondrial complexes

iv

Abbreviations

PCa Prostate cancer
MAO A Monoamine oxidase A
AP Anterior prostate
DP Dorsal prostate
LP Lateral prostate
DRE Digital rectal exam
PSA Prostate specific antigen
ADT Androgen deprivation therapy
Pten Phosphatase and tensin homolog
BLI Bioluminescence imagaing
ROS Reactive oxygen species
EMT Epithelial-to-mesenchymal transition
MAOIs Monoamine oxidase inhibitors
ADP Adenosine diphosphate
ATP Adenosine triphosphate
KO Knock out
FCCP Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

v

Abstract
Prostate cancer (PCa) emerges as the second leading cause for male cancer death in the
Western world. In 2019, there will be approximately 174,650 new prostate cancer diagnoses and
31,620 prostate cancer deaths (Siegel et al., 2019).

Monoamine oxidase A (MAO A) is a
mitochondrial enzyme that degrades monoamine neurotransmitters and is overexpressed in
prostate cancer (Peehl et al., 2008). MAO inhibitors effectively inhibit PCa cell proliferation and
tumor growth (Gaur et al., 2019; Peehl et al., 2008; Wu et al., 2014). Phenelzine is an irreversible
monoamine oxidase A and B university inhibitor and clinically available antidepressant. Recent
studies reveal that phenelzine is effective as an anti-cancer agent. An ongoing phase II clinical trial
is evaluating phenelzine for prostate cancer patients based on the scientific research from the Shih
laboratory.
As the cancer field moves towards the discovery of mitochondria-related molecular
mechanisms, the role of altered mitochondrial function in a wide range of cancers continues to
expand. Here, by using a prostate-specific Pten gene deletion mouse model detectable by luciferase
imaging, we show that 2-weeks of phenelzine treatment resulted in decreased MAO A activity
(69.5% decrease vs control) in prostate. The reduction of prostate cancer tumorigenesis is
demonstrated by decreased prostate weight (36.3% decrease vs control) and decreased prostate-
specific luciferase signal (86.1% decrease vs control). In ex vivo Pten KO mouse prostate,
phenelzine inhibited both basal and maximal mitochondrial respiration and reduced ATP
production. The overall coupling state between electron transport chain and ATP synthesis was
also decreased by phenelzine treatment. Further, phenelzine decreased protein expression of
mitochondrial Complex I (33.7% decrease vs control) and Complex V (35.4% decrease vs control)
vi

significantly. In contrast, phenelzine increased Complex II driven respiration. We conclude that
MAO inhibitor phenelzine reduces tumorigenesis through a mitochondrial mechanism that
involves upregulation of Complex II enzyme activity.

1

Chapter 1. Introduction
1. 1 Cancer and prostate cancer
1.1.1 Cancer, also called malignancy, is a group of complex diseases caused by multiple genetic
alterations from carcinogens and environmental causes. They can be characterized by the
uncontrollable cells differentiation and as malignancy increases they may metastasize and spread
throughout the body. There are a wide range of different cancers, including cancers from breast,
skin, lung, prostate and lymphoma. Symptoms always vary and often depends on the type of
cancer. Prostate cancer (PCa) is the second leading cause of cancer death among men in Western
world and the incidence of PCa is climbing continuously (Qi et al., 2015). Prostate in human body
looks like a walnut-shaped gland, which produces the seminal fluid that nourishes and transports
sperm, which can be divided into different anatomical regions: transition, central, periurethral and
peripheral zone. The peripheral zone is the largest zone among others, comprising approximately
70% of the glandular tissue, and 70-80% of prostate cancer are located in this peripheral zone
(Violette and Saad, 2012).

Fig. 1 Representative prostate anatomy. A. Human prostate can be divided into 3 zones: central, transition, and
peripheral zones. B. Mouse prostate consists of four pairs of lobes around the urethra: AP (anterior prostate), DP
A B
2

(dorsal prostate), LP (lateral prostate) and VP (ventral prostate). SV, seminal vesicle; U, urethra (Peng and Joyner,
2015).
Screening in the early stages of some types of cancers can help patients and their doctors
intervene at early less aggressive stages of prostate hyperplasia. One way to diagnose prostate
cancer is the digital rectal exam (DRE), which needs doctor to put finger into the rectum to feel
the prostate gland. Another way for diagnosis of prostate is by testing blood prostate specific
antigen (PSA), which is an essential component in the prostatic glandular fluid and is detectable
in blood. PSA is present in the blood serum of men with healthy prostates but elevated in the
prostate cancer or other prostate disorders (Catalona et al., 2017). PSA is not only an indicator of
prostate cancer, but also useful on detecting prostatitis or the possibility of benign prostatic
hyperplasia (Velonas et al., 2013). The United States Food and Drug Administration (FDA)
approved the use of the PSA test in conjunction with a digital rectal exam (DRE) to screen men
for prostate cancer. In general, most clinicians consider PSA levels of 4.0 ng/mL and lower as
normal but there are many exceptions that cause PSA levels to fluctuate. Higher PSA may indicate
infection, inflammation or cancer of prostate (Walsh, 1997) and may be further diagnosed by
imaging tests, such as a trans-rectal ultrasound, x-rays, or cystoscopy. If the clinician suspects PCa,
a prostate biopsy will be collected and examined by a pathologist for microscopic tissue hallmarks
of cancer.
The traditional therapies for prostate cancer involve prostatectomy, radiation therapy,
chemotherapy, hormone therapy like androgen deprivation therapy (ADT), or any combination
treatment. For patients diagnosed with low level prostate cancer, treatment might not necessary,
doctors may recommend activity surveillance instead. For high histological grade PCa, it is
difficult to be treated and it can be incurable and potentially lethal. A better understanding of the
3

molecular basis of prostate cancer progression will improve therapeutic options to reduce or
eliminate the development of aggressive types of prostate cancer.
1.1.2 Phosphatase and tensin homolog (Pten) is encoded by the Pten gene in humans (Steck et al.,
1997). Mutations of this gene are an essential step during the progression of many serious cancers.
Pten is a tumor suppressor gene and function through the action of its phosphatase protein product,
in which phosphatase is participating in the prevention of rapid cells growth and differentiation
(Chu and Tarnawski, 2004). The deletion of Pten has been reported in many primary and advanced
PCa patients (Li et al., 1997). Losing function of Pten leads to PI3K/AKT signaling pathway
downstream activation and other regulatory pathways involving in cancer progression. Thus Pten
function losing is one of the most common genetic alterations observed in human prostate cancer
(Chalhoub and Baker, 2009). Recent studies preferably using either transgenic mouse models or
combine with modified gene activation or deleted gene model in recapitulating the development
of human cancer.
There are few evidence to show that the Pten gene deleted throughout mouse could result
in the death of embryos, scientists thus have developed a mouse model with ARR2PB-Cre-
mediated Pten deletion. The benefit of this model is to effectively mimic human disease
pathogenesis in regards of prostate adenocarcinoma by specifically deleting prostatic epithelia
Pten (Wang et al., 2003; Wu et al., 2001). In short, this conditional Pten knockout (KO) model
leads the 2 months male mouse from hyperplasia to cellular atypia and prostatic intracellular
neoplasia (PIN) and further progress to adenocarcinoma at the age between 3–6 months (Liao et
al., 2018). This mouse model has been widely used to evaluate various genes in vivo functions that
participate in PCa development.
4

Fig. 2 Modeling prostate cancer in the Pten conditional knockout mouse model.
In this study, the mouse model was designed to knockout prostate-specific Pten gene and
combine with a conditional luciferase reporter gene (Liao et al., 2007). Thus, the growth of cancer
can be monitored by noninvasive bioluminescence imaging (BLI) of luciferase when the luciferin
substrate is administered.
1.2 Monoamine oxidase and prostate cancer
Monoamine oxidases (MAOs) catalyze oxidative deamination of monoamine
neurotransmitters and dietary amines, including serotonin, norepinephrine, dopamine,
phenylethylamine, tyramine, resulting in the production of their aldehyde, then quickly convert to
acid, ammonia and hydrogen peroxide (Shih et al., 1999). Hydrogen peroxide is a major source of
reactive oxygen species (ROS) that predispose cancer cells to DNA damage and initiate tumor
progression (Trachootham et al., 2009). There are two isoenzymes of monoamine oxidase, MAO
A and MAO B, which are encoded by two different genes located on the X chromosome (Bach et
al., 1981; Shih, 2018). They have different distribution, substrate and inhibitor specificities. The
abnormality of MAO has widely been associated with neurological and psychological disorders
such as depression, anxiety, Alzheimer’s disease, autism, etc. (Trachootham et al., 2009; Youdim
et al., 2006).
5

Although MAO A was primarily considered a neurotransmitter regulator, recent studies
reveal that MAO A plays an essential role in tumorigenesis. The molecular mechanisms of MAO
A regulated tumor growth are active areas of research. High levels of MAO A activity result in
some cancers like prostate cancer (Peehl et al., 2008) and renal cell carcinoma (Hodorová et al.,
2012). Shih’s laboratory found that inhibits MAO A or knock down of MAO A gene inhibited the
growth or even eliminated PCa tumor cell in mice xenografts. They also show that MAO A
inhibition regulates epithelial-to-mesenchymal transition (EMT) and destabilizes the transcription
factor HIF1α to reduce prostate cancer (Wu et al., 2014).
1.3 Monoamine oxidase inhibitor and cancer
Monoamine oxidase inhibitors (MAOIs) are a class of drugs used as to treat depression,
Parkinson’s disease and several psychiatric disorders. There are two subclasses of MAOIs that can
reversibly or irreversibly inhibit MAO, thus reducing the breakdown of serotonin, dopamine and
norepinephrine, resulting in increased levels of neurotransmitters. The benefits of the increases are
improved mood and anti-panic effect. However, MAO inhibitors may have novel therapeutic value
for cancer either alone or in combination with current chemotherapies. Clorgyline, for example, a
selective and irreversible inhibitor of MAO A, is cytotoxic to drug-resistant human glioma cells.
Using clorgyline alone or as a combined treatment with temozolomide resulted in decreasing
glioma progression (Kushal et al., 2016), slowing down the tumor growth in VCaP mouse
xenograft model (Flamand et al., 2010). Another MAO inhibitor, phenelzine, can decreases tumor
progression in prostate cancer cells as well (Lin et al., 2017) and is undergoing a Phase II clinical
trial for prostate cancer patients. These prior investigations indicate that MAO inhibitors may
6

provide a novel strategy for cancer treatment and urge us to study MAO mechanisms for
therapeutic development.
1.4 Mitochondrial function in cancer and mitochondrial respirometric assay
1.4.1 It has long been considered that glycolysis is the major metabolic process for cancer cells to
produce energy for cell proliferation and thus oncogenesis (Vander Heiden et al., 2009). The terms
mitochondrial “function” and “dysfunction” are often used in cell biology and bio-energetics but
the exact definitions may vary depending on the objectives of the study. The predominant
physiological mitochondrial function is to generate ATP by a series reactions called oxidative
phosphorylation. Additional mitochondrial functions include generating and detoxifying reactive
oxygen species, involved in regulating apoptosis, regulating cytoplasmic and mitochondrial matrix
calcium, synthesizing and catabolizing metabolites and transporting organelles within the cell
(Brand and Nicholls, 2011). What attracts scientists’ attention is the perspective of metabolism, in
which it becomes clearer that some metabolites of mitochondria are sufficiently driving
oncogenesis and endowing malignant cells with metabolic plasticity (Dang et al., 2010; Fendt et
al., 2013). Abnormality in any of these processes can be termed mitochondrial dysfunction. Any
alterations in mitochondrial function have been identified as the contributing factors in diseases
that are degenerative, and altered mitochondrial functions are becoming increasingly crucial for a
better understanding of the cancer cell metabolic phenotype. For example, mitochondrial capacity
to produce higher ROS favors the accumulation of potential oncogenic DNA defects and activate
the possible oncogenic signaling pathways (Sabharwal and Schumacker, 2014). Increased
oxidative stress as a driver of prostate cancer resulted from imbalance between levels of ROS and
7

the protective mechanism has been demonstrated by strong clinical evidence (Khandrika et al.,
2009).
Within each mitochondrion is a system of enzymes present in the inner membrane that
comprise the electron transport chain coupled to the oxidative phosphorylation enzyme ATP
synthase. Complex I, or NADH dehydrogenase, function in the transfer of electrons from NADH
to the respiratory chain, is one of the main sites of producing superoxide. Complex II (succinate
dehydrogenase) is another electron transport pathway works same as Complex I but produce less
energy compared with Complex I- induced energy. It is the only enzyme involved in both the TCA
cycle and ETC. Importantly, SDHB has been specifically implicated as a tumor suppressor and
inhibition or loss of SDHB results EMT and mitochondrial dysfunction (Aspuria et al., 2014).
Complex III, or cytochrome c reductase, is necessary for producing the proton gradient. Complex
IV (cytochrome c oxidase) is the last enzyme in the ETC of mitochondrial oxidative
phosphorylation. It is located within the inner membrane. Complex V, also called ATP synthase,
produces ATP in the presence of a chemo electrical (proton) gradient across the inner
mitochondrial membrane.
8

Fig. 3 Schematic representation of the electron transfer, ATP synthesis, and proton leak. Transfer of electrons through
the respiratory chain is connected to the protons transport. The energy resulted from electrochemical gradient is used
to generate ATP. There is also a process called proton leak, which will dissipates the electrochemical gradient.

Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2)
undergo a series of oxidation and reduction reactions by those mitochondrial enzymes, known as
the electron transport chain. Reactions produce a flow of electrons from the substrates to the final
electron acceptor, oxygen, and the energy is released for pumping protons from matrix to the
intermembrane space. As a result, either different proton concentration or different electric
transmembrane potential is created across the inner membrane of mitochondria. ATP synthase is
located in the inner mitochondrial membrane and uses the energy resulted from the proton flow to
synthesize ATP. The oxidative reactions of substrates and the uses of oxygen maintain the electron
flow and are coupled to ATP synthesis.
9

However, the leakage of the proton from intermembrane to matrix can diminish the proton
gradient, resulting in less than 100% efficiency of substrate oxidation-driven ATP synthesis,
thereby releasing energy that is not coupled to ATP synthesis. In other words, due to the proton
leak, the consumption of oxygen is used to compensate the loss of proton gradient (Jonckheere et
al., 2012). Based on this model, ex vivo determination of oxygen consumption (respirometry) in
isolated mitochondria provides a useful tool to investigate the mitochondria-related diseases or
mitochondria targeting drugs in great details.
1.4.2 Mitochondrial respirometric assays include two assays: coupling and electron flow assay.
Assays can be performed using isolated mitochondria from dissected tissues like muscle, brain,
liver etc. Mitochondrial coupling assay provides substrate specific information as coupling
efficiency and maximal respiration. In our study, baseline mitochondrial oxygen consumption was
measured at the beginning. State 3 respiration was stimulated by the addition of 4mM ADP, State
4 respiration was induced with the addition of 2.5µg/ml Oligomycin (State 4o), and State 3u which
refers to 4uM FCCP-induced maximal uncoupler-stimulated respiration was sequentially induced.
Respiratory control ratio (RCR) (state 3/state 4) is used as an overall index of the condition of
isolated mitochondria. The number of RCR indicates whether mitochondrial ETC has a tight
coupling state with ATP production. By comparing the different RCRs, we can know the changes
of the mitochondria.
For electron flow assay, the initial substrate mix of pyruvate/malate + FCCP allows for the
measurement of Complex I-driven maximal respiration. Then the addition of rotenone
subsequently inject with succinate allows for the evaluation of OCR of Complex II-driven
respiration. Because the Ascorbate/TMPD is an electron donor to Cytochrome C/Complex IV, the
10

injection of Antimycin A, which is an inhibitor of Complex III, and followed by ascorbate/TMPD
reveal the OCR of respiration driven by Complex IV. Coupling combined with electron flow assay
could be used in tandem to evaluate the changes in mitochondrial function due to pharmacologic
intervention.

11

1.5 Aim of study
We proposed that phenelzine would inhibit the development of prostate cancer and weaken
cancer mitochondrial efficient electron coupling. To test this hypothesis, we used the Pten KO
mice as model of prostate cancer and approximately 6 months of age when adenocarcinoma has
developed and administered phenelzine (10 mg/kg) or saline vehicle daily via oral gavage for 14
days. At the end of the treatment period, prostate regions were imaged via luciferase, prostate
weights and prostate MAO A activity of phenelzine-treated mice were compared with vehicle-
treated controls. Mitochondria were isolated from fresh prostate tissue and the energy-transducing
capacity of prostate mitochondria was measured via Seahorse microrespirometry. Further, the
expression of mitochondrial complex proteins of prostate were investigated.

12

Chapter 2. Materials and Methods
2.1 Animals and chemicals
2.1.1 Animals
The use of animals for the study was approved by the Institutional Animal Care and Use
Committee at the University of Southern California (Protocol 20212). All the experiments were
performed with 6 months old male Pten knock-out (Pten KO) mice (ARR2PB-Cre and floxed Pten
(Pten
f/f
) alleles on C54BL/6xDBA2/129 background) weighing 25–40 g. Our mouse PCa model
has designed to combine prostate-specific Pten gene knockout with a conditional luciferase
reporter gene (Liao et al., 2007). Mice were housed under controlled conditions of temperature
(22°C), humidity, and light (14 h light, 10 h dark) with water and food available ad libitum. After
2 weeks of daily phenelzine (10mg/kg) via oral gavage, mice were sacrificed. The dose of
phenelzine (10 mg/kg body weight) was chosen as representative of a standard systemic MAO
inhibitor therapy used clinically and in previous studies.
2.1.2 Chemicals
Phenelzine sulfate salt (phenelzine): Sigma-Aldrich, USA. Percoll: P1644; Sigma-Aldrich.
Phosphatase Inhibitor Cocktail: PhosSTOP; Roche Applied Science. Protease Inhibitor Cocktail:
Roche Applied Science, obtained from Sigma-Aldrich (St. Louis, MO). 5-hydroxytryptamine
binoxalate (5-HT): PerkinElmer, Boston, MA. All other compounds were stored properly in accord
with the recommendations from the manufacturer. All the working solutions were prepared fresh
the day of experiment according to detailed protocol.

13

2.2 Bioluminescence imaging (BLI)
Mice were anesthetized with a mixture of 1.5% isoflurane/air using an Inhalation
Anesthesia System (VetEquip, Inc., Pleasant Hill, CA), and were given a single i.v. injection of
luciferin d at 50 mg/kg mouse body weight (unless otherwise specified). After waiting for ~ 7 min
to allow proper distribution of luciferin, the mice were placed in the chamber of IVIS 200 optical
imaging system (Xenogen Corp.) with continuous 1% to 2% isoflurane exposure. A region of
interest (ROI) was manually selected over relevant regions of signal intensity. The area of the ROI
was kept constant within experiments and the intensity was recorded within a ROI. Photons were
collected, and images were analyzed using Living Image software v. 2.50 (Xenogen). Signal
intensity was quantified as photon count rate per unit body area per unit solid angle subtended by
the detector (units of photons/s/cm
2
/steradian).
2.3 MAO A catalytic activity/inhibition assay
MAO-A catalytic activity was determined in control and phenelzine treated mice by
radio-assay as described previously (Wu et al., 2014). In this assay, 5-hydroxytryptamine
binoxalate (5-HT) served as substrate. Appropriate amount of protein (~1 mg prostate
homogenate) was incubated with 1 mM 14C-5-HT diluted in the assay buffer for 20 minutes at
37°C. The reactions were terminated by adding the ice-cold 6 N HCl. The resulting products
were extracted using benzene/ethyl acetate (1:1) and then were centrifuged. Radioactivity of
reaction products in organic phase was determined by the liquid scintillation spectroscopy. For
inhibition activity assay, sample homogenates were pre-treated with MAO inhibitor (in this
project, phenelzine) for 20 minutes at 37°C, then the MAO A assays were followed.
14

2.4 Mitochondrial isolation
Prostate mitochondria were isolated from mice as previously described (Irwin et al., 2011).
Mice were sacrificed, and the prostate was rapidly dissected and weighted, and homogenized at
4°C in 5ml mitochondrial isolation buffer [MIB; pH7.4, containing sucrose (160 mM), EDTA
(10mM), Tris-HCl (100mM), Phosphatase Inhibitor Cocktail and Protease Inhibitor Cocktail].
Prostate homogenates were pull up into one sample for each group and were centrifuged at 3,100
× g for 5 min at 4°C. The pellet was resuspended in MIB, homogenized and centrifuged again at
3,100 × g for 5 min. The supernatants from two times-centrifugations were combined and crude
mitochondria were pelleted by 10 min centrifugation at 21,000 × g at 4°C. Pellet was re-suspended
in 6ml 15% Percoll made in MIB, spin again at 21,000 × g for 10 min. Layer the loose pellet over
premade 23/40% Percoll discontinuous gradient, and centrifuged at 31,000 × g for 10 min. After
centrifugation, the purified mitochondria were collected at the 23/40% interface and then washed
by centrifugation at 16,700 × g for 13 min using 10 ml MIB.

Fig. 4 Purified mitochondria at the interface of 23% and 40% premade Percoll gradient.
Collect loose pellet at the bottom and transfer into 1.5ml Eppendorf tube, wash
mitochondria in MIB by centrifugation at 9000 × g for 8 min 4 C. The resulting mitochondria
15

samples were resuspended in MIB to appropriate concentration and were used immediately for
protein reading, respiratory activity measurements or stored at -80 C for future experiments.
2.5 Respiratory measurement
Mitochondrial oxygen consumption was measured using Seahorse XF96 Analyzer (Agilent
Technologies, Ins.). Isolated prostate mitochondria were diluted in mitochondrial assay solution
[MAS; 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM
EGTA, and 0.2 % w/v fatty acid-free BSA, pH 7.2 at 37 C] with 10mM succinate, 2uM rotenone,
10mM pyruvate, 2mM malate, 4uM FCCP as substrate to yield a final concentration of 20µg/250µl.

Fig. 5 2µg prostate mitochondria under microscope (x10)
In coupling assay, parameters involving non-mitochondrial oxygen consumption
(minimum rate measurement after Antimycin injection), basal respiration (last rate measurement
before first injection subtracted by non-mitochondrial respiration rate), maximal respiration
(maximum rate measurement after FCCP injection subtracted by non-mitochondrial respiration
rate), proton leak (minimum rate measurement before Oligomycin injection subtracted by non-
16

mitochondrial respiration rate), and ATP production (last rate measurement before Oligomycin
injection subtracted by minimum rate measurement after Oligomycin injection) can be calculated.
Stimulated of state 3 respiration was initiated by the addition of ADP. State 4 respiration was
estimated by the respiration rate following depletion of ADP. The rate of oxygen consumption was
calculated based on the slope of the response of mitochondria to the successive administration of
substrates. The respiratory control ratio (RCR) was determined by dividing the rate of oxygen
consumption/min (OCR) for state 3 by OCR for state 4 respirations.
In the electron flow assay, for Complex I, its inhibitor rotenone was injected. For Complex
II, substrate succinate was injected. Calculating the OCR rate before and after these injections
provides the data of respiration driven by Complex I or II.
2.6 Western blot analysis
About 30µg/well protein were loaded into each well of 12% SDS-PAGE gels (Bio-Rad
Laboratories, Hercules, CA). Gel were electrophoresed with Tris/glycine running buffer, and
transferred to Immun-Blot PVDF Membrane (Cat. 1620177; Bio-Rad Laboratories, Inc). The blots
were incubated with MitoProfile Total OXPHOS Rodent WB Antibody Cocktail consisting of
anti-Complex I subunit NDUFB8 20kDa; anti-Complex II subunit 30kDa; anti-Complex III
subunit core II 48kDa; anti-Complex IV COXI 40kDa; anti-ATP synthase subunit alpha 55kDa
(1:1000; Mitosciences). Depending on the molecular weight of probed target proteins, anti-β-actin
(1:1000; AbCam) was used as a loading control for either prostate lysates or mitochondria.
Antigen-antibody complex were visualized with Pierce SuperSignal chemiluminescent substrates
(Thermo Scientific, Waltham, MA) and captured by ChemiDoc Touch Imaging System (Bio-Rad
Laboratories). All band intensities were quantified using Image Lab software (Bio-Rad
17

Laboratories).
2.7 Statistical analysis
T-test was used for statistical analysis and p-value was determined using Microsoft Excel
and Prism 6 (GraphPad, Inc, USA). Differences were considered statistically significant at * p <
0.05 and ** p < 0.01.
18

Chapter 3. Results
3.1 Phenelzine reduces prostate weight in Pten KO mice
Pten KO mice ~ 6 months old were treated with 10mg/kg phenelzine (Day 1, mean body
weight: 31.68 ± 4.79 g) or 0.9% saline as controls (average weight: 31.79 ± 5.08 g) daily oral
gavage for 2 weeks. The day after the last treatment, mice were sacrificed. Prostate, brain, liver
and heart were dissected and weighed. After 14 days of treatment, as shown in Fig. 6A, mouse
body weight from two groups were not significantly different (Day 14, mean body weight: 30.06
± 4.39g in phenelzine vs 31.43 ± 5.58g in controls). Fig. 6B showed no significant difference
indicating no general toxicity compared to saline control. Phenelzine effectively decreased prostate
size as measured by wet weight (107.88 ± 36.13 mg, n=26) compared to controls (169.43 ± 49.73
mg, n=26), reduced 36.3% (Fig. 6C), while no changes were seen in other tissues (brain and heart)
from two groups (data not shown).

19

Fig. 6 6-months-old Pten KO male mice were treated with 10mg/kg phenelzine in 0.9% saline (n=22) or 0.9% saline
as controls (n=21) for 2 weeks. Prostate was then dissected and weighted. A. Mice body weight after treatment. B.
Mice daily body weight in two groups during the treatment. C. Prostate weight after treatment. Data represent the
mean ± SD, *p<0.05, **p<0.01

B
C A
20

3.2 Phenelzine reduces the MAO A activity of prostate from Pten KO mice
After phenelzine treatment, MAO A catalytic activity was determined in prostate from Pten
KO mice.
Phenelzine treated group decreased MAO A activity (0.15 ± 0.02 nmol/20min/mg)
compared to controls (0.50 ± 0.11 nmol/20min/mg), reduced 69.5%, indicating phenelzine was
effective in inhibiting MAO A catalytic activity.
21

Fig. 7 Phenelzine treated group showed lower MAO A activity. 6 months old Pten KO male mice were treated orally
with 10mg/kg phenelzine or saline as controls for 2 weeks. Mice were then sacrificed, prostates were homogenized.
MAO A radio-assay was performed with homogenates as described in Material and Method section. Data represent
the mean ± SEM, *p<0.05, **p<0.01

22

3.3 Phenelzine inhibits prostate tumorigenesis
Our previous work shows that loss of MAO A in the Pten KO mouse model of prostate
cancer significantly reduced cancer formation, epithelial cell proliferation, and cancer stem cells
compared with Pten KO prostate (Liao et al., 2018), which suggested that phenelzine could reduce
prostate tumorigenesis as well.
The progression of cancer in mice was monitored by bioluminescence imaging (BLI) at the
end of the treatment using a stably expressed Firefly luciferase reporter. Sometimes, the emitting
light will be absorbed by the skin or fur, thus the signal from BLI will be attenuated (Rice et al.,
2001). We shaved mice dark fur on the site of prostate in order to reduce the attenuation of the
light. Mice were anesthetized immediately following luciferin i.v. administration and imaged.
Images shown are from 1 mouse in control group and 3 mice in phenelzine treated group. As shown
in Fig. 8, a strong bioluminescent signal from an area corresponding to the prostate in control
group (~ 10-fold higher signal), whereas the phenelzine treated mice had a relatively weak signal
intensity (2.13E+07 vs 1.16E+08 p/sec/cm
2
/sr). BLI imaging showed that the signal decreased by
phenelzine treatment.
As shown in Fig. 9, prostates from Pten KO mice had enlarged APs and VPs compared to
phenelzine treated mice. Most importantly, APs in Pten KO prostates were usually fused to the
adjacent seminal vesicle and the range of APs were generally larger than controls, which is
indicative of local invasion resulted from cancer. In summary, phenelzine abrogated cancer
development in mice prostate.
23

Fig. 8 Male 6 months-old Pten KO mice were anesthetized. After i.v. injection with luciferin (40 mg/kg), the
chemiluminescence was imaged by an IVIS imaging system for 1 minutes. One representative mouse in control group
and 3 of phenelzine treatment mice are shown. Control mouse shows higher signal compared with treated mice. The
scale bar that accompanies these images displays counts.

24

Fig. 9 Phenelzine treated attenuated the cancer morphology. After one day of the treatment, mice were sacrificed and
intact prostates dissected. Representative images from two groups were presented. Different prostate lobes were
circled with blue dashed lines and labeled with letters. A, anterior prostate; V, ventral prostate; S, seminal vesicle.
Red arrow: blood vessels.

25

3.4 Phenelzine reduces the mitochondrial aerobic metabolism of prostate from Pten KO mice
This experiment not only examines the overall mitochondrial condition before and after
treatment but also determines the possible target of phenelzine on electron transport chain as well
as ATP synthesis.
45% decrease in basal respiration (p<0.02) shows lower energetic demand of the
mitochondria in treated group. With the addition of oligomycin, mitochondrial ATP production
was decreased 44% (p<0.04), which demonstrated the phenelzine affect the ATP synthase. Since
proton circuit across the inner membrane of mitochondria is the mechanism that drives oxidative
phosphorylation and ADP phosphorylation, 51% decrease in proton leak (p<0.001) and 44%
decrease in maximal respiration (p<0.02) confirmed the influence on respiratory chain complexes
by phenelzine. All data were analyzed by Excel, detailed calculation was described in Materials
and Methods, and expressed as OCRs (pmol/min/well).
Overall, the purified prostate mitochondria from the phenelzine-treated group exhibited
decreased consumption of oxygen compared with controls. In the same animals, there were no
significant changes in mitochondrial respiration parameters of brain and liver (data not shown)
generally indicating tissue-specific effect in the Pten KO prostate cancer mouse model. The effect
of phenelzine induced decline in aerobic respiration was further studied by examining the
expression of proteins involved in oxidative phosphorylation and ATP synthesis.
Next, we examined the prostate mitochondrial respiratory control ratio (RCR), the increase
in respiration rate in response to ADP, considered the best general measure of mitochondrial
26

function in isolated mitochondria. We observed a downward trend in RCR that did not reach
significance following treatment with phenelzine, suggesting that the mitochondrial coupling state
of respiration with ATP production, meaning the cancerous tissue may have a decreased ability to
produce ATP to support or maintain growth.
Importantly, the electron flow assay phenelzine-treated mitochondria revealed a significant
increase (25.2%) in succinate stimulated Complex II-driven respiration compared with the control
group, which lead us to investigate the expression of mitochondrial proteins of the respiratory
chain.

27

A
B
28

Fig. 10 Phenelzine treatment reduced prostate mitochondrial aerobic metabolism. A. Illustrative example of
mitochondrial respiration measurement performed on mitochondria isolated from phenelzine treated and control
prostate. Basal respiration reveals the energetic demand of the mitochondria under different initial conditions; ATP
production reveals the partial of basal respiration was used; H
+
(proton) leak reveal the remaining basal respiration
that not coupled to ATP production; Maximal respiration reveals the maximum OCR that mitochondria can achieve
after FCCP collapsing the proton gradient and disrupting the mitochondrial membrane potential. B. Significant
difference in respiratory parameters from groups. C. Respiratory control ratio (RCR) revels the mitochondrial coupling
state. Result shows that RCR was decreased in phenelzine treated group. Data are presented as the average of 5-8
replicate wells ±SEM. ** indicates p < 0.01, * indicates p < 0.05.

C
29

Fig. 11 A representative graph of electron flow experiment using mitochondria isolated from treated and control mice.
Pten KO mice (male, 6 months) were orally treated with phenelzine 10mg/kg (n=3) or saline as controls (n=3) for 2
weeks. After treatment, mice were sacrificed, prostates were homogenized, the lysates were pooled from 3 mice, and
mitochondria were isolated. 2µg mito/well of prostate mitochondria were attached to XF96 plate and the experiment
was performed in the presence of pyruvate/malate/FCCP. Complex I respiration was initiated, followed by sequential
addition of rotenone, succinate, Antimycin A and TMPD/Ascorbate. Oxygen consumption rates were converted to
pmol/min. Succinate-stimulated Complex II respiration was significantly increased by 25.2%. Complex II, also known
as succinate dehydrogenase is a known tumor suppressor (Aspuria, 2014).

30

3.5 Phenelzine decreases the expression of mitochondrial OXPHOS complexes in Pten KO
prostate
The expression levels of the OXPHOS complexes were evaluated by quantitative
immunoblot analysis of lysates from Pten KO mouse prostates 14 days after treatment with
phenelzine (10mg/kg in saline) or vehicle control. In Fig. 12A, the protein expression of OXPHOS
complexes are shown. Representative subunits from Complex II, III and IV were not significantly
altered. Complex I (Fig. 12B), subunit NDUFB8 and Complex Vα (Fig. 12C) expression were
significantly reduced by 33.7% and 35.4%, respectively after 2 weeks of daily oral phenelzine
10mg/kg. This result corroborates with the result from coupling assay (Fig. 10B) in which ATP
production was decreased by phenelzine.

31

Fig. 12 Phenelzine-induced decline potentiation of protein bioenergetics-related profile. Mice were
orally treated with 10mg/kg phenelzine and 0.9% saline as control for 2 weeks. After 14 days treatment, mice were
sacrificed and protein were isolated from mice prostate.30µg/well mitochondria were loaded each well. Result shows
phenelzine induced a significant decline of Complex I and V. A. Representative protein expression of OXPHOS
complexes. B. Complex I, subunit NDUFB8, accessory subunit of the NADH dehydrogenase; C. Complex V, subunit
α, contained within the extra-membranous, producing ATP from ADP; D. Complex II, subunit SDHB, one of the four
subunits of succinate dehydrogenase; E. Complex III, core II subunit, is necessary for the formation of the complex;
F. cytochrome c oxidase, subunit IV (COXIV), one of subunits of Complex IV

A B
C D
E
F
32

Chapter 4. Conclusion and Discussion
MAO A inhibitors were previously developed as effective therapeutic agents for treating
panic disorders and depression. Recent studies reveal the value of MAOIs as anti-cancer agents.
In this study, we investigated the role of phenelzine in the development of PCa and mitochondria
functions, using Pten KO mouse models, which share a similar genetic defect with human PCa
patients.
First, after 2 week-treatment, prostate weight showed significant decreased by phenelzine
but no change on mouse body weight indicating the decrease on prostate may due to the tumor
reduction (Fig. 6). BLI results confirmed this hypothesis that the luciferase signal from treated
group were lower than controls (Fig. 8). Meanwhile, unlike Pten KO prostates, the prostate glands
in treated mice were surrounded by intact basement membranes, with no epithelial cells invading
into adjacent stromal areas (Fig. 9). These suggested the development of cancer was stopped by
phenelzine treatment.
Mitochondria from phenelzine treated group showed overall decreased consumption of
oxygen and changed main parameters that describe key aspects of mitochondrial oxidative
phosphorylation from coupling assay (Fig. 10). Decreased RCR of treated group indicates
downregulation of the respiratory machinery by phenelzine in cancerous tissue. Electron flow
assay revealed that a potential target of phenelzine efficacy is succinate dehydrogenase (Complex
II) an important mitochondrial enzyme of both the citric acid cycle and the electron transport chain
(Fig. 11). Though the OXPHOS protein expressions were generally decreased by treatment (Fig.
33

12), functionally increased Complex II-driven respiration will lead us to further explore Complex
II enzyme activity in future studies to identify the molecular mechanisms elicited by MAO A
inhibition, resulting in reduced prostate cancer in the Pten KO mouse model.
Collectively, these studies are indicative of the therapeutic potential of MAO A inhibitors
for prostate cancer. Using the Pten KO model, this study confirms our previous work using various
xenograph mouse models and provides new insights on the mechanisms of MAO A inhibitors on
mitochondrial function. These preclinical findings highlight the promise of targeting these
mechanisms to stop cancer.

34

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Abstract (if available)

Abstract Prostate cancer (PCa) emerges as the second leading cause for male cancer death in the Western world. In 2019, there will be approximately 174,650 new prostate cancer diagnoses and 31,620 prostate cancer deaths (Siegel et al., 2019). Monoamine oxidase A (MAO A) is a mitochondrial enzyme that degrades monoamine neurotransmitters and is overexpressed in prostate cancer (Peehl et al., 2008). MAO inhibitors effectively inhibit PCa cell proliferation and tumor growth (Gaur et al., 2019

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Creator Li, Jiachun (author)

Core Title Monoamine oxidase inhibitors regulate tumorigenesis and mitochondrial function in a prostate cancer mouse model

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/29/2019

Defense Date 04/29/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag mitochondrial function,monoamine oxidase,OAI-PMH Harvest,phenelzine,prostate cancer,tumorigenesis

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Advisor Shih, Jean Chen (committee chair), Cadenas, Enrique (committee member), Zhang, Yong (committee member)

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