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NMI: a near infrared conjugated MAO-A inhibitor as a novel targeted therapy for colorectal and other cancers

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NMI: a near infrared conjugated MAO-A inhibitor as a novel targeted therapy for colorectal and other cancers

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Content llश्रीll
NMI: A NEAR INFRARED CONJUGATED MAO-A INHIBITOR AS A NOVEL
TARGETED THERAPY FOR COLORECTAL AND OTHER CANCERS
by
VED HARSHAD KOPARDE
A Thesis Presented to the
FACULTY OF THE ALFRED E. MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
[PHARMACEUTICAL SCIENCES]
August 2024
Copyright 2024

ii
Acknowledgements
First and foremost, I'd want to thank Dr. Jean Shih, my adviser, for her important counsel,
support, and patience during this journey. Your thoughts and encouragement motivated me to
advance academically and personally.
I want to express my heartfelt gratitude to my committee members, Dr. Ian Haworth and Dr.
Tiger Zhang, for their time, effort, and useful feedback. Your experience was crucial in
shaping this research.
I am deeply grateful to my family and friends for their constant support and understanding.
Special thanks to my colleagues and lab mates, Dr. Hui-Ju Tseng, Unnati Shah, and Ronak
Shethia, for their camaraderie and for making the study process more enjoyable.
In the end, I'd like to thank the Alfred E. Mann School of Pharmacy for providing the
vital resources and infrastructure to make this research possible.
Thank you for being a part of this adventure. Your contributions and encouragement have
been invaluable, and I am eternally thankful.
Thank you.

iii
Table of Contents
a. Acknowledgements……………………………………………………………………ii
b. List of Tables…………………………………………………………………………...v
c. List of Figures…………………………………………………………………………vi
d. Abstract………………………………………………………………………………vii
1. Chapter I……………………………………………………………………………….1
1.1 Introduction: Role of MAO-A in cancer…………………………………………..1
1.2 MAO-A mediates the progression of prostate cancer……………………………..4
1.3 Monoamine oxidase inhibitors as new anti-cancer therapeutics.………………….5
2. Chapter II………………………………………………………………………………8
NMI: Novel Approach to Cancer Treatment
2.1 Heptamethine dyes for tumor targeting
activity…………………………………………………………………………………….8
2.2 NMI structure and
properties………………………………………………………........................................10
2.3 NMI inhibits the growth of prostate cancer………………………………..................12
2.4 NMI and clorgyline treatment of glioma in vitro and
vivo…………………………………………….………………………………………....12
3. Chapter III……………………………………………………………………………14
Colorectal Cancer: Signaling Pathways, Potential use of MAO-A Inhibitors as a
Therapy and NMI as a therapy
3.1 Progression of CRC and important associated signaling
pathways………………………………………………………………………….15
3.2 PI3K/AKT pathway and prognosis of
CRC…………………………................................................................................15
3.3 The role of MAO-A in
CRC…………………………………………........................................................16
3.4 MC-38 and CT-26 cancer cell
lines………………………………………………………………………………17

iv
3.5 Mediation of Tumor Associated Macrophages for immunotherapy in cancer
is controlled by MAO-A………………………………………………................18
3.6 Suppression of MAO-A reduces MC-38 cancer through macrophage
reprogramming ………………………………………………………..................19
3.7 Goals of the Thesis………..……………………………………………………...19
4. Chapter IV……………………….…………………………………………………...21
Potential NMI interacting proteins in CT-26 and MC-38 cells
4.1 Experimental Procedures and Results……………………………………………21
4.1.1 Cell culture………………………………………………….....................21
4.1.2 NMI and clorgyline treatment………………………………....................21
4.1.3 Separation of NMI binding proteins by SDS-PAGE……………………..22
4.1.4 Mass spectrometry of NMI interacting proteins ……………....................22
4.2 Results…………………………………………………………………………....22
4.2.1 Detection of 65 kDa NMI-Interacting Proteins in CT-26 and MC-38
Colon Cancer Cells…………………………………….…………………22
4.2.2 Proteins in the 65 kDa band in MC-38…………………………………...23
4.2.3 Myosin 9 .………………………………………………………………...25
4.2.4 ERM Proteins.……………………………………………………………25
4.2.5 Importin beta 1.…………………………………………………………..26
4.2.6 Tubulin 3 beta …………………………………………………………....27
4.2.7 Fatty Acid Synthase ……………………………………………………...28
4.2.8 Dynamin 1………………………………………………………………..28
4.2.9 Exportin 2………………………………………………………………...29
4.2.10 HECW2 ……………………………………………………….................29
4.2.11 Programmed death 6 interacting protein………………………………....30
4.2.12 Plectin-1……………………………………………………….................30
4.2.13 Filamin-A………………………………………………………………...31
4.2.14 ZMAT3...………………………………………………………................31
5. Chapter V…………………………………………………………………………….33
Summary………………………………………………………………………….….33
e. References…………………………………………………........................................35

v
List of Tables
1. Expression levels of MAO A and B in different cancer types compared
to normal tissue………………………………………………………………………...4
2. MAOIs and their influence on various kinds of cancers………………………………..7
3. Heptamethine dyes combined with drugs for cancer targeting
and treatment……………………………………………………………......................9
4. List of proteins interacting with NMI in MC-38 cells with the number of
peptides in each sample……………………………………………………………….24

vi
List of Figures
1. In PTEN/MAO knockout mice, the lack of MAO reduces growth and
EMT…………………………………………………………………………………...5
2. MHI-148 dye structure………………………………………………………………..9
3. Structure of NMI……………………………………………………………………..10
4. SDS gel Composite and Raw stained membrane of CT-26
and MC-38 cells……………………………………………………………………..23
5. Predicting MAO-A inhibition by NMI disrupts PI3K/AKT/mTOR pathway,
reducing key proteins in MC-38 cells…………….………………………………….32

vii
Abstract
Monoamine oxidase A (MAO-A) has been linked to a variety of cancers because it produces
reactive oxygen species (ROS), which contribute to oxidative stress and mutagenesis.
Prostate, glioma, and colorectal cancers show upregulated expression of MAO-A. NearInfrared MAO-A Inhibitor (NMI), a heptamethine dye conjugated MAO-A inhibitor, targets
with higher specificity to cancer cells, and can function as both a non-invasive diagnostic tool
and a therapeutic agent. Wu et al. (J Am Chem Soc 2015;137:2366-2374), study on prostate
cancer found that MAO A inhibition by NMI decreases the prostate cancer growth, making
NMI a novel therapeutic agent. Irwin et al. (Pharm Res 2021; 38:461–47), studied the NMIbound proteins in glioma using gel electrophoresis and found two significant bands at around
~65kDa and less than 10kDa. This thesis aims to study the range of proteins bound by NMI
in CRC cell line MC-38 by gel electrophoresis followed by mass spectrometry. Gel
electrophoresis results revealed that NMI can bind to proteins at a specified molecular
weight, resulting in a single band at 65 kDa. Mass spectrometry data shows that NMI
decreases moesin; myosin 9; radixin; lamin B1; importin beta 1; tubulin 3, beta exportin-2;
prelamin A/C; dynamin-1; fatty acid synthase; tubulin 3-beta associated with CRC cell
growth and survival. These data require confirmation, but suggests that NMI could be a
potential therapeutic agent for cancers.

1
Chapter I
1.1 Introduction: Role of MAO-A in cancer
Mary Bernheim identified the first monoamine oxidase enzyme in 1928 and labeled it
tyramine oxidase. Monoamine oxidases are members of the flavin protein family and, like
most members of this family, are presumed to function as flavin amine oxidoreductases.
There are basically two kinds of monoamine oxidases: monoamine oxidase A (MAO-A) and
monoamine oxidase B (MAO-B). Although MAO-B is present in blood platelets, most of the
MAO-A in the body is found within cells in the gastrointestinal system, lungs, liver, and
placenta. Both types of monoamine oxidase catalyze the oxidative deamination of biogenic
amines like dopamine, but the amine oxidases play different roles and have different
physiology. Of the two types of monoamine oxidase, MAO-A is probably the more
important. 1
MAO-A plays a key role in the maintenance of functional neurotransmitter levels. As such, it
has been credibly linked to a host of neurological disorders. The most direct pathway from
MAO-A to a specific disorder seems to involve the enzyme converting too much serotonin
into 5-hydroxyindoleacetic acid (the pathologically elevated substance found in the
cerebrospinal fluid of depressed patients with neurodegenerative illness). Neuroanatomical
studies provide a clear basis for linking elevated MAO-A levels and activity to increased
expression and pathologically consequential activity of pro-apoptotic proteins (leading to
unnatural death of dendrite-hugging neurons) and of proteins that normaly degrade the Tau
protein found in the "tangles" that riddle the brains of Alzheimer's patients and those with
other forms of dementia. 1

2
MAO-B can be classified as a mitochondrial enzyme that is dependent on flavin adenine
dinucleotide (FAD). It helps to catalyze the oxidative deamination of certain amines. Two
substrates that MAO-B is preferentially selective for are benzylamine and 2-
phenylethylamine, but it also degrades other monoamines like tyramine and dopamine. One
of the first and most selective MAO-B inhibitors that was a significant improvement over
earlier drugs was selegiline (formerly L-deprenyl), which has been used for nearly 40 years to
treat Parkinson's disease. The enzyme is abundant in glial brain cells, specifically in the area
surrounding dopaminergic synapses. In the synaptic cleft, it primarily regulates the amounts
of biogenic amines, their release, and their storage. When it oxidizes monoamine substrates,
MAO-B produces reactive oxygen species (ROS) and hydrogen peroxide (H2O2). Another
way to think about it is that MAO-B "burns" amine neurotransmitters to produce energy (a
high amount of "energy" is also found in H2O2).1
MAO plays an important role in the development of cancer as a direct or indirect
consequence of its ability to produce H2O2 through the oxidative deamination of a variety of
amines. H2O2 is a source of oxidative stress that is capable of damaging DNA and is thought
to be a contributor to the mutagenesis that initiates oncogenesis. Either amplification of the
MAO gene or overexpression of the enzyme is found in several human tumors, including
those of the brain, prostate, and liver. Tumorigenesis is a multistep process that most likely
depends on MAO catalysis for the initial oxidation of certain amines to genotoxic products
that pay for the engine of cell division. Although increased production of H2O2 by MAO
increases mutagenesis, the implications of net increases or decreases in individual amines for
signaling and neuronal functions as potential effects of altered expression of the isoenzymes
(MAO-A and MAO-B) in various types of cancers are less well understood.2

3
High-grade MAO-A expression has also been linked to renal cell carcinoma.1 Furthermore,
studies have indicated that human glioma tissues and cell lines show markedly elevated levels
of MAO-A. When MAO-A was starved to near extinction, in vitro invasion rates decreased
sharply, and glioma tumors regressed when clorgyline—an irreversible and highly selective
MAO-A inhibitor—was applied. Non-small cell lung cancer (NSCLC) tissues had
significantly higher expression of MAO-A protein and mRNA than healthy lung tissues.
Moreover, higher MAO-A levels were associated with N2 lymph node metastases and the
aggressive clinical stage of lung cancer. Cancer invasion and metastasis occur in a series of
steps, one of which is the epithelial to mesenchymal transition (EMT). MAO-A might be
involved in the promotion of non-small cell lung cancer (NSCLC) via signaling pathways
which decreases the expression of E-cadherin, which is a protein that normally helps cells
stick to one another in a very organized and orderly way, which is how they tend to behave
when they are part of an epithelial layer. Another way is that MAO-A may increase the
expression of N-cadherin, which is somewhat like E-cadherin but promotes a more
"mesenchymal" kind of behavior where the cells start to move around a lot more. In breast
cancer, higher expression levels of the isoenzyme MAO-A correspond to poorer clinical
outcomes. Investigating the association of clinical data has shown that monoamine oxidase A
is negatively correlated with patient survival in multiple cancer types, including breast
cancer, as observed in the GSE9893 cohort. Interestingly, a typical feature of human breast
tumor cell lines that have acquired resistance to anticancer drugs is overexpression of MAOA. This linkage of MAO-A to prognosis is especially pronounced with high-grade, estrogen
receptor-negative breast cancers, where the poor recurrence-free survival of patients is
alarmingly subpar. In neuroendocrine gastroentero-pancreatic tumors, increased monoamine
oxidase A expression makes the disease more aggressive.1 3 4 5 6

4
In addition, recent research has indicated that the MAO-A gene can be a target for certain
innovative anticancer therapies, such as those that involve valproic acid. Valproic acid
induces the expression of the MAO-A gene via a signaling mechanism that has recently come
to light. That signaling mechanism starts when the valproic acid activates the gene for Akt, a
protein kinase that goes on to activate a set of factors known as the Forkhead box O (FoxO)
transcription factors. These are proteins that function in a cellular context—here, in the
context of cancer that grows in a tissue with a lot of oxidative stress—to regulate the
expression of a set of target genes, one of which is the MAO-A gene.
4 The Table 1, shows
the expression of MAO-A and MAO-B in different types of cancers.
Table 1. Expression levels of MAO A and B in different cancer types compared to normal tissue. Data on Prostate
Cancer is based on Chen and Wu 2023. Data on Breast Cancer and Lung Cancer is based on Aljanabi et al. 2021.
Data on Colorectal Cancer is based on Wang et al. 2021. Pancreatic Cancer data is based on Schmich et al. 2023.
Glioma Cancer data is based on Kushal et al. 2015.
Cancer Type MAO-A Expression MAO-B Expression
Prostate Cancer Higher Higher
Breast Cancer Higher Lower
Colorectal Cancer Higher Lower
Pancreatic Cancer Higher Not well-documented
Lung Cancer Higher Higher
Glioma Higher Lower
1.2 MAO-A mediates the progression of prostate cancer

5
It is known that the deletion of PTEN leads to increased AKT phosphorylation, a wellestablished tumor-promoting factor that enhances both the proliferation and migration of
prostate cancer (PCa) cells. Surprisingly PCa cell line investigations demonstrate that MAOA
promotes proliferation of cells via the AKT/FOXO1/TWIST1 pathway. Thus, Liao et al.
studied whether MAOA deletion affected AKT phosphorylation in PCa formation. The PCa
cell line was established from a primary tumor in the mouse prostate and carries a double loss
of the tumor suppressors Pten and p53. A lentiviral system to introduce shRNAs that
specifically target MAO A into the cells was able to decrease MAO A enzymatic activity by
greater than 50% in the mouse cell line. Inoculation of the MAO-A knocked down cells into
the flank of an immunocompetent mouse did not cause any tumor growth. Conversely, when
PCa control cell lines inoculated into the flank of an immunocompetent mouse, the mouse
developed a tumor at the site of inoculation. Figure 1 reveals that MAOA elimination
reduces the level of AKT phosphorylation produced by Pten deletion in prostate epithelial
cells, supporting the significance of MAOA in AKT cascades in PCa development.7
Fig 1. In PTEN/MAO knockout mice, the lack of MAO reduces growth and epithelial . Liao et al. 2020
1.3 Monoamine oxidase inhibitors as new anti-cancer therapeutics
MAO-A PI3K
AKT
mTOR
ROS
EMT and Proliferation

6
Pargyline and phenelzine, two MAO-A inhibitors, reduce the neuroendocrine differentiation
and autophagy activation of prostate cancer cells. This led to consider the potential of these
inhibitors as treatment agents for neuroendocrine tumors. However, the study also points out
that MAO-A is probably not the only target through which these two compounds carry out
their anti-cancer effects. Combination of isoniazid (a known MAO-A inhibitor) and
heptamethine cyanine dyes does effectively reduce metastatic prostate cancer.
However, in MDA-MB-231 cells, clorgyline, an irreversible, selective MAO-A inhibitor,
triggered a mesenchymal to epithelial transition. From a scientific standpoint, this behavior is
explained by clorgyline-mediated activation of the epithelial protein marker E-cadherin in
breast cancer cells. Investigations found that clorgyline inhibits both the E-cadherin-β-catenin
and β-catenin/p-GSK3β complexes. In conclusion, the transition from a mesenchymal to an
epithelial-like cell state in clorgyline-treated MDA-MB-231 cells demonstrates that MAO-A
is an important regulator of EMT in breast cancer.
On the other hand, clorgyline has been shown to reduce the growth of gliomas that are
resistant to temozolomide (TMZ). This effect was due in part to a decrease in the invasion of
tumor cells and to the cytotoxicity produced by clorgyline. Therefore, a low-dose regimen of
TMZ in combination with an MAO-A inhibitor like clorgyline may be an
effective therapy against brain cancer. 8 Many other MAO-A and MAO-B inhibitors like
Phenelzine, Moclobemide, Selegiline, Procarbazine, Rasagiline and Linezolid are being used
to treat different types of cancers are illustrated in the Table 2.
Table 2. MAOIs and their influence on various kinds of cancers. Phenelzine data obtained from Chen and Wu 2023
and Wang et al. 2021. Clorgyline data obtained from Chen and Wu 2023. Moclobemide data obtained from Wang et
al. 2023. Rasagiline data obtained from Ayoup et al. 2021. Selegiline, Procarbazine and Linezolid data obtained from
Meier-davis et al. 2012

7
Inhibitor Tumor Type Molecular Mechanism References
Phenelzine
Prostate Cancer,
Colon Cancer,
Melanoma
Reduces MAO-A, slowing tumor growth and
improving the immune system's response.
2,9
Moclobemide Colon Cancer,
Melanoma
Durable MAO-A inhibitor boosts the T-cell
responsiveness and decreases immunosuppression
promoting macrophages.
9
Clorgyline Prostate Cancer The irreversible MAO-A inhibitor enhances
apoptotic activity and lowers metastasis.
2,10
Selegiline Various Cancers
Specifically blocks MAO-B, modifies
immunological responses, and decreases growth of
tumors.
11
Procarbazine Hodgkins
Lymphoma
Blocks both the MAO-A and MAO-B, which
influences cancer cell activity and proliferation.
11
Rasagiline Various Cancers
Specifically lowers MAO-B and decreases
inflammatory action in the tumor
microenvironment.
8
Linezolid Various Cancers Mild reversible MAO blocker slows growth of
tumors and improves treatment alternatives.
11

8
Chapter II
NMI: Novel Approach to Cancer Treatment
2.1 Heptamethine dyes for tumor targeting activity
In fluorescence imaging for cancer detection, the most commonly used near-infrared (NIR)
dyes are heptamethine cyanines. These dyes are generally attached to either small molecules
(such as medicinal drugs or ligands) or large macromolecules (like polymers or proteins) to
aid in the selective targeting of tumors. Without such preparations, however, numerous
heptamethine cyanine dyes—like IR-780, IR-783, IR-786, and IR-808 (also known as MHI148)—are used for targeting tumors and exhibiting anticancer activity just by virtue of their
chemical structure. Figure 2, shows the general structure of heptamethine dyes with
molecules denoted “X” and “Y” which are the substituted by different functional groups for
the dyes MHI-148, IR-780, IR-783, and IR-786. Moreover, the significant NIR absorption of
heptamethine cyanine dyes in the 650–950 nm range coupled with their low toxicity makes
them exceptional candidates for use in photothermal therapy (PTT) when combined with
standard chemotherapy techniques. 12,13
Various small molecules, including tumor-targeting ligands and potent drugs, have been
conjugated to commercial cyanine dyes for use in biomedical treatments in different types of
cancers (refer Table 3). However, the physicochemical properties of the dyes, including their
distinct hydrophobicity, polarity, and net surface charge, can impart a variety of nonspecific
interactions that impact the specificity of the resulting conjugates. To counter this issue, the
conjugation chemistry used with these dyes has been diversified, allowing for the
construction of new dye-drug candidates.

9
It was demonstrated that the formation of noncovalent/covalent albumin conjugates, which
can be trapped in tumors via receptor-mediated endocytosis of albumin, was responsible for
the tumor accumulation and persistence of heptamethine cyanine dyes. Albumin is an ideal
transporter protein for cyanine dyes because it is biodegradable, nontoxic, non-immunogenic,
and biocompatible. To limit the release of off-target payload after systemic injection,
albumin-cyanine dye complexes, in which albumin serves as an active targeting carrier for
imaging agent transport to tumor sites, may remain stable in the systemic circulation.
Because albumin improves the heptamethine cyanine dyes' water solubility and tumor
targetability, it plays a critical role in delivering these dyes to the tumor tissue and boosting
their availability.13

Fig 2. MHI-148 dye structure. Cooper et al. 2021
Table 3. Heptamethine dyes combined with drugs for cancer targeting and treatment. Cooper et al 2021
Type of Tumor Cancer cell lines Heptamethine Cyanine
Dye
Conjugate
Lung Cancer A549, NCIH-460, H358,
A549-DR
IR-783, IR-780, MHI-148 Methotrexate, Erlotinib
Breast Cancer MCF-7, MDA231, LTED IR-783, IR-780, MHI148,DZ-1
Methotrexate, FTS,
Genistein, Erlotinib
Hepatoma SMMC-7721, HepG2 IR-783, MHI-148, IR-780 Dasatinib, Methotrexate
Prostate Cancer PC-3, LNCap, DU-145, C4-
2
MHI-148, IR-780, IR-783,
IR-786
Isoniazid, MAO-A
inhibitor, Gemtabicine

10
Glioblastoma GL26, LN18, U251, T98T,
Primary PDX, U87
IR-780, IR-783,IR-786,
DZ-1
Rucaparib, Dasatinib,
Gemtabicine, MAO-A
inhibitor, Crizotinib
2.2 NMI structure and properties
Fig 3. Structure of NMI. Wu et al. 2015
NMI, which stands for near-infrared (NIR) dye conjugate MAO A inhibitor, is a brand-new
MAO A inhibitor that our lab has created (Fig 3). A potential theranostic chemical, NMI
serves as both a targeted cancer imaging probe and an anticancer drug. It is made up of two
primary parts: the near-infrared fluorescent heptamethine cyanine dye, MHI-148, and the
clorgyline moiety, an irreversible inhibitor of monoamine oxidase A (MAO A). This
combination enables NMI to act as an anticancer therapy as well as a targeted cancer imaging
probe. The near-infrared fluorescence of the MHI-148 dye renders in the visualization and
identification of cancer cells, and the drug's anticancer effects are further enhanced by the
clorgyline moiety's inhibition of MAO A activity. NMI is a good option for cancer detection
and treatment because of the synergy between MHI-148 and the clorgyline moiety, which
Clorgyline MHI-148 Dye

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improves the drug's capacity to localize within mitochondria, target cancer cells, decrease
MAOA activity, and demonstrate considerable cytotoxicity against cancer cells.14
Using widely available starting components it was synthesized in eight steps using a scalable
technique to yield hundreds of milligrams of product. It was demonstrated that the conjugate
localizes in mitochondria, targets PCa cells, and inhibits MAOA activity in the low
micromolar IC50 range. Its effectiveness in inhibiting the growth of three PCa cell lines with
elevated levels of MAOA—LNCaP, C4-2B, and MAOA-overexpressing PC-3—has been
demonstrated to surpass that of clorgyline, a recognized MAOA inhibitor. Tumor hypoxia,
likely caused by an activated HIF1α/OATPs signaling axis, is responsible for the increased
uptake and retention of NMI in tumors. This is corroborated by our observation that the
tumor-targeting effect of NMI may be amplified in PCa that express high levels of MAOA,
which is linked to increased tumor hypoxia. Indeed, the increase in HIF1α immunostaining in
cancer samples with enhanced expression of MAOA parallels the larger accumulation of
NMI in MAOA-overexpressing PC-3 tumors compared to a control tumor. 14 15
Our research team has studied its effectiveness in treating cancer in detail, with a particular
focus on prostate and glioma (brain) cancers. NMI outperformed clorgyline itself in
anticancer studies conducted both in vitro and in vivo. In orthotropic mice models, it
demonstrated strong cytotoxicity against glioma cells, including glioma stem cells, and
efficiently suppressed tumor growth, even in the presence of temozolomide (TMZ)
resistance, which is frequently seen in glioblastoma (GBM). It has been demonstrated that
NMI affects glioma cells as well as the tumor microenvironment. This finding provided more
evidence in favor of the theory that decreased tumor growth is a result of NMI's high
accumulation and MAOA suppression in tumor cells. .
14,16

12
NMI has the ability of serving as a therapeutic agent for CRC. Its use in therapy seems to
have many facets. NMI may potentially serve as a targeting agent for imaging the cancer
cells. But beyond that, NMI may have anti-cancer effects that are significant and that work
through a mechanism that is not fully understood. It seems likely that the production of the
targeted heptamethine dye is a vital part of how NMI exerts anti-cancer effects.
2.3 NMI inhibits the growth of prostate cancer
Wu et al. studied the efficacy of NMI as a MAO-A inhibitor by treating it on prostate cancer.
Genes responsible for the growth of tumor which were downregulated in the prostate cancer
after NMI treatment were also investigated. NMI targets LNCaP cells and enters them,
ultimately reducing the level of the MAO-A enzyme. When tested in vitro, NMI reduces the
viability of LNCaP, C4-2B, or C4-2B+AR cells. In vivo studies indicated that NMI reduced
tumor growth and MAO-A expression, as well as downregulating many oncogenic and cell
cycle regulatory genes in tumors, including MYC, FOS, NFKB1, JUN, and many others. In
vitro studies indicated that the reduction in MAO-A protein expression was correlated with a
reduction in migration and invasion of these cells. After treatment with NMI, the genes TP53,
CDKN1A, and CDKN2A, which are responsible for tumor suppression and cell cycle
regulation, were seen to have their expression increased. Along with these genes, the
expression of other genes like Bcl2, VIM, and MET was suppressed. The product of the MET
gene and its pathways play an important role in regulating prostate cancer cell growth and
survival. It was found that treating the mice with NMI inhibited tumor growth and reduced
expression of prostate-specific antigen. Furthermore, significant decrease in tumor weight
was recorded .
15
2.4 NMI and clorgyline treatment of glioma in vitro and vivo suppresses
development of tumors

13

NMI was tested for effectiveness against Temozolomide (TMZ) resistant glioblastoma
patient-derived (PDC) cells. The patient-derived cells, U251R, were then implanted in mice
and the effectiveness of the NMI was again tested, both as a single agent and in combination
with TMZ. Results showed that the NMI negatively influences the tumor growth and
significantly increases the survival rate for the mice that had the U251R cells implanted in
their brains.
Experiments of angiogenesis, proliferation, and innate immunity tissue staining on the NMI
and Clorgyline treatments were carried out. CD31 was chosen to evaluate the NMI and
Clorgyline effects on angiogenesis. CD31 is a marker for the endothelial cells, which are the
cells that form blood vessels. The results show that the density of microvessels is reduced in
tissues treated with NMI and Clorgyline compared to control tissues. Although the control
tissues show some appearance of blood vessel structures, the structures in the NMI and
Clorgyline tissues appear very faint. These results suggest that NMI and Clorgyline both
reduce angiogenesis in tumors.
Both Clorgyline and NMI, two inhibitors of the A isoform of MAO, have been shown to
enhance the innate immune response in their treated mice. This enhancement is apparently
achieved by increasing the levels of two pro-inflammatory factors, TNF-α and IL-6, and is
presumably important because these two factors have been shown in previous research to act
as major drivers of the immune response following an attack by a pathogen.16

14
Chapter III
Colorectal Cancer: Signaling Pathways, Potential Therapeutic Use of
MAO-A Inhibitors and NMI
The American Cancer Society uses incidence data from central cancer registries (through
2020) and mortality data from the National Center for Health Statistics (through 2021) to
estimate the number of new cancer cases and deaths in the United States each year and to
assemble the most recent data on population-based cancer occurrence and outcomes. It is
anticipated that there will be 611,720 fatalities due to cancer and 2,001,140 cases of cancer
diagnosed in the US in 2024. Cancer mortality decreased until 2021, saving almost 4 million
lives since 1991 as a result of declining smoking rates, early cancer identification for certain
types of cancer, and enhanced adjuvant and metastatic cancer treatment choices.
Nevertheless, rising incidence for six of the top ten malignancies poses a danger to these
achievements. Between 2015 and 2019, the incidence rates increased by 0.6% to 1% for
cancers of the pancreas, breast, and uterine corpus, and by 2% to 3% for cancers of the
prostate, female liver, kidney, human papillomavirus-associated mouth carcinomas, and
melanoma. Additionally, among young persons, the incidence rates of CRC (age <55) and
cervical cancer (age 30-44) increased by 1%–2% yearly. Among the late 1990s, CRC ranked
fourth for cancer-related deaths among people under 50 years of age for both men and
women, but it currently ranks first for males and second for females.17
The second most frequent cause of cancer-related deaths in the US is CRC. A 1%–2% yearly
rise in the number of people under 55 since the mid-1990s has contributed to the slowdown in
the incidence of CRC, which had been declining at a rate of 3%–4% per year during the

15
2000s. As a result, the percentage of cases among those under 55 rose from 11% in 1995 to
20% in 2019. Since roughly 2010, the incidence of regional-stage disease has grown by 2%–
3% yearly in those under 65, while the incidence of distant-stage disease has increased by
0.5%–3% annually in the same age group. This has reversed the general trend toward earlier
stage diagnosis that took place between 1995 and 200518
3.1 Progression of CRC and important associated signaling pathways
The causes of cancer and CRC, in particular, are not fully understood, but they are known to
involve multiple, successive, and mostly genetic changes. Many of the pathways implicated
in these signaling events are known to go awry in CRC. They include: Epidermal growth
factor receptor/EGFR, RAS, AXIN, RAF, Notch1, The p110α catalytic subunit of PI3K
(PIK3CA), PTEN, SMADs, Jagged1, TGFβR1/2, and β-catenin (CTNNB1). EGFR, RAS,
and AXIN are known to have increased numbers of mutations that give rise to CRC, while
changes in the worrisome pathways above tend to alter gene expression in favor of CRC cell
growth, survival, and capacity for invasion.
3.2 PI3K/AKT pathway and prognosis of CRC
The intracellular signaling pathway known as PI3K/Akt performs several essential cellular
functions. It controls the kinds of things that cells do when they are told to do something, like
migrate, differentiate, or grow. The signaling pathway that we are talking about in this case is
the one activated by the EGFR. This pathway is among those that are often described as
“cancer pathways” because they are too frequently seen in malignancies. The PI3K pathway
leads directly to Akt / Protein Kinase-B. Although different, PI3K and Akt are often lumped
together in discussions of cancer.

16
The way in which PI3K affects the growth and spread of tumors is via Akt, via
threonine/serine protein kinase (i.e., is a kinase that acts on serine and threonine residues).
Akt is therefore the downstream effector of PI3K. In human CRC (and with evidence from
several tumor types), phosphorylation of Akt has been closely associated with cell growth
and survival in that it represses apoptosis. Conversely, the molecules that we have studied
repress the PI3K/Akt pathway (and some that inhibit mTOR, a downstream target of Akt) are
being evaluated as a novel way to treat cancer. Another mechanism that activates PI3K is to
stimulate it with RAS or via receptor tyrosine kinase (RTK), which itself has been stimulated
by an external ligand.
PI3K activation phosphorylates phosphoinositol 4,5 bisphosphate (PIP2), producing
phosphoinositol 3,.4,.5 trisphosphate (PIP3). PIP3 is the second messenger whose cell
signaling function is mostly responsible for the oncogenic activity of PI3K pathway leading
to development and dissemination of CRC, therefore this conversion has utmost importance.
PIP3 has a half-life in biological systems that is longer than the half-life of PIP3's synthesis
from PIP2 by the action of PI3K. By the dephosphorylation of PIP3, the tumor suppressor
protein phosphotase and tensin homologue protein (PTEN) inhibits the PI3K pathway. .
3.3 MAO-A role in CRC
In CRC, the EMT is predominantly regulated through the activation of the signaling
pathways Stat3/ZEB1 and IL-13/IL-13Rα1/STAT6/ZEB1. In these pathways, factor
Stat6/Stat3 is responsible for regulating the expression and activity of MAO-A. Investigation
into the role of MAO-A in CRC has shown that MAO-A, which is known to be expressed
constitutively in limited cell types and to be induced by certain stimuli, is present in increased
amounts in colorectal tissues and CRC-derived cells. A series of experiments have shown that
increased MAO-A in colorectal cells leads to generation of ROS, which has been linked in

17
several studies to increased cell invasion and migration in CRC. These pieces of evidence
indicate that MAO-A could serve as a key molecular target for the prevention of CRC
metastasis. The reason why is quite interesting. The authors attribute this immunosuppressive
effect to MAO-A being a key molecule in the regulation of the ROS signaling pathway in
cells within the tumor microenvironment, transmitting a signal that affects the polarization of
tumor-associated macrophages (TAMs) toward an immunosuppressive phenotype.6 19
3.4 MC-38 and CT-26 Cancer Cell lines
This part explains about the cell lines used for this study.
The cancerous CT26 cells were initially obtained from BALB/c mice that had been treated
with N-nitroso-N-methylurethane (NMU) to induce cancer. CT26 now produces a colon
adenocarcinoma, a kind of cancer that originates in the epithelial cells of the colon, known
for its robust growth and rapid spread. The CT26 cell model has been particularly valuable in
the preclinical studies to develop and test various drugs and biologics designed to kill cancer
cells or otherwise eliminate them. Analysis of gene expression has revealed that CT26 cells
express cancer-related genes at high levels and that they are especially influenced in
pathways associated with cancer cell metabolism and cell cycle control. Intriguingly, mRNA
levels for Rb1, a well-known regulator of the cell cycle, are up-regulated eightfold in CT26
cells, while the levels of a second key regulator, Foxo3, are dramatically low. Moreover, the
increased expression of Ezh2 in CT26 cells undoubtedly adds to their aggressive and
metastatic behavior. Even though Wnt10a is expressed at high levels, only one target gene of
the Wnt signaling pathway, Birc5, is present.
20
In 1975, a team of scientists established the MC38 cell line from a colon tumor in a female
C57BL/6 mouse that had been treated with the carcinogen dimethylhydrazine. The derivation
of this cell line from 3-D tissue culture has made it a useful model to study colon carcinoma,

18
anti-tumor immunity, and various immunotherapy regimens. The complete immune system of
the mouse and the syngeneity of the cell line with the host allow for a relevant study of these
important proto-oncogenes and their associated pathways. The most prominent mutations are
in the tumor suppressor genes TP53 and PTEN. The alterations in the TP53 gene lead to
amino acid substitutions, and the mutations in the PTEN gene result in a "loss of function."
Other mutations appear in the TGF-beta signaling pathway, most notably in the TGFB2 gene.
Some mutations occur that lead to partial or complete inactivation of other pathway members,
such as ACVR2A and SMAD4. These gene changes contribute to an oncogenic pathway.
Most of the cells in this line exhibit high microsatellite instability. Changes in BRAF are
common and indicate high levels of this protein. BRAF helps drive several activities in the
cell that lead to higher 'gain of function' phenotypes, which means these cells more easily
become cancerous. These changes occur in spite of intact signals from the Wnt-beta-catenin
pathway. The AXIN2 gene is occasionally mutated in these cells, which also confers high
Wnt signal activity to help drive oncogenesis. The most frequent driver mutations in human
CRCs define common CRC subtypes, such as poorly differentiated tumors. The MC38 cell
line is an excellent model for studying such subtypes because it contains the driver mutations
found in humans. Moreover, the line expresses CRC tissue antigens at a high level, three of
which—TAD2, RQCD1, and SPAG9—are expressed by the CT26 cell line.
21 22
3.5 Mediation of Tumor Associated Macrophage for immunotherapy in
cancer is controlled by MAO-A
The study by Wang et al. 2021 explains the importance of MAO- A in MC-38 cells and
treating MC-38 murine models with phenelzine a MAO-A inhibitor and immunotherapy
helps to reduce the growth of cells in the murine models and also initiates a anti-tumor
response by CD8+ cells. Both human and murine TAMs show increased levels of MAO-A,

19
which aligns with and augments their immunosuppressive properties. In the mouse models, it
was observed that TAM-mediated immunosuppression was significantly and consistently
reduced in MAO-A-deficient animals. This decrease in TAM-mediated
immunosuppressioncorrelated well with an upturn in anti-tumor immunity, a surge which is
often marked with CD8 T cells. The presence of serotonin in the T-cells helped to increase
the anti-tumor response and also prevents the tumor escape from the immune activity. 9
3.6 Suppression of MAO-A reduces MC-38 cancer through macrophage
reprogramming
In cancer models of the MC-38 cell line, the development of tumors and survival rates were
positively affected when MAO-A was suppressed. The authors of the study correlated the
observed tumor growth and survival rates with the polarization state of the TAMs. When they
suppressed MAO-A, they saw an increase in the pro-inflammatory cytokines, like TNF-α and
IL-6, and a decrease in the pro-cancer markers, like the expression of CD206, in the TAMs.
When the macrophages were in this "better" state, there was a decrease in the development of
tumors and an increase in the survival of the mice bearing these tumors.
9,19
3.7 Goals of the Thesis
Research has indicated that MAO-A in mouse colon cancer type MC-38 cells maintains the
homeostasis of the immune system, and when MAO-A is inhibited, it leads to a sluggish
cancer growth in mice. The combination of a drug like phenelzine with an anti-PD-1 antibody
gives the best results, reactivating the immune system much more effectively than either drug
could alone. In prostate cancer type cells, NMI hinders the expression of a handful of
proteins, like BCl-2, FOS, VIM, MYC, TP53, NFKB1, which are responsible for the cancer

20
cell migration, proliferation, and EMT via blocking the MAO-A and disrupting the
PI3K/AKT/mTOR pathway.
NMI is an effective MAO inhibitor and a good target for cancers linked to hyperactivated
MAO activity, such as CRC. It is suspected that NMI may hinder the proliferation of CT-26
and MC-38 cells through inhibition of certain proteins in the PI3K/AKT/mTOR pathway. The
aim of this study is to identify the proteins interacting with NMI by using SDS-PAGE and
mass spectroscopy for studying the protein eluted on the gel. If so, this would be a good
starting point for future investigations of the untapped potential of NMI as an anti-cancer
drug.

21
Chapter IV
Potential NMI binding proteins in CT-26 and MC-38 cell lines
Based on the previous research, NMI inhibits prostate and glioma growth and deregulates
genes such as Bcl-2, MYC, JUN, NFKB1, etc. Further we have shown that NMI binds 65
kDa proteins in glioma.
23 Here we investigate NMI binding protein in colon cancer.
4.1 Experimental Procedures and Results
Cell culture
MC-38 and CT-26 cells were acquired from Dr. Heinz Joseph's lab. We cultured the cells in
plates in DMEM (Dulbecco's Modified Eagle's Media) supplemented with 10% Fetal Bovine
Serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. In a humidified incubator at 37°C
and 5% CO2, passage the cells at least three times before treating them with any medications.
NMI and Clorgyline treatment
The cultivated cells were placed in a 6-well plate and allowed to adhere to the plate's bottom.
After 24 hours, the cells had adhered effectively and were prepared for drug therapy. In the
control treatment, the cells were fed only DMEM medium enhanced with 10% FBS. For the
NMI treatment, the cells were treated with 10 μM of NMI. For the Clorgyline treatment, the
cells were treated with 1 μM of Clorgyline. For the combination treatment with NMI and
Clorgyline, first, 1 μM of Clorgyline was applied to mouse colon cancer CT-26 and a second
tumor cell line, MC-38, and then a Clorgyline+NMI solution (10 μM Clorgyline and 100 μM
NMI) was applied. Following a 24-hour drug treatment cycle, the cells were subjected to
SDS-PAGE.

22
Separation of NMI binding proteins by SDS-PAGE
After administering the required medication, we cleaned and lysed the cell pellet with RIPA
lysis buffer. Sample is centrifuged to remove any leftover debris and to extract the
supernatant. We calculated the protein concentration using the BCA assay and prepared a
sample containing 25 µg of protein, based on the estimated protein concentration. 4x dye to
the mixture to visualize the bands, then heated it to 95°C for denaturation. 10% SDS PAGE
gel was used keeping in mind the approximate molecular weights of the proteins of interest.
The total run time was less than 1.5 hours. After the gel run, the bands were imaged with a
near-infrared imaging system.
Mass spectrometry of the NMI interacting proteins separated from SDS-PAGE
Mass spectrometry was used to analyze the proteins from the gel bands of MC-38 cells that
had been treated with NMI and Clorgyline. Poochon Scientific performed the analyses, and a
list of proteins was returned.
4.2 Results
4.2.1 Detection of 65 kDa NMI-Interacting Proteins in CT-26 and MC-38
Colon Cancer Cells
CT-26 and MC-38 cells were cultured in a six-well plate and treated for 24 hours with
Clorgyline, NMI, and the combination of NMI + Clorgyline. Cell lysates from these treated
cells were then prepared and subjected to SDS-PAGE followed by Coomassie Blue staining.
Staining revealed that CT-26 and MC-38 cells treated with Clorgyline displayed no visible
bands. By contrast, bands of equal intensity were observed for the NMI-treated cells and for
the combination of NMI and Clorgyline as shown in Figure 4.

23

Fig 4. SDS gel Composite and Raw stained membrane of CT-26 and MC-38 cells
Clorgyline does not have any effect on the cell lines, therefore it shows no band. MAO-A is
very negligible in the cell lines. Both the cell lines show the bands at 65kDa.
4.2.2 Proteins in the 65 kDa band in MC-38 cells
NMI has the ability to bind proteins, which can subsequently be detected using imaging
techniques. The 65 kDa band detected in gel electrophoresis can be likely due to proteins
with molecular weights similar to 65 kDa, such as MAO A (60 kDa) and albumin (66 kDa),
and heptamethine dyes are capable of binding albumin with a half-life of up to three weeks.23
It is critical to understand that the 65 kDa band shown in gel electrophoresis may not reflect a
single protein, but rather can be many proteins that are either comparable in size or form
complexes. These proteins may migrate during electrophoresis, producing a single band that
can be identified by mass spectrometry. Proteins may experience a variety of Post
Translational Modifications that affect their apparent molecular weight on SDS-PAGE,
thereby allowing proteins to migrate at or around 65 kDa even if their theoretical molecular
weight differs. Mass spectrometry detects proteins and identifies them using peptide
sequences or the number of peptides present in the single band. Mass spectrometry can detect

24
peptides independently of their original position on the gel, therefore the proteins found in the
65 kDa band do not have to be 65 kDa themselves. 24,25
The preliminary findings of the analysis of the SDS-PAGE samples from the cell lysate using
mass spectroscopy had 20 of which were identified as most important. These proteins are
responsible for the signaling pathway in MAO-A and PI3K/AKT/mTOR pathway. NMI
treatment increase/decrease the levels of these proteins. Enolase 1 was the predominant
protein found in both protein samples. Myosin 9, a significant player in muscle contraction
and an important mediator for disease progression, had a reduced levels in NMI-treated MC38 cells compared to controls. ZMAT3, a key splicing factor, exerts a tumor-suppressive
effect was increased. The treatment of colon cancer cells with NMI lowers levels of radixin,
which is thought to enhance the progression of tumors, and moesin, which has been
implicated in the increased expression of PD-L1 in tumor cells. The treatment also reduces
levels of an important protein, tubulin 3 beta, that has been detected in invasive CRCs.
Finally, NMI has also been shown to decrease another important protein, importin beta 1,
that helps in the nuclear translocation of PI3K alpha. NMI also lowers several proteins
associated with poor outcomes in CRC. These include Fatty acid synthase, Dynamin-1,
Exportin-2, Lamin B-1, Programmed cell death 6 interacting protein, Plectin-1, and
Filamin A, which may have roles in carcinogenesis and cancer progression. Properties of
these proteins based on the literature are described below. Table 4 depicts the number of
peptides that were found in control and the NMI treated cells.
Table 4. List of proteins interacting with NMI in MC-38 cells with the number of peptides in each sample
Protein Number of peptides in
Control
Number of peptides in
NMI treated cells
Myosin-9 158 156
Ezrin/radixin/moesin 25/24/41 20/16/35

25
Importin beta 1 60 57
Tubulin 3 beta 73 68
Fatty Acid Synthase 80 71
Dynamin 1 13 12
Exportin 2 63 60
HECW2 20 16
Programmed death 6
interacting protein
10 5
Plectin-1 87 77
Filamin A 58 53
ZMAT3 10 15

4.2.3 Myosin-9
The key protein involved in the PI3K/AKT/mTOR pathway is myosin-9. Although it was
previously thought that MYH9 functioned as a tumor suppressor, more recent research has
shown that MYH9 actually confers resistance to radiation therapy and chemotherapy while
simultaneously promoting the development of tumors. In fact, MYH9 performs two protumorigenic functions in certain cancers. It interacts with several well-known oncogenic
proteins and signaling pathways. For instance, in CRC, MYH9 interaction with EGFR, for
instance, is important because the signaling from this receptor promotes EMT, a process that
enhances the invasive and migratory capacities of cancer cells. Embryonic human fibroblasts
were used to demonstrate that MYH9 promotes the expression of EMT markers, like
vimentin, and represses the expression of epithelial markers, like E-cadherin.
26 Our results
suggest that, the PI3K/AKT/mTOR pathway can be disrupted because of MAO-A blockage
by NMI, impacting levels and downregulation of Myosin-9.
4.2.4 ERM Proteins

26

The ezrin/radixin/moesin (ERM) protein family has been shown to play a critical role in
connecting the actin cytoskeleton to various plasma membrane proteins. Among these are
several transmembrane proteins that serve as drug targets in cancer cells—most notably, Pglycoprotein, which, when overexpressed, contributes to the well-documented phenomenon
of multidrug resistance (MDR). Research has uncovered a novel role for moesin in MDR by
showing that moesin gene silencing significantly impaired the plasma membrane localization
of P-glycoprotein in a human breast cancer cell line. Silencing the genes of ezrin, radixin, and
moesin significantly reduced the level of PD-L1 on the cell surface. These results suggest that
ezrin and radixin in LS180 cells likely serve as scaffold proteins that mediate the plasma
membrane localization of PD-L1. This may occur via a mechanism of posttranslational
modification. Furthermore, our data indicate a decrease of ezrin, radixin and moesin when the
cells were treated by NMI.
27
4.2.5 Importin beta 1
The normal growth and development of organisms depend on the signaling of PI3Kα, a
pathway that is frequently overexpressed in various human cancers, including CRC. PI3Kα is
a lipid kinase that phosphorylates the inositol ring of phosphatidylinositol (PtdIns) to generate
PtdIns(3,4,5)P3. The conversion of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to
PtdIns(3,4,5)P3 occurs when the p110α catalytic subunit is recruited by regulatory p85
subunits to activated receptor tyrosine kinases (RTKs). This process, taking place at the
plasma membrane or at microtubule-bound endosomes in the cytoplasm, is referred to as
canonical agonist-stimulated PI3Kα signaling. PtdIns(3,4,5)P3 then binds to effector proteins
that contain pleckstrin homology (PH) domains, among other motifs, and activates
downstream signaling pathways that promote cell survival and growth like AKT (AKT

27
serine/threonine kinase), PDK1 (pyruvate dehydrogenase kinase 1), and SIN1 (stressactivated protein kinase–interacting 1).
AKT can act at diverse intracellular sites to phosphorylate downstream substrates that affect
cell survival, proliferation, growth, and metabolism. The phosphorylation of downstream
substrates is regulated by PDK1 and the mammalian target of rapamycin complex 2. SHIP1
and PTEN, two lipid phosphatases that dephosphorylate PtdIns(3,4,5)P3 at the 3 and 5′
positions of the inositol ring, respectively, inhibit PtdIns(3,4,5)P3 synthesis. About 15% and
30% of human CRCs have activating mutations in the genes encoding p110α, PIK3CA
(phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and PTEN protein
expression loss, respectively. The principal regulator of PtdIns(3,4,5)P3 levels in the nucleus
of CRC cells is the nuclear translocation of PI3Kα, which occurs via the importin β nuclear
import pathway.
28 Importin beta 1 levels decrease can be due to NMI causing of the AKT
pathway blockage, with PI3Kα downregulation.
4.2.6 Tubulin 3 beta protein
Beta-3 tubulin, one of the nine b-isoforms of tubulin involved in the formation of
microtubules, is well-known for its essentiality in cell growth, division, motility, signaling,
and shape maintenance. Initial studies found that tubulin 3 beta overexpression was a big
reason why some cancers developed resistance to the taxanes, a class of chemotherapeutic
agents that induce cell death by targeting tubulin. When 3 beta was overexpressed, it showed
a strong correlation with tubulin-targeting drug resistance and unfavorable clinical outcomes.
Cells with less 3 beta expression and more alpha-tubulin/collagenase were found to be more
sensitive to the taxanes.
The aggressive nature of spreading cancers seems to be linked to the expression of certain
proteins, and one of those is tubulin 3 beta. About 64.78% of CRCs examined had the protein

28
at the invasive edge, suggesting it is playing a role in the biology of this tumor. And it's not
just colorectal tumors; it's pretty clear that tubulin 3 beta is part and parcel of many different
kinds of aggressive tumors, possibly linked to their lossy nature.
29 Results show that, NMI
treatment decrease the levels of tubulin 3 beta, indicating that metastasis could be hindered
due to NMI.
4.2.7 Fatty Acid Synthase
Fatty acid synthase (FASN) is a pivotal enzyme in the creation of new fatty acids, and it is
often overproduced in cancer cells. This overproduction facilitates an additional influx of
fatty acids into the cells. Studies have shown that FASN overexpression is associated with
enhanced cell growth, metastasis, and an increased likelihood of recurrence, along with a
poor overall prognosis. Furthermore, FASN has been found to be expressed at significantly
high levels in CRC. Its expression has been tied not only to the advent of the disease but also
to its progression.
Studies have shown that when CRC cells are subjected to Fatty Acid Synthase inhibition,
they suffer a decrease in ATP production. We know from cell bioenergetics that AMPactivated protein kinase (AMPK)—which measures the levels of AMP and ATP within the
cells—becomes activated when ATP levels are low. AMPK governs the metabolism of both
malignant and healthy cells since it is a strong negative regulator of the mTOR pathway,
which is directly related to cell invasion, metastasis, and proliferation.
Consequently, there is a good chance that enhanced expression of Fatty Acid Synthase in
CRC cells could increase ATP synthesis, which would then act to inhibit AMPK and activate
mTOR and thus push CRC cells to grow and metastasize.
30 MC-38 cells treated with NMI
have lower Fatty Acid Synthase, which can be a result of inhibition of AKT/mTOR pathway.
4.2.8 Dynamin 1

29
It is likely that PI3K/Akt activation contributes to the reduced impact of TRAIL by
diminishing the expression of the DR4 and DR5 TRAIL receptors. Insulin-like growth factor1 and epidermal growth factor are two growth hormones that inhibit TRAIL-induced
apoptosis through the Akt pathway. Akt inhibits apoptosis by phosphorylating components of
the cellular apoptotic regulatory circuit, such as BAD and Forkhead transcription factors, and
activating NF-kB. When TRAIL becomes operational, calcium stores in the endoplasmic
reticulum are released via the ryanodine receptor. This pathway activates Dynamin 1,
TRAIL-DR endocytosis, calcineurin-mediated dephosphorylation, and increased resistance to
TRAIL-induced apoptosis. NMI treatment could suppress PI3K/AKT, which can activate the
TRAIL apoptotic pathway, decreases dynamin 1, and increases caspase 8 levels.31,32
4.2.9 Exportin 2
Colon cancer tumorigenesis involves the protein Exportin 2. Studies show that when
clorgyline was used to treat MC-38 cells, those cells exhibited a significantly higher level of
Exportin 2. On the other hand, when an NMI treatment was administered to the MC-38 cells,
the abundance of Exportin 2 in those cells was markedly reduced. These results add to and
are consistent with findings from other laboratories that identified colon cancer cell lines,
such as HT29, HCT-116, and SW480, to have elevated levels of Exportin 2 expression. The
other laboratories also showed that in these cell lines, when the gene for Exportin 2 was
knocked down, there was a significant suppression of apoptosis, cell cycle arrest, colony
formation, and cell proliferation. Consequently, the decreased concentration of Exportin 2 in
MC-38 cells when given NMI could be an element in the decline of cell proliferation.33
4.2.10 HECW2
Strong expression of HECW2 (HECT, C2, and WW domain-containing E3 ubiquitin protein
ligase 2) in CRC tissues and cells was detected. When HECW2 was knocked down in CRC

30
cells, it disrupts the progression of the cancer and its resistance to chemotherapy were greatly
reduced. Conversely, overexpression of HECW2 in CRC cells enhanced the cancer's growth
and its resistance to standard chemotherapeutic agents. HECW2 seems to accomplish all of
this by tagging lamin B1 for degradation in the ubiquitin-proteasome system, which in turn
helps to activate the AKT/mTOR signaling pathway.
34 Lamin B1 numbers increase
among MC-38 cells treated with NMI, indicating that HECW2 levels could be reduced due
to AKT/mTOR pathway inhibition.
4.2.11 Programmed death 6 interacting protein
The tissues of CRC show elevated amounts of the Programmed death 6 interacting protein.
The studies that led to this discovery began by examining the previously identified protein
PDCD6, which was found to play a role in regulating cell death. Investigators had established
that PDCD6 is overexpressed in many cancers, including malignancies of the breast, ovary,
and skin. In studies where researchers manipulated the amounts of PDCD6 (by
overexpressing or knocking down the protein), they found that it behaved like an oncogene.
Correlating those experimental findings with the clinical data, they proposed that PDCD6 is
an oncogenic protein that plays a significant role in the pathogenesis of CRC.
35 Decrease in
concentrations of Programmed Death 6 interacting protein are detected in MC-38 cells
exposed to NMI, demonstrating that NMI may be able to disable the pathway of this protein
via an alternative approach, causing a favorable outcome.
4.2.12 Plectin-1
The cytolinker and scaffolding protein plectin-1 is an extremely potent inducer of many
cancer hallmarks, especially in human malignancies. Its fiber-like, intertwined structure lends
plectin-1 the ability to bind many other proteins directly and in a context-dependent manner.

31
Furthermore, plectin-1's intricate protein interactome enables it to play many roles in as-yetundetermined cellular processes that fuel tumorigenesis. Plectin-1 also uses its interactome to
exert many functions in signal transduction, a process that the protein appears to carry out
preferentially when in the presence of certain cancer types.
36 Plectin-1 is reduced by NMI
medication, which may enhance the likelihood of successful outcome.
4.2.13 Filamin-A
Filamin-A was first discovered as a non-muscle actin-binding protein, and it organizes
filamentous actin into stress fibers and orthogonal networks. Filamin-A offers a broad variety
of cytoplasmic and nuclear signaling proteins a scaffold to work on, and it also serves to tie a
variety of transmembrane proteins to the actin cytoskeleton. Numerous investigations, some
of which have a quite different focus, have found that filamin-A participates in many
different pathways and associates with a variety of non-cytoskeletal proteins that have
different functions.
A large number of mutations and abnormal expressions of filamin-A in humans have been
tied to cancer and inherited disorders. This mechanism could account for the reasons behind
such associations, since the proteolysis of filamin-A by calpain is known to be controlled by
the phosphorylation of serine 2152, a site that is heavily modified by the pro-survival
oncogenic protein kinase AKT. It is found that a prominent PI3K inhibitor that works
clinically to prevent certain types of cancers from progressing is also effective at cleaving
filamin-A, and modulating its expression.37. Our drug NMI, significantly lowers filamin-A
levels which can be due to AKT inhibition.
4.2.14 ZMAT3
The gene ZMAT3, or WIG-1, is regulated by the p53 protein, and its function is largely
unknown. However, it has been proposed that ZMAT3 overexpression might suppress tumor

32
growth and provoke the death of neoplastic cells in a manner that is contingent upon the p53
protein.
38 NMI can increase the ZMAT3 expression indicating tumor suppression activity.
Fig 5. Predicting MAO-A inhibition by NMI disrupts PI3K/AKT/mTOR pathway, reducing key proteins in MC-38
cells.
Figure 5 demonstrates a possible mechanism, by which NMI may play a role in decreasing
the levels of the key proteins. NMI may disrupt PI3K/AKT/mTOR pathway, by inhibiting
MAO-A , which may eventually lead to reducing the levels of proteins like Myosin-9, Eosin,
Radixin, Moesin, Programmed death 6 interacting protein, Fatty acid synthase,
Importin beta 1, Plectin-1, Dynamin-1, Exportin-2 and Filamin-A.

33
Summary
This report reviewed the literatures on the use of us MAO-A inhibitors and NMI on cancer
therapy. The data show that NMI can inhibit the MAO-A enzyme in cell line models as well
as in animal models of cancer.
Our findings on NMI (Near-infrared Monoamine Oxidase Inhibitor) indicate that it has the
potential to be a substantial therapeutic agent for CRC, particularly in MC-38 cells. Our
findings show that NMI selectively binds to proteins around 65 kDa, affecting multiple
critical proteins involved in cancer cell proliferation, migration, and survival.
NMI therapy reduced the expression of several key proteins, including Myosin-9, which is
implicated in the PI3K/AKT/mTOR pathway and is known to promote proliferation of
tumors. The inhibition of ezrin/radixin/moesin (ERM) proteins, which are essential for
plasma membrane integrity and drug resistance, demonstrates NMI's potential to reduce
cancer cell invasiveness and improve treatment efficacy. Furthermore, the decrease in
importin beta 1 and tubulin 3 beta, both associated with aggressive cancer behavior and poor
prognosis, demonstrates NMI's ability to disrupt essential cellular processes involved in
tumor growth and metastasis.
The considerable decrease in fatty acid synthase and dynamin-1 indicates that NMI may
affect metabolic pathways required for cancer cell survival and proliferation. The lowering of
exportin-2 and the programmed death 6 interacting protein, both of which are related with
increased cell proliferation and tumor progression, lends evidence to NMI's therapeutic
potential in CRC treatment. Furthermore, the decrease in HECW2 and plectin-1, as well as
the increase in the tumor-suppressive protein ZMAT3, demonstrate NMI's multifaceted
approach to targeting a number oncogenic pathways.

34
Overall, our preliminary findings suggest that NMI could be an effective anti-cancer drug by
altering numerous key proteins and processes in CRC. These findings lay a solid framework
for future research and development of NMI as a targeted therapy for colorectal and maybe
other malignancies with high MAO-A activity. More research is needed to confirm these
findings and understand the specific mechanisms by which NMI exerts its anti-cancer
benefits, opening the way for future clinical applications.

35
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Abstract (if available)

Abstract Monoamine oxidase A (MAO-A) has been linked to a variety of cancers because it produces reactive oxygen species (ROS), which contribute to oxidative stress and mutagenesis. Prostate, glioma, and colorectal cancers show upregulated expression of MAO-A. Near Infrared MAO-A Inhibitor (NMI), a heptamethine dye conjugated MAO-A inhibitor, targets with higher specificity to cancer cells, and can function as both a non-invasive diagnostic tool and a therapeutic agent. Wu et al. (J Am Chem Soc 2015;137:2366-2374), study on prostate cancer found that MAO A inhibition by NMI decreases the prostate cancer growth, making NMI a novel therapeutic agent. Irwin et al. (Pharm Res 2021; 38:461–47), studied the NMI bound proteins in glioma using gel electrophoresis and found two significant bands at around ~65kDa and less than 10kDa. This thesis aims to study the range of proteins bound by NMI in CRC cell line MC-38 by gel electrophoresis followed by mass spectrometry. Gel electrophoresis results revealed that NMI can bind to proteins at a specified molecular weight, resulting in a single band at 65 kDa. Mass spectrometry data shows that NMI decreases moesin; myosin 9; radixin; lamin B1; importin beta 1; tubulin 3, beta exportin-2; prelamin A/C; dynamin-1; fatty acid synthase; tubulin 3-beta associated with CRC cell growth and survival. These data require confirmation, but suggests that NMI could be a potential therapeutic agent for cancers.

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Creator Koparde, Ved Harshad (author)

Core Title NMI: a near infrared conjugated MAO-A inhibitor as a novel targeted therapy for colorectal and other cancers

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2024-08

Publication Date 08/23/2024

Defense Date 08/23/2024

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

Tag colorectal cancer,monoamine oxidase-A,myosin-9,NMI,OAI-PMH Harvest,SDS-PAGE

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Haworth, Ian (committee member), Shih, Jean (committee member), Zhang, Tiger (committee member)

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