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Potential therapeutic effect of monoamine oxidase (MAO) inhibitor on human neuroblastoma
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Potential therapeutic effect of monoamine oxidase (MAO) inhibitor on human neuroblastoma
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Content
POTENTIAL THERAPEUTIC EFFECT OF
MONOAMINE OXIDASE (MAO)
INHIBITOR ON
HUMAN NEUROBLASTOMA
by
Tianhan Dong
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
(Molecular Pharmacology and Toxicology)
May 2016
2
Acknowledgments
I am grateful to my advisor, Dr. Jean C. Shih, for her guidance in research and thesis.
She always supported and instilled confidence. She provided extensive training on how
to be a scientist and motivated me to think creatively and broaden my horizon.
I thank the members of my master’s degree committee, Dr. Roger Duncan and
Dr. Yong Zhang, for their guidance in writing thesis. I also thank the members of Dr.
Shih’s lab for assistance: Yi Cheng Lin, Bin Qian, Shikha Gaur, Vijaya Pooja Vaikari
and Rucha Deo. I am especially grateful to Jami Pei-Chuan Li for guidance and Frank
Hong for assistance with writing thesis.
Finally, I would like to express gratitude to my family for their love and support,
without which I would not have been able to study and work and persevere the
challenges faced while pursuing a master’s degree.
3
Table of Contents
Acknowledgments............................................................................................................2
Abstract .............................................................................................................................. 4
List of Figures .................................................................................................................... 5
List of Tables ..................................................................................................................... 6
Chapter 1: Introduction ................................................................................................... 7
1.1 Neuroblastoma .................................................................................................................. 7
1.2 Monoamine Oxidase ....................................................................................................... 11
1.3 Objectives of the Project ................................................................................................. 14
Chapter 2: Results ........................................................................................................... 15
2.1 The mRNA expression levels of MAO A and MAO B in neuroblastoma ..................... 15
2.2 MAO A/B catalytic activity in human neuroblastoma cells ........................................... 17
2.3 Inhibition of MAO A activity by clorgyline or deprenyl in neuroblastoma CHLA-255
and SK-N-SH cell lines ................................................................................................... 20
2.4 Effect of various MAO inhibitors on MAO A catalytic activity and the viability of
neuroblastoma cells ......................................................................................................... 22
2.5 Effect of doxorubicin on reducing the viability of neuroblastoma cells ......................... 26
2.6 Effect of combining MAO inhibitor clorgyline (or phenelzine, deprenyl, pargyline)
with doxorubicin on viability of neuroblastoma CHLA-255 cells ................................ 28
2.7 Effect of combining MAO inhibitor clorgyline (or phenelzine, deprenyl, pargyline)
with doxorubicin on viability of neuroblastoma SK-N-SH cells .................................... 31
Chapter 3. Discussion and Conclusion .......................................................................... 33
Chapter 4. Materials and Methods ................................................................................ 37
4.1 Reagents .......................................................................................................................... 37
4.2 Cell cultures .................................................................................................................... 37
4.3 Database .......................................................................................................................... 38
4.4 MAO catalytic activity assay .......................................................................................... 38
4.5 MTT assay ...................................................................................................................... 39
4.6 Statistical analysis ........................................................................................................... 39
References ........................................................................................................................ 40
4
Abstract
Neuroblastoma is one of the most common solid tumor in childhood and the most
commonly diagnosed in infancy. Doxorubicin is the current standard treatment for
neuroblastoma. Side effects such as heart damage caused by doxorubicin limited its use
for treatment. Ample evidence has indicated that monoamine oxidase A (MAO A) plays
an important role in cancer development. This thesis studied the role of MAO A in
neuroblastoma and its potential for treatment. Analysis of Oncomine database showed
that the expression level of MAO A mRNA is higher in malignant compared to benign
neuroblastic tumors, indicating that MAO A may play a role in neuroblastoma. The
MAO A catalytic activity was found in three neuroblastoma cell lines: CHLA-255, SK-
N-SH and SK-N-BE2. CHLA-255 and SK-N-SH were used in further studies as they
expressed higher MAO A activity than SK-N-BE2. MAO A in these two cell lines was
further characterized by its sensitivities to four inhibitors clorgyline, deprenyl, pargyline
or phenelzine. Using MTT assay, we found that all four inhibitors itself were not
effective on the neuroblastoma cell viability. Doxorubicin itself was cytotoxic to these
two cell lines; in CHLA-255, all four MAO inhibitors enhanced the cytotoxicity of
doxorubicin while in SK-N-SH cells, only clorgyline enhanced the cytotoxicity of
doxorubicin. Furthermore, clorgyline significantly enhanced the cytotoxicity of low
dose doxorubicin. These results indicate that MAO inhibitors enhance the cytotoxicity
of doxorubicin; thus, the combination of MAO inhibitor and doxorubicin may provide a
more effective treatment for neuroblastoma.
5
List of Figures
Figure 1. Expression levels of MAO A and MAO B mRNA in benign and malignant
neuroblastic tumors.………………………………………………...……………………16
Figure 2. Inhibition of MAO A activity by clorgyline or deprenyl in neuroblastoma
CHLA-255 and SK-N-SH cells. …………………………………………..…………..... .21
Figure 3. Effect of four MAO inhibitors on MAO A activity in CHLA-255 and SK-
N- SH cell………………………………………………………………………………..23
Figure 4. IC
50
of doxorubicin on the viability of CHLA-255 and SK-N-SH cells……...27
Figure 5. Effect of doxorubicin with or without 1 µM MAO inhibitors on the viability
of CHLA-255 cells………………..…………………………………………….………30
Figure 6. Effect of doxorubicin with or without 1 µM MAO inhibitors on the viability
of SK-N-SH cells………………………………………………………………………..32
6
List of Tables
Table 1. MAO A and MAO B catalytic actives in three human neuroblastoma cell
lines: CHLA-255, SK-N-SH and SKN-BE2………….………..…................................18
Table 2. Characteristics of three human neuroblastoma cell lines: CHLA-255, SK-
N-SH and SK-N-BE2……………………………………………………….……….....19
Table 3. The Effect of MAO inhibitors on MAO A activity and cell viability in
neuroblastoma CHLA-255 and SK-N-SH cell line……………………...…………......24
7
Chapter 1: Introduction
1.1 Neuroblastoma
Neuroblastoma is a cancer that arises during the early developmental stage of nerve cells
(called neuroblasts). It is an embryonic tumor that is probably derived from primitive
cells of the sympathetic nervous system. Neuroblastoma develops when normal
neuroblasts fail to mature, but instead keep growing and dividing. As a disease of
developing tissues, neuroblastoma occurs most often in infants and young children with
the median age of 17 months at diagnosis (London et al., 2005). Nearly 90% of
neuroblastoma cases are diagnosed before the age of 5 years and are very rare in people
over the age of 10 years. By far, neuroblastoma is one of the most common solid cancer
in childhood and most common cancer diagnosed during infancy, accounting for about
7- 10% of all childhood cancers (Schmidt et al., 2000). There are about 700 new cases in
the United States each year, which has remained about the same for many years (Evans,
Blore, Hadley, & Tanindi, 1971).
8
The etiology of neuroblastoma is unclear at present. Lifestyle-related risk factors
such as diet, physical activity and alcohol, and additionally environmental agents, which
play a major role in adult cancers, may not increase the chance of developing
neuroblastoma (Brodeur, 2003). Recent advances in biology and genetics have allowed
researchers to find important differences that distinguish malignant neuroblastoma cells
from normal neuroblasts and benign neuroblastoma. Furthermore, they also investigate
abnormality in the DNA of neuroblastoma cells. In rare cases, neuroblastoma in children
is inherited from their parents, with most of these children showing alterations in the
ALK oncogenes. Still, most neuroblastoma are not caused by heredity. Genes that affect
the rate of cell growth, like the oncogene MYCN, seem play an important role in the
development of neuroblastoma (Sugimoto et al., 2013).
Neuroblastoma is also one disease that exhibits extreme heterogeneity, which can
be classified into three categories: low, intermediate and high risk. A tumor composed
primary of neuroblasts is referred to as neuroblastoma; tumor composed of mature
ganglion cells and other mature tissues is referred to as ganglioneuroma, and a tumor
with both immature and mature cell types is called ganglioneuroblastoma (Lonergan,
Schwab, Suarez, & Carlson, 2002). Ganglioneuroma is considered benign while the
immature neuroblasts in neuroblastoma and ganglioneuroblastoma are indicative of the
malignant properties of the tumor. This classification of neuroblastoma helps to select
9
appropriate therapy for patients: low risk tumors can have good outcome though
observation only or surgery while high-risk disease is difficult to cure even with most
intensive chemotherapy (Maris, Hogarty, Bagatell, & Cohn, 2007). Chemotherapy
plays an important role in neuroblastoma treatment since most patients present with
metastases at the time of diagnosis require intensive and systemic chemotherapy (Ora
& Eggert, 2011). Current chemotherapy for patients with metastatic neuroblastoma
includes alkylating agents (i.e. cyclophosphamide), platinum analogues (i.e. cis-
platinum), vinca-alkaloids (i.e. vincristine) and anthracyclines (i.e. doxorubicin). They
already have well-established efficacy against neuroblastoma, serving as standard
treatment options (Bagatell et al., 2011; Kushner, Kramer, Modak, & Cheung, 2011;
Kushner, Kramer, Modak, Qin, & Cheung, 2010; Rubie et al., 2010). However, these
chemotherapeutics can also affect normal cells, resulting in serious side effects, which
include hair loss, mouth sores and an increased risk of infection (Maris et al., 2007).
Thus, complementary or alternative treatment methods for neuroblastoma are required
to lessen side effects in the future.
Doxorubicin is a drug that intercalates DNA to block the process of DNA
replication and inhibits the biosynthesis of macro-molecules (Tacar, Sriamornsak, &
Dass, 2013). As one of the most common cancer treatment options, doxorubicin is
widely used to treat metastatic neuroblastoma. But, along with the side effects listed
10
above, doxorubicin has its unique side effects: it can cause severe heart damage to
neuroblastoma patients. Currently, physicians are trying to reduce this risk as much as
possible by limiting the doses of doxorubicin administered (Maris et al., 2007). This
suggests that it is worthwhile to identify a method through which the therapeutic
efficacy of doxorubicin could be enhanced, especially at low doses.
11
1.2 Monoamine Oxidase
Monoamine oxidase (MAO), is an enzyme that catalyzes the oxidative deamination of
monoamine neurotransmitters and dietary amines, resulting in the production of
aldehyde, ammonia and hydrogen peroxide (Shih, Chen, & Ridd, 1999). MAO A and
MAO B are two isoenzymes of monoamine oxidase, which are encoded by two different
genes located on the X chromosome (Bach et al., 1988). They share 70% amino acid
identity but have different distribution, substrate and inhibitor specificities (Grimsby,
Chen, Wang, Lan, & Shih, 1991). MAO A is predominantly present in
catecholaminergic neurons while MAO B is mainly found in serotonergic and glial cells
(Shih et al., 1999). They also have different substrates. Serotonin (5-HT),
norepinephrine (NE) and epinephrine are mainly oxidized by MAO A while
phenylethylamine (PEA) is mainly oxidized by MAO B. They also share a common set
of substrates such as dopamine (DA). MAO A can be irreversibly inhibited by low dose
clorgyline while MAO B is irreversibly inhibited by low dose deprenyl (Shih et al.,
1999).
It is important for neurotransmitters such as serotonin, norepinephrine and
dopamine to be degraded after completing the neurotransmission. MAO, protects the
12
body by oxidizing amines and preventing their entry into the circulation. The MAO
abnormality has been associated with several neurological and psychological disorders
such as depression, anxiety, Atkinson’s disease (PD), Alzheimer’s disease, autism, etc.
(Youdim, Edmondson, & Tipton, 2006). For instance, low MAO A activity would lead to
antisocial behaviors in humans while low MAO B activity with increased level of PEA
is linked to alcoholism and other stress- related disorders. MAO A and MAO B knock
out mice differ in neurotransmitter metabolism and behaviors. There is elevated brain
levels of 5-HT, noradrenaline and dopamine in MAO A knock mice (Cases et al, 1995)
and an elevated level of 2-phenylethylamine in MAO B knock out mice (Grimsby et al.
1997). Both MAO A and MAO B knock out mice showed increased sensitivity to stress,
which is similar to the phenotype after administering non-selective MAO inhibitors.
Intriguingly, the number of serotonin releasing neuroendocrine cells is correlated
with tumor progression (Shinka et al., 2011). In addition, increased expression of MAO
A is correlated with high-grade aggressive prostate cancer and it also contributes to the
phenotype (Peehl et al., 2008; Zhao, Nolley, Chen, Reese, & Peehl, 2008). Recently, it
was determined that MAO A mediates prostate tumorigenesis and cancer metastasis
through inducing epithelial-to-mesenchymal transition (EMT) and augments hypoxia
13
response to increase tumor migratory, invasive and metastatic potential (Wu et al.,
2014). Accumulating evidence indicates that MAO A may represent an important
prognosticator of cancer and further development of targeted MAO A inhibitors are
anticipated.
There are a wide range of MAO inhibitors, including reversible or irreversible
inhibitors of MAO A, MAO B, or both MAO A and MAO B. The therapeutic value of
these MAO inhibitors has been proven in several medical conditions such as affective
disorders, neurodegenerative disease, stroke, and aging (Xu et al., 2015). In addition,
MAO inhibitors are also being studied in the area of cancer treatment. Clorgyline, which
is a selective and irreversible inhibitor of MAO A, is cytotoxic to drug-resistant human
glioma cells when used alone or in combination with low does temozolomide (Kushal et
al., 2016) Another MAO A inhibitor, phenelzine, also decreases tumor progression
when combined with docetaxel in prostate cancer cells and is currently undergoing a
Phase II clinical trial for prostate cancer patients. Furthermore, pargyline, which is an
MAO B and A inhibitor, inhibited the proliferation of human breast cancer cells (Hyung
Tae lee et al., 2013). These studies indicate that MAO inhibitors alone or in
combination with other drugs may provide a novel strategy for cancer treatment.
14
1.3 Objectives of the Project
The objectives of this study are to understand the role of MAO A in human
neuroblastoma using two cell lines CHLA-255 and SK-N-SH. And to investigate if
MAO inhibitors could be used alone or in combination with the current therapeutic
doxorubicin. If yes, an improved therapeutic strategy may be developed.
15
Chapter 2: Results
2.1 The mRNA expression levels of MAO A and MAO B in
neuroblastoma
The expression levels of MAO A and MAO B mRNA in human malignant and benign
neuroblastic tumors were investigated. We used the Oncomine platform to analyze
Albino_Brain dataset. Tumor samples included neuroblastoma and ganglioneuroma.
Neuroblastoma and ganglioneuroma are tumors arising from sympathetic nervous
system, which arose from primitive sympathogonia, and are referred to collectively as
neuroblastic tumors. Ganglioneuroma is composed entirely of mature ganglion cells and
other mature tissues, thus is considered benign (Lonergan et al., 2002). The expression
level of MAO A mRNA is significantly higher in neuroblastoma compared to
ganglioneuroma (Fig 1A) while MAO B mRNA expression is lower in neuroblastoma
compared to ganglioneuroma (Fig 1B). Thus, the MAO A mRNA level is higher and
MAO B mRNA is lower in malignant compared to benign neuroblastic tumors. The
result indicates that MAO A may play a role in neuroblastoma.
16
Figure 1. Expression levels of MAO A and MAO B mRNA in benign and malignant
neuroblastic tumors. Ganglioneuroma is benign neuroblastic tumor whereas
neuroblastoma is malignant neuroblastic tumor. Data were obtained from Oncomine
(Albino brain, USA) and analyzed using Prism 6 (GraphPad, Inc.,USA).
-1
0
1
2
3
Fold change=1.55
P= 0.0351
Ganglioneuroma Neuroblastoma
log 2 median-centered
intensity (MAOA)
AlbinoBrain_ Cancer AlbinoBrain_ Cancer
-1
0
1
2
3
log 2 median-centered
intensity (MAOB)
Fold change=0..70
NS
P= 0.36
Ganglioneuroma Neuroblastoma
B A
17
2.2 MAO A/B catalytic activity in human neuroblastoma cells
The MAO A and MAO B catalytic activities of three human neuroblastoma cell lines
CHLA-255, SKN-SH and SKN-BE2 were determined. All three neuroblastoma cell
lines had both MAO A and MAO B catalytic activities (Table 1). Compared to SK-N-
BE2 cell line, CHLA-255 and SK-N-SH cell lines had higher MAO A and MAO B
catalytic activities. Thus, CHLA-255 and SK-N-SH were selected for further study.
Characteristics of these two cell lines are listed in Table 2. The primary tumor site of
these two cell lines is brain, while the metastatic site of CHLA-255 is brain and SK-N-
SH is bone marrow. MYCN, which is a key oncogene in neuroblastoma progression, is
amplified in CHLA-255 but not in SK-N-SH cell lines.
18
Table 1. The MAO A and MAO B catalytic activity of three human neuroblastoma cell
lines: CHLA-255, SK-N-SH and SK-N-BE2.
MAO A Activity
(nmol/20min/mg)
MAO B Activity
(nmol/20min/mg)
Substrate 5-HT PEA
CHLA-255 38.87 6.89
Human neuroblastoma SK-N-SH
cell lines
19.47 10.26
SK-N-BE2 2.06 2.04
MAO A specific substrate, 5-HT, or MAO B specific substrate, PEA, worked as
substrate; cells were incubated at 37 ℃ for 20 minutes, and then MAO radioassay was
performed as described in Materials and Methods section.
19
Table 2. Characteristics of three human neuroblastoma cell lines: CHLA-255, SK-N-SH
and SK-N-BE2.
Human neuroblastoma Gender Primary tumor site Metastatic site Phenotype MYCN amplification
CHLA-255 NA Brain Brain adherent +
SK-N-SH Female Brain Bone marrow Adherent, epithelial -
SK-N-BE2 Male Brain Bone marrow
Mixed, adherent and
+
suspension
NA: not determine
20
2.3 Inhibition of MAO A activity by clorgyline or deprenyl in
neuroblastoma CHLA-255 and SK-N-SH cell lines
To characterize the MAO A activity in both in CHLA-255 and SK-N-SH cell lines,
MAO A activity inhibition assay was performed. To inhibit MAO A catalytic activity,
cells were treated with MAO A specific inhibitor clorgyline. As a control, MAO B
specific inhibitor deprenyl was used. Both CHLA-255 and SK-N-SH cell lines were
more sensitive to clorgyline compared to deprenyl (Fig 2). CHLA-255 with higher
MAO A catalytic activity compared to SK-N-SH was more sensitive to clorgyline than
SK-N-SH cell line (IC50 of clorgyline on CHLA-255 cell line is 1.4 x10
-13
M while is
4.1 x10
-12
M on SK-N-SH cell line). Thus, the presence of MAO A activity in these two
cell lines was confirmed.
21
Figure 2. Inhibition of MAO A activity by clorgyline or deprenyl in neuroblastoma
CHLA-255 and SK-N-SH cell lines. Cells were pre-incubated with increasing doses of
clorgyline or deprenyl at 37 ℃ for 20 minutes, then incubated with 5-HT for another 20
minutes at 37 ℃. MAO radioassay was performed as described in Material and Method
section
Clorgyline
IC
50
10
.13
M
Deprenyl
IC
50
10
.7
M
CHLA7255
Substrate: 5HT
Substrate: 5HT
SK.N.SH
Deprenyl
IC
50
10
-7
M
Clorgyline
IC
50
10
-12
M
A
B
CHLA-255
22
2.4 Effect of various MAO inhibitors on MAO A catalytic activity and
the viability of neuroblastoma cells
The inhibitory effect of clorgyline, deprenyl, pargyline or phenelzine on MAO A
activity and cell viability in the CHLA-255 and SK-N-SH cell lines was studied.
Phenelzine is another non-selective and irreversible inhibitor of both MAO A and MAO
B and pargyline is an irreversible selective MAO B inhibiting drug. These two MAO
inhibitors were approved by FDA and increasingly used in in vitro studies.
Suppression of the MAO A activity by four MAO inhibitors were compared in
CHLA-255 (Fig 3 A) and SK-N-SH (Fig 3 B) cell lines. CHLA-255 was most sensitive
to clorgyline, followed by phenelzine, deprenyl and pargyline. SK-N-SH was also most
sensitive to clorgyline, followed by phenelzine, deprenyl and pargyline.
The suppressive effect of four MAO inhibitors on the viability of neuroblastoma
CHLA-255 and SK-N-SH cells was examined using MTT assay. Based on MTT assay,
the order of sensitivity of CHLA-255 cells to four MAO inhibitors was clorgyline >
phenelzine > deprenyl > pargyline while the sensitivity of SK-N-SH cells was
phenelzine > clorgyline > deprenyl > pargyline (Table 3).
23
Figure 3. Effect of four MAO inhibitors on MAO A activity in neuroblastoma CHLA-
255 and SK-N-SH cell lines.
-18 -14 -10 -6 -2
0
20
40
60
80
100
120
MAO A activity [% of control]
Log [M]
CHLA-255
Substrate: 5-HT
-18 -14 -10 -6 -2
0
20
40
60
80
100
120
Log [M]
MAO A activity [% of control]
SK-N-SH
Pargyline
Phenelzine
Clorgyline
Deprenyl
Substrate: 5-HT
A
B
24
Table 3. Effect of MAO inhibitors on MAO A activity and cell viability in
neuroblastoma CHLA-255 and SK-N-SH cell lines.
25
When the two IC
50
values of each MAO inhibitor for MAO A activity assay and
MTT assay were compared, we found that the effective concentrations (IC
50
) of MAO
inhibitors are much higher for MTT assay than MAO A activity assay. This suggests
that MAO A inhibitor itself is not effective in suppressing the viability of neuroblastoma
cells. Therefore, combining MAO inhibitor with current therapeutics for neuroblastoma
may be necessary for more effective treatment.
26
2.5 Effect of doxorubicin on reducing the viability of neuroblastoma
cells
Doxorubicin (Adriamycin), belongs to a class of drugs that interacts with DNA, and
inhibits macromolecular biosynthesis in cancer cells. It has well-established efficacy
against neuroblastoma and is one of the standard therapies for neuroblastoma patients.
Next, we studied the effect of doxorubicin on suppressing the viability of CHLA-255
and SK-N-SH cells using MTT assay.
Effect of doxorubicin on the viability of CHLA-255 and SK-N-SH cells after 24
hours and 48 hours treatment was determined. As shown in Fig 4, 48 hours treatment
showed better inhibition of cell viability compared to 24 hours treatment. From here on,
we used incubation at 37 ℃ for 48 hours as the treatment condition. After 48 hours
treatment, the IC
50
of doxorubicin in CHLA-255 cell line was 3.6 x 10
-6
M while the
IC
50
was 8.9 x 10
-6
M in SK-N-SH cell line.
27
Figure 4. IC
50
of doxorubicin on cell viability in neuroblastoma CHLA-255 and SK-N-
SH cell lines. 5000 cells per well were seeded in 96 well plate, and incubated with
increasing concentrations of doxorubicin. After 48 hours of treatment at 37 ℃, cell
viability was measured using MTT assay.
-12 -10 -8 -6 -4 -2
0
20
40
60
80
100
120
Doxorubicin [M]
Cell Viability ( % of control)
48
hour
24 hour
CHLA-255
-12 -10 -8 -6 -4 -2
0
20
40
60
80
100
120
Doxorubicin [M]
Cell Viability ( % of control)
24 hour
48 hour
SKN-SH
A
B
C
!
Cell line CHLA-255 SKN-SH
IC
50
[M]
24 hours’ treatment 1.0 x 10
-4
2.4 x 10
-4
48 hours’ treatment 3.6 x 10
-6
8.9x 10
-6
28
2.6 Effect of combining MAO inhibitor clorgyline (or phenelzine,
deprenyl, pargyline) with doxorubicin on viability of neuroblastoma
CHLA-255 cells
We next studied whether combining an MAO inhibitor with doxorubicin would improve
the efficacy against neuroblastoma cells. In CHLA-255 cells, 1 µM clorgyline by itself
caused less than 10% inhibition of viability. Doxorubicin at 10
-8
, 10
-7
, 10
-6
M caused
0, 30%,60% inhibition on cell viability. This shows dose-dependent inhibitory pattern of
doxorubicin in this cell line (Fig 5 A). Co-treatment with 1 µM clorgyline increased the
cytotoxicity of doxorubicin. The cytotoxicity increased from 2% to 34% (p < 0.001)
when clorgyline was combined with doxorubicin (10
-8
M) while it increased from 33%
to 55% (p < 0.01) when combining with doxorubicin (10
-7
M) (Fig 5 A).
In the case of phenelzine, 1 µM concentration casued less than 10% inhibition of
viability of CHLA-255 cells. Co-treatment with 1 µM phenelzine increased the
cytotoxicity of doxorubicin. The cytotoxicity increased from 2% to 23% (p < 0.01)
when phenelzine was combined with doxorubicin (10
-8
M) (Fig 5 B).
For deprenyl, 1 µM concentration caused less than 10% inhibition of viability of
CHLA-255 cells. Co-treatment with 1 µM deprenyl increased the cytotoxicity of
29
doxorubicin. Inhibition on cell viability increased from 2% to 18% (p < 0.01) when
phenelzine was combined with doxorubicin (10
-8
M) (Fig 5 C).
For pargyline, 1 µM concentration also caused less than 10% inhibition of
viability of CHLA-255 cells. Co-treatment with 1 µM pargyline increased the
cytotoxicity of doxorubicin. It increased from 2% to 15% (p < 0.01) when
pargyline was combined with doxorubicin (10
-8
M) (Fig 5 D).
The above results showed that combining each of these four MAO inhibitors
with doxorubicin caused greater cytotoxicity compared to doxorubicin alone and
MAO inhibitor alone in CHLA-255 cells. Among the four MAO inhibitors,
combination of 1 µM clorgyline with 10
-8
M doxorubicin had most significant
improvement in cytotoxicity. This indicates that MAO inhibitor has the ability to
enhance the cytotoxicity of low dose (10
-8
M) doxorubicin.
30
Figure 5. Effect of doxorubicin with or without 1 µM MAO inhibitor on cell
viability in CHLA-255 cell line. 5000 cells were seeded per well in the 96 well
plate and incubated with doxorubicin (10
-6
, 10
-7
and 10
-8
M) combined with 1 µM
clorgyline (A), deprenyl (B), pargyline (C) and1 phenelzine (D). Cell viability was
determined using MTT assay. * p<0.05, ** p<0.01and*** p<0.001.
#
p<0.05 and
##
p<0.01.
Clorgyline(1µM) — + — + — + — +
Doxorubicin(M) — — 10
78
10
77
10
76
0
20
40
60
80
100
120
1 2 3 4 5 6
Cell*Viability*(*%*of*Control)
CombinationofClorgylineandDoxorubicinfor48hours
##
NS
***
###
**
Deprenyl(1µM) — + — + — + — +
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationofDeprenylandDoxorubicinfor48hours
**
*
NS
##
###
Doxorubicin(M) — — 10
68
10
67
10
66
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationofPargylineandDoxorubicinfor48hours
Pargyline(1µM) — + — + — + — +
###
**
*
NS
##
Doxorubicin(M) — — 10
88
10
87
10
86
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationPhenelzineandDoxorubicinfor48hours
Phenelzine(1µM) — + — + — + — +
**
*
NS
##
#
Doxorubicin(M) — — 10
78
10
77
10
76
A B
C D
31
2.7 Effect of combining MAO inhibitor clorgyline (or phenelzine,
deprenyl, pargyline) with doxorubicin on viability of neuroblastoma
SK-N-SH cells
Similar to CHLA-255 cells, 1 µM clorgyline caused less than 10% inhibition on
the viability of SK-N-SH cell line after 48 hours treatment (Fig 6 A). At 10-8 M
and 10-7 M, doxorubicin alone slightly inhibited of cell viability of SK-N-SH cell
(< 10%) (Fig 6 A). Co-treatment with 1 µM clorgyline increased the cytotoxicity of
doxorubicin. Clorgyline enhanced the cytotoxicity of 10-8 M doxorubicin from 2%
to 20% (p < 0.01), and increased the cytotoxicity of 10-7 M doxorubicin from 6%
to 27% (p < 0.01).
Contrasting the results obtained with CHLA-255 cells, only clorgyline
enhanced the cytotoxicity of doxorubicin in SK-N-SH cells while deprenyl,
phenelzine and pargyline all caused almost no enhancement (Fig 6).
Taken together, data shown in Figure 5 and Figure 6 provide evidence that
MAO inhibitors can improve the cytotoxicity of doxorubicin in human
neuroblastoma CHLA-255 and SK-N-SH cells in vitro. Furthermore, most
significant combined effect was observed when MAO inhibitor (1 µM) was co-
treated with low dose (10
-8
M) doxorubicin, suggesting that MAO inhibitor may be
used to lower the drug dose of doxorubicin in neuroblastoma clinics.
32
Figure 6. Effect of doxorubicin with or without 1 µM MAO inhibitor on cell
viability in SK-N-SH cell line. 5000 cells were seeded per well in the 96 well plate,
and incubated with doxorubicin (10
-6
, 10
-7
and 10
-8
M) combined with 1 µM
clorgyline (A), deprenyl (B), pargyline (C) and1 phenelzine (D). Cell viability was
determined using MTT assay. * p < 0.05, ** p < 0.01 and *** p < 0.001,
#
p < 0.05
and
##
p < 0.01.
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationPargylineandDoxorubicinfor48hours
*
NS
NS
#
NS
Pargyline (1µM) — + — + — + — +
Doxorubicin (M) — — 10
88
10
87
10
86
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationPhenelzineandDoxorubicinfor48hours
NS
NS
NS
NS
NS
Phenelzine (1µM) — + — + — + — +
Doxorubicin (M) — — 10
78
10
77
10
76
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationofClorgylineandDoxorubicinfor48*hours ##
NS
**
##
**
Clorgyline (1µM) — + — + — + — +
Doxorubicin (M) — — 10
78
10
77
10
76
0
20
40
60
80
100
120
Cell*Viability*(*%*of*Control)
CombinationDeprenylandDoxorubicinfor48hours
Deprenyl(1µM) — + — + — + — +
NS
NS
NS
#
Doxorubicin (M) — — 10
68
10
67
10
66
A B
C D
33
Chapter 3. Discussion and Conclusion
Doxorubicin is the main chemotherapy treatments for neuroblastoma patients but
an urgent need exists to reduce side effects by reducing dosage (Peehl et al., 2008).
Combining with another drug may provide a potential strategy to lower the amount
of doxorubicin that needs to be taken, thus decreasing the side effects of cancer
treatment compared to single-agent chemotherapy (Zhao et al., 2008). In this study,
we, for the first time, reported the role of MAO A in human neuroblastoma and
showed that combination of MAO inhibitors and doxorubicin provides potential
strategy for the treatment of neuroblastoma.
First we compared the expression level of MAO A and MAO B mRNA in
malignant and benign neuroblastic tumor (Fig 1). The mRNA expression level of
MAO A is higher in malignant compared to benign neuroblastic tumor, indicating
that MAO A may play a role in neuroblastoma. Three human neuroblastoma cell
lines, i.e. CHLA-255, SK-N-SH and SK-N-BE2, have both MAO A and MAO B
catalytic activities (Table 2). Among these three cell lines, CHLA-255 and SK-N-
SH showed higher MAO A and MAO B activities, therefore we chose these two
cell lines for further experiments.
34
Then MAO A activity inhibition assay was performed to confirm the MAO
A activity in both in CHLA-255 and SK-N-SH cell lines (Fig 2). The results
showed that both CHLA-255 and SK-N-SH cell lines were more sensitive to MAO
A specific inhibitor clorgyline compared to MAO B specific inhibitor deprenyl.
This confirms MAO A activity in these two cell lines.
Next, we compared the inhibitory effect of four MAO inhibitors clorgyline,
deprenyl, phenelzine and pargyline on MAO catalytic activity and viability of
CHLA-255 and SK-N-SH cells (Table 3). The MAO A activity in both cell lines
were most sensitive to clorgyline, followed by phenelzine, deprenyl and pargyline.
In MTT assay, the order of sensitivity of CHLA-255 cells to four MAO inhibitors
was clorgyline > phenelzine > deprenyl > pargyline while the sensitivity of SK-N-
SH cells was phenelzine > clorgyline > deprenyl > pargyline.
We found that, compared to MAO A activity assay, the IC
50
value of four
MAO inhibitors were much higher in MTT assay. This suggesting the MAO
inhibitor itself is not effective in inhibition of neuroblastoma cell viability.
Combining with current therapeutics may be necessary for more efficient treatment.
Doxorubicin is one of the major chemotherapeutics for treating
neuroblastoma. Doxorubicin inhibits the viability of CHLA-255 and SK-N-SH
cells (Fig 4). As single
35
agent, in CHLA-255 cell line, 10
-8
M of doxorubicin had no effect on cell viability,
10
-7
M doxorubicin caused ~30% inhibition and 10
-6
M doxorubicin caused ~60%
inhibition on cell viability. This indicates that there is a dose-dependent inhibition
by doxorubicin in this cell line (Fig 5). In SK-N-SH cell line, both 10
-8
M and 10
-7
M doxorubicin caused less than 10% inhibition of viability (Fig 6).
Compared to doxorubicin treatment, combining doxorubicin with MAO
inhibitor caused greater inhibition of viability in CHLA-255 cells. While 1 µM
clorgyline by itself had less than 10% inhibition of viability, co-treatment with 1
µM clorgyline increased the cytotoxicity of doxorubicin. The cytotoxicity
increased from 2% to 34% (p < 0.001) when clorgyline was combined with
doxorubicin (10
-8
M) while it increased from 33% to 55% (p < 0.01) when
combining with doxorubicin (10
-7
M) (Fig 5 A). Combining each of other three
MAO inhibitors phenelzine, deprenyl and pargyline with doxorubicin also has
better effect on cytotoxicity compared to doxorubicin alone and MAO inhibitor
alone in CHLA-255 cells (Fig 5 B: phenelzine, C: deprenyl, D: pargyline). Among
the four MAO inhibitors, combination of 1 µM clorgyline with 10
-8
M doxorubicin
had most significant improvement in cytotoxicity (Fig 5 A).
Similar to CHLA-255 cells, 1 µM clorgyline caused less than 10% inhibition
on the viability of SK-N-SH cell line after 48 hours treatment (Fig 6 A). Co-
treatment with 1 µM clorgyline increased the cytotoxicity of doxorubicin.
36
Clorgyline enhanced the cytotoxicity of 10
-8
M doxorubicin from 2% to 20% (p <
0.01), and increased the cytotoxicity of 10
-7
M doxorubicin from 6% to 27% (p <
0.01). Contrasting the results obtained with CHLA-255 cells, only clorgyline
enhanced the cytotoxicity of doxorubicin in SK-N-SH cells while deprenyl,
phenelzine and pargyline all caused almost no enhancement (Fig 6). Furthermore,
most significant combined effect was observed when MAO inhibitor (1 µM) was
co-treated with low dose (10
-8
M) doxorubicin, suggesting that MAO inhibitor may
be used to lower the drug dose of doxorubicin in neuroblastoma clinics.
In conclusion, we showed MAO A play a role in neuroblastoma progression
and MAO inhibitor has ability to inhibit viability of neuroblastoma cells. MAO
inhibitor also can enhance the cytotoxicity of doxorubicin in neuroblastoma cells.
The above finding suggests the potential therapeutic effect of MAO inhibitor for
human neuroblastoma. The mechanism through which MAO inhibitor enhance the
cytotoxicity of doxorubicin in neuroblastoma is unknown. Also, the function of
MAO B in neuroblastoma progression needs to be determined in the future.
37
Chapter 4. Material and Methods
4.1 Reagent
MAO inhibitors were used as followed: N-Methyl-N-propargyl-3-(2,4-
dichlorophenoxy) propylamine hydrochloride (clorgyline), R- (−)-Deprenyl
hydrochloride (deprenyl), Phenelzine sulfate salt (phenelzine) and Pargyline
hydrochloride (pargyline). All MAO inhibitors were obtained from Sigma-Aldrich,
USA. Doxorubicin were also obtained from Sigma-Aldrich, USA. Thiazolyl Blue
Tetrazolium Bromide (MTT kit) (Sigma-Aldrich, USA) was used in MTT assay
while dimethyl sulfoxide (DMSO) used in MTT assay was obtained from Sigma-
Aldrich, USA.
4.2 Cell cultures
Human neuroblastoma cell lines CHLA-255, SKN-SH and SKN-BE2 were kindly
provided by Dr. Yves A. DeClerck, Keck School of Medicine, University of
Southern California, Los Angeles, CA, USA. CHLA-255 cell line was cultured in
10% fetal calf serum in Iscove's Modified Dulbecco's Medium (ThermoFishe,
USA) supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin in a
humidified incubator at 37°C and 5% CO
2
. Cell line SKN-SH and SKN-BE2 were
cultured in Roswell Park Memorial Institute 1640 media (ThermoFisher, USA),
38
also supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin in a
humidified incubator at 37°C and 5% CO
2
.
4.3 Database
The mRNA expression was ascertained from publicly-accessible Gene Expression
Omnibus (GEO) databases that appeared online on AlbinoBrain_cancner using the
Oncomine platform (https://www.oncomine.org/resource/login.html). For
comparison, the mRNA expression of tumors and their normal counterpart from an
identical study conducted using the same methodology were analyzed.
4.4 MAO catalytic activity assay
MAO catalytic activity was determined by radio-assay as described previously
(Wu et al., 2014). For the MAO A catalytic activity assay, 5-hydroxytryptamine
(5-HT) served as the substrate. Cells were incubated with 1 mM
14
C-5-HT in the
assay buffer for 20 minutes at 37℃. The reaction products were extracted and
radioactivity was determined by liquid scintillation spectroscopy. For MAO B
catalytic activity assay, cells were incubated with 100 µM
14
C-phenylethylamine
(PEA) for 20 minutes at 37℃, in the assay buffer. The reaction products were
extracted and radioactivity was determined. For inhibition activity assay, cells
were pre-incubated with different compounds at increasing concentrations for 20
39
minutes at 37°C. Then
14
C-labeled 5-HT or PEA were added into the mix, reacted
for another 20 minutes at 37°C, followed by extraction of reaction products and
determination of radioactivity.
4.5 MTT assay
Briefly, MTT assay were performed by seeding 5,000 cells per well in 96-well
plate and cultured 24 hours before the addition of compounds. Different
compounds at different concentrations were added and incubated for 24 hours or
48 hours. Then 5 mg/ml MTT kit were added into each well and incubated at 37 ℃
for 4 hours. Adding 50 µL DMSO for solubilizing the MTT crystals in each well.
The intensity is measured at 590 nm.
4.6 Statistical analysis
Student’s 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, ** p < 0.01 and *** p < 0.001.
#
p < 0.05 and
##
p < 0.01.
40
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Abstract (if available)
Abstract
Neuroblastoma is one of the most common solid tumor in childhood and the most commonly diagnosed in infancy. Doxorubicin is the current standard treatment for neuroblastoma. Side effects such as heart damage caused by doxorubicin limited its use for treatment. Ample evidence has indicated that monoamine oxidase A (MAO A) plays an important role in cancer development. This thesis studied the role of MAO A in neuroblastoma and its potential for treatment. Analysis of Oncomine database showed that the expression level of MAO A mRNA is higher in malignant compared to benign neuroblastic tumors, indicating that MAO A may play a role in neuroblastoma. The MAO A catalytic activity was found in three neuroblastoma cell lines: CHLA-255, SK-N-SH and SK-N-BE2. CHLA-255 and SK-N-SH were used in further studies as they expressed higher MAO A activity than SK-N-BE2. MAO A in these two cell lines was further characterized by its sensitivities to four inhibitors clorgyline, deprenyl, pargyline or phenelzine. Using MTT assay, we found that all four inhibitors itself were not effective on the neuroblastoma cell viability. Doxorubicin itself was cytotoxic to these two cell lines
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Potential therapeutic effect of monoamine oxidase (MAO) inhibitor on human neuroblastoma
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Molecular Pharmacology and Toxicology
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