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Accepted Manuscript
The impact of Catechol-O-methyl transferase knockdown on the cell proliferation ofhormone-responsive cancers
Mai F. Tolba, Hany A. Omar, Fatima Hersi, Ane C.F. Nunes, Ayman M. Noreddin
PII: S0303-7207(19)30091-7
DOI: https://doi.org/10.1016/j.mce.2019.03.007
Reference: MCE 10417
To appear in: Molecular and Cellular Endocrinology
Received Date: 9 June 2018
Revised Date: 24 February 2019
Accepted Date: 17 March 2019
Please cite this article as: Tolba, M.F., Omar, H.A., Hersi, F., Nunes, A.C.F., Noreddin, A.M., The impactof Catechol-O-methyl transferase knockdown on the cell proliferation of hormone-responsive cancers,Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.03.007.
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The impact of Catechol-O-methyl transferase knockdown on the cell proliferation of hormone-
responsive cancers
Mai F. Tolba1,2,3§*, Hany A. Omar,4,5,6§*, Fatima Hersi6, Ane C. F. Nunes7, Ayman M. Noreddin2,3,4
1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University,
Cairo 11566, Egypt; 2Chapman University School of Pharmacy, Irvine 92618, CA, USA; 3Department of
Medicine, University of California Irvine, CA, USA; 4College of Pharmacy, University of Sharjah,
Sharjah 27272, United Arab Emirates;5 Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef, 62511 Egypt;6Sharjah Institute for Medical Research,
University of Sharjah, United Arab Emirates; 7 Division of Nephrology and Hypertension, School of
Medicine, University of California Irvine, CA, USA
§Contributed equally to this manuscript
*Correspondence should be addressed to:
Mai F. Tolba, Ph.D.
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo 11566,
Egypt. Email: [email protected], [email protected]. Phone: +(20)240-51120.
And
Hany A. Omar, Ph.D.
Department of Pharmacy Practice and Pharmacotherapeutics, College of Pharmacy, University of
Sharjah, Sharjah 27272. Email: [email protected]. Phone: +(971)65057411.
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Abstract
Estrogen (E2) plays a central role in the development and progression of hormone-responsive cancers.
Estrogen metabolites exhibit either stimulatory or inhibitory roles on breast and prostate cells. The
catechol metabolite 4-hydroxyestradiol (4-OHE2) enhances cell proliferation, while 2-methoxyestradiol
(2ME) possesses anticancer activity. The major metabolizing enzyme responsible for detoxifying the
deleterious metabolite 4-OHE2 and forming the anticancer metabolite 2ME is Catechol-O-Methyl
Transferase (COMT). The current work investigated the relationship between the expression of COMT
and cell proliferation of hormone-responsive cancers. The results showed that COMT silencing enhanced
the cell proliferation of ER-α positive cancer cells MCF-7 and PC-3 but not the cells that lack ER-α
expression as MDA-MB231 and DU-145. The data generated from our study provides a better
understanding of the effect of COMT on critical signaling pathways involved in the development and
progression of BC and PC including ER-α, p21cip1, p27kip1, NF-κB (P65) and CYP19A1. These findings
suggest that COMT enzyme plays a tumor suppressor role in hormone receptor-positive tumors which
opens the door for future studies to validate COMT expression as a novel biomarker for the prediction of
cancer aggressiveness and treatment efficacy.
Keywords: Catechol-O-methyl transferase (COMT); breast cancer; prostate cancer; proliferation, tumor
suppressor
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1.Introduction
Estrogen (E2) plays a central role in the development and progression of hormone-responsive
cancers[1,2]. Two types of solid tumors with major public health impact fall into this category. Breast
cancer (BC) is the most commonly diagnosed cancer in women worldwide and is the leading cause of
cancer deaths among women after lung cancer [3]. According to statistical evidence, the 5-year survival
for metastatic BC is 25%. Prostate cancer (PC) is the most frequently diagnosed solid tumor in men in
USA and is ranked as the third cause of cancer deaths in men. The 5-year survival rate is only 29% for
metastatic PC [3]. A better understanding of the molecular mechanisms involved in the tumor progression
is highly warranted and will ultimately lead to developing effective treatment modalities for these
debilitating cancers.
The mechanisms of E2-induced carcinogenesis are complex and involve both estrogen receptor
(ER)-dependent and receptor-independent effects [2,4]. ER-α plays an important role in the receptor-
dependent stimulation of breast tissue growth and cell proliferation [4,5]. Clinical studies supported that
the blockade of ER-α in BC through the administration of tamoxifen significantly reduced BC incidence
[6]. Tamoxifen also suppresses the recurrence rate of BC in patients with ER-positive tumors [7,8]. ER-α
is also associated with enhanced proliferation, inflammation, and carcinogenesis in prostate tissues [2].
The ER-independent effects of E2 mainly involve the effect of E2 metabolites[9]. Due to their duplicitous
nature, E2 metabolites exhibit either stimulatory or inhibitory roles on breast or prostate cells through
various mechanisms [2,4,10]. For example, continuous exposure of the epithelial breast cells MCF-10F to
the catechol E2 metabolite 4-hydroxyestradiol (4-OHE2) triggers mutations, cell transformation and
carcinogenesis [11,12]. Similar outcomes were demonstrated in the prostate epithelial cells BPH1 with
long-term exposure to catechol E2 metabolites 4-OHE2 or 2-OHE2 [13,14]. The catechol metabolites 2-
OHE2 and 4-OHE2 are produced through the oxidative metabolism of E2 by cytochrome P450 (CYP)-
1A1 and CYP1B1, which are constitutively expressed in breast and prostate tissues [15,16]. On the other
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hand, 2-methoxyestradiol (2ME) is another E2 metabolite that possesses a potent cytotoxic activity
against various cancer types [17]. The mechanisms for 2ME anticancer activity is mainly through the
induction of apoptosis and the inhibition of hypoxia-inducible factor-1α which is a key mediator in
angiogenesis signaling [18]. 2ME is formed endogenously through the sequential hydroxylation and O-
methylation of E2 at the 2-position [17]. The major metabolizing enzyme in breast and prostate cells that
is responsible for the detoxification of the deleterious catechol metabolites (2-OHE2, 4-OHE2) and the
formation of the anticancer metabolite 2ME is Catechol-O-Methyl Transferase (COMT) [19]. This
enzyme is present in two distinct isoforms, a soluble isoform COMT (S-COMT) and a membrane-bound
isoform COMT (MB-COMT)[19]. Overexpression of COMT in uterine leiomyoma cells attenuates ER-α
signaling through the increased production of 2ME [20]. The role of COMT in tumorigenesis is supported
by epidemiological studies which indicated that polymorphisms of the COMT encoding gene leading to
low enzyme activity are associated with higher risk of hormone-responsive tumors, such as BC [21],
ovarian cancer [22] and PC [23]. However, studies and meta-analyses reports on the association between
COMT polymorphisms and PC risk are controversial [23-28]. Based on these observations, COMT seems
to play a critical role in BC and PC tumorigenesis. It is imperative to approach a better understanding of
the effect of COMT expression on cancer proliferation. The impact of COMT expression on BC or PC
growth has not been investigated before. The goal of this study is to characterize the relationship between
COMT expression and the proliferation of human BC and PC cells.
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2. Material and Methods
2.1. Materials
Silencer®Select COMT specific siRNA (CAT#4390824, ID# s41) and Lipofectamine RNAiMAX
transfection reagents were purchased from Thermo Fisher Scientific Inc (Hanover Park, IL). Phenol red-
free OPTIMEM was purchased from Gibco™, Thermo Fisher Scientific Inc (Hanover Park, IL). 17β-
Estradiol (E2), 2-hydroxyestradiol (2OHE2), DMSO and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide] were purchased from (Sigma-Aldrich, St. Louis, MO).
2.2. Cell culture
All cell lines were obtained from the American Type Culture Collection (Manassas, VA). MCF-
7 and PC-3 cells were cultured in RPMI-1640 medium (Gibco™, Thermo Fisher Scientific Inc,
Hanover Park, IL) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
DU-145 cell line was cultured in EMEM medium (ATCC, Manassas, VA) and MDA-MB231
cell line was cultured in low glucose DMEM medium (Gibco™, Thermo Fisher Scientific Inc,
Hanover Park, IL) with the same supplements. All the cell cultures were routinely tested to
confirm the absence of mycoplasma infection using EZdetect™ kit (HiMedia Laboratories Pvt.
Ltd., Mumbai, India). All cells were cultured at 37°C in a humidified incubator containing 5%
CO2. The cells were used at low passage numbers less than 25.
2.3. Short interfering (si)RNA mediated-COMT knockdown
The cells were transfected with Silencer® Select COMT specific siRNA or scrambled siRNA
using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific Inc, Hanover
Park, IL) as per the manufacturer’s instructions. The knockdown of the corresponding protein
was confirmed by immunoblotting.
2.4. Western blotting
Cell lysates were collected at 48h after siRNA transfection and were prepared for Western blotting of
COMT (Dilution 1:500; Sigma-Aldrich, St. Louis, MO, CAT# C6870-Lot: 108K4754), ER-α (Dilution
1:1000; Cell Signaling Technology Inc, Danvers, MA, CAT# 13258S - Lot:1), ER-α (p-S167) (Dilution
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1:1000; Abcam plc, Cambridge, MA, CAT#: ab131105 - Lot: GR94539-16) and β-actin. Western blot
analysis was performed as previously described [29]. Image analysis was done using Image Lab™
software (Bio-Rad Laboratories, Irvine, CA).
2.5.MTT proliferation assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for cell
viability analysis as described before [30]. Briefly, 24h after transfection the cells were detached using
TrypLE Select and seeded in 96-well plates at a density of 4000/well for 24h endpoint and 2000/well for
48h time points. The cells were seeded in phenol red-free RPMI-1640 supplemented with 10% charcoal-
stripped FBS. Specific wells were dedicated for scrambled control cells (n=12) and other sets of wells
were dedicated for COMT-siRNA cells (n=12). Six wells of scrambled control and COMT-siRNA cells
were maintained in E2-free medium and another set of six wells of each group was maintained in E2
(0.01µM) supplemented medium. At 24h and 48 h time points, the medium was removed and replaced by
200 µL new media containing MTT (0.5mg/mL) then the incubation was resumed for 2 h, at 37°C in CO2
incubator. After that, the supernatants were discarded, and the produced violet formazan crystals were
solubilized in DMSO (200 µL/well). The optical density was measured at 590 nm using a microplate
reader. Results are expressed as percent cell viability relative to scrambled control. The experiments were
performed 3 independent times.
2.6. Testing the effect of entacapone (ENT) on cell proliferation
The reported IC50 of ENT for COMT inhibition is 151 nM [31]. In the current study, MCF-7 and PC-3
cells were exposed to ENT 1 µM and the exposure to ENT –alone or combined with 2OHE2 was
continued for 48h. At the end of exposure, MTT assay was performed as described previously [30].
Results were expressed as percent cell viability relative to untreated control.
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2.7. Testing the effect of COMT product 2-methoxyestradiol (2ME) on cell viability
The effect of the COMT product 2ME on the cell viability was assessed by exposing the different cell
lines to serial concentrations 0.1, 0.3, 1, 3 and 10 µM of 2ME for 48h. At the end of exposure MTT assay
was performed as described previously [30]. Results were expressed as % viability relative to untreated
control. The calculation of the IC50 value was performed using sigmoidal concentration– response curve
fitting model (Graph Pad, Prizm software).
2.8. Quantitative Real-Time polymerase chain reaction assessment of P21Cip1, P27kip1 and CYP19A1
The isolation of total cellular RNA was achieved using RNeasyMini Kit® (Qiagen Inc., Valencia, CA,
USA). The reverse transcriptions were performed using iScript cDNA Synthesis Kit, (Bio-Rad
Laboratories Inc, Hercules, CA, USA) as per the manufacturer's instructions. The produced cDNA was
then amplified and quantified using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc,
Hercules, CA, USA) and Bio-Rad CFX96™ Real-Time PCR System. Primer sequences were as follows:
P21Cip forward primer: 5-CGA CTG TGA TGC GCT AAT GG-3, reverse primer: 5-CCA GTG GTG
TCT CGG TGA CA-3. P27 kip forward primer: 5′-GTCAAACGTAAACAGCTCGAAT-3′, reverse
primer: 5′-TGCATAA TGCTACATCCAACG-3′. CYP19A1: forward primer:5′-
AAATCCAGACTGTTATTGGTGAGAG-3′, reverse primer: 5′-
GTAGCCATCGATTACATCATCTTCT-3′. Data were analyzed using the ∆∆CT method. Changes in the
gene expression were calculated as fold change compared to the scrambled control group and were
normalized to the expression of β-actin.
2.9. ELISA assays for NF-κB p65 (pS536)/Total NF-κB protein expressions
The expression level of NF-κB p65 (pS536)/Total NF-κB proteins were assessed using SimpleStep
ELISA kit purchased from Abcam (Cambridge, MA). The assay was performed per the manufacturer's
recommendations and the generated optical density was measured at 450 nm.
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2.10. ELISA assays for COMT protein expression
Cell lysates were diluted 20X using sample diluent buffer before their incorporation to the ELISA
reaction. The protein levels of COMT were assessed using Human COMT AccuSignalTM ELISA Kit
(Rockland Immunochemicals Inc., Limerick, PA) as per the manufacturer's protocol and the generated
optical density was measured at 450 nm. The total protein levels were measured using BCA protein assay
(Pierce Biotechnology, Rockford, IL) and used for normalization of COMT levels which was expressed
as pg/mg protein.
2.11. Docetaxel (DOC) IC50 determination
The IC50 of DOC was calculated by exposing the different cell lines to serial concentrations 0.01, 0.1, 1,
10 and 100 nM of DOC in phenol red-free medium supplemented with charcoal-stripped FBS and the
exposure was continued for 48h. Parallel exposures were done to compare the potency of DOC alone or in
the presence of 2-OHE2 (0.01µM) in the medium. MTT assay was performed at the end of the exposure
duration as described previously[30]. The IC50 calculation was performed using sigmoidal concentration–
response curve fitting model (Graph Pad, Prizm software).
2.12. Statistical analysis
Data are presented as means ± SD. Individual groups were compared using the two-tailed independent
Student's t-test. However, the multiple group comparisons were done via one-way analysis of variance
(ANOVA) followed by the Tukey-Kramer test for post-hoc analysis. Statistical significance was accepted
at a level of P< 0.05. All statistical analyses were performed using GraphPad InStat software, version
3.05 (GraphPad Software, Inc. La Jolla, CA, USA). Graphs were sketched using GraphPad Prism
software, version 5.00 (GraphPad Software, Inc).
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3. Results
3.1. siRNA- mediated knockdown of COMT in BC and PC cells
Transfection of the cells with COMT specific siRNA led to a significant reduction of COMT expression
at the protein level by 54-56%compared to cells transfected with scrambled siRNA (p<0.001) (Fig.1) as
confirmed by Western blotting. These cells were named as COMT-si cells. It is noteworthy that the ratio
of S-COMT to MB-COMT was the highest in MCF-7 cells followed by PC-3 cells, while MDA-MB231
and DU145 cells showed lower expression (Fig. 1A, B).
3.2. Effect of COMT silencing on cancer cell proliferation
Assessment of MCF-7 cell proliferation 24 h post COMT silencing showed significantly increased cell
proliferation by 28% (P<0.001) in COMT-si group versus the scrambled control group (Fig. 1C). The
proliferation of MCF-7 cells was significantly enhanced by 54% (P<0.001) at 48h post COMT-silencing
compared to scrambled control. The proliferation of MDA-MB231 cells was not significantly altered at
24h post COMT-silencing, while it was reduced by 9% (P<0.05) 48h after COMT silencing (Fig.1 C).
The proliferation of PC-3 cells did not show significant changes at 24h post silencing, while it was
significantly enhanced by 41.5% (P<0.01) at 48h post-COMT silencing compared to scrambled
control(Fig.1C). No significant change was observed in DU-145 cell proliferation after COMT-silencing
(Fig.1C).
COMT silencing with concomitant exposure to E2 (0.01µM) significantly boosted MCF-7 cell
proliferation by 62% (P<0.001) versus scrambled siRNA group at 48h post-silencing (Fig. 2A). Similarly,
the proliferation of COMT-si PC-3 was enhanced under E2-exposure conditions by 44.8% (P<0.001)
compared to scrambled control group at 48h post-silencing (Fig.2A). It is noteworthy that the cells were
detached for seeding 24 h after silencing then incubation with E2 was done for 24h only. Treatment of the
ER +ve cell line MCF-7 with E2 for different exposure times 24, 48 and 72h indicated that E2 treatment
significantly enhanced cell proliferation starting from 48h exposure. Treatment with E2 0.01 or 0.1 µM
for 48h significantly increased the cell growth by 30 and 28.8% (P<0.05) respectively compared to
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control. The exposure of MCF-7 cells to E2 0.001, 0.01 and 0.1 0.1 µM for 72h significantly increased
the cell proliferation by 25.7, 34.4 and 45.1% (P<0.05) compared to control (Fig. 1, Supplementary
Material).
3.3. Effect of Entacapone (ENT) on cell proliferation
The reported IC50 of ENT for COMT inhibition is 151 nM [31]. In the current study, the cells were
exposed to ENT 1 µM and the exposure was continued for 48h. Entacapone did not elicit any changes in
the proliferation of MCF-7 cells. However, exposure to ENT alone significantly boosted the proliferation
of PC-3 cells by 23 % (P<0.01) compared to control. Concomitant exposure of PC-3 cells to ENT and
COMT substrate 2-OHE2 significantly increased the proliferation by 32 % (P<0.01) compared to control
(Fig.2B).
3.4. Effect of COMT silencing on ER-α (p-S167)
COMT silencing resulted in a significant increase in ER-α phosphorylation at S167 by 2.9 and 1.7-fold
(P<0.001) in MCF-7 and PC-3 cells compared to the scrambled control counterparts (Fig.2C, D)
3.5. Effect of COMT product 2-methoxyestradiol (2ME) on cell viability
The effect of the COMT product 2ME on the cell viability was assessed by exposing the cells to serial
concentrations 0.1, 0.3, 1, 3 and 10 µM of 2ME for 48h. Treatment of BC cells with 2ME for 48h showed
cytotoxic activity with IC50 of 0.25±0.06 and 0.4±0.04 µM in MCF-7 and MDA-MB231 cell lines (Fig.
3A). PC cells showed lower sensitivity compared to BC cells on treatment with 2ME. The IC50 of 2ME
was 1.96± 0.14 and 2.29±0.6 µM in PC-3 and DU-145 cell lines (Fig.3B).
3.6. Effect of COMT silencing on p21Cip1 and p27kip1 gene expression
The expression of p27kip1 was significantly down-regulated by 79% (P<0.001) in MCF-7 cells and by
35.5% (P<0.05) in MDA-MB231 cells after COMT silencing (Fig.4A). PC-3 cells showed a significant
down-regulation of p27kip1 by 39.9% (P<0.05) in COMT knockdown group compared to scrambled
control (Fig.4 A).
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COMT silencing resulted in a significant increase in p21Cip1 mRNA transcripts in MDA-MB231 cell line
by 1.99 folds (P<0.05) compared to scrambled control. No change in p21Cip1 levels was observed in MCF-
7 cells after COMT-knockdown (Fig.4B). COMT silencing in PC-3 cells significantly down-regulated
p21Cip1 levels by 78% (P<0.01) compared to scrambled control. The level of p21Cip1 mRNA transcripts
was significantly (P<0.001) boosted in DU-145 cells by 2.3 folds after COMT silencing (Fig.4B).
3.7. Basal COMT expression and NF-κB (p65) phosphorylation in wild-type (WT) cell lines
Western blot analysis helped to assess the expression of S-COMT and MB-COMT isoforms in each cell
line in a semi-quantitative way, while the ELISA assay provided an accurate quantitation of the total level
of COMT protein in each of the tested cell lines. The results of ELISA determinations were in line with
Western blot analysis and confirmed that MCF-7 cell line possesses the highest level of COMT protein.
MCF-7 WT cells showed the highest basal COMT expression level. The level was significantly (P<0.001)
higher by 3.2-fold compared to MDA-MB231 and DU-145 cells and by 2.97-fold compared to PC-3 cells
(Fig.5A). The triple negative BC cell line MDA-MB231 showed the highest level of S536
phosphorylation of NF-κB (p65) subunit. The level was significantly higher by 1.4, 1.43 and 1.6 folds
compared to MCF-7, PC-3 and DU-145 (Fig.5B).
3.8. Effect of COMT silencing on NF-κB (p65) phosphorylation (S536)
COMT silencing in BC cells resulted in a significant increase in NF-κB p-65 (S536) phosphorylation in
MCF-7 by 2 folds (P<0.001) compared to scrambled control. No change was noticed in MDA-MB231
cells. COMT basal level was significantly higher in MCF-7 WT cells compared to MDA-MB231 and was
inversely correlated with basal NF-κB (p65) phosphorylation. Therefore, silencing COMT in MCF-7
resulted in more prominent effects on NF-κB activation and cell proliferation (Fig.5C). On the other hand,
the S536 phosphorylation of NF-κB (p65) was significantly increased in PC-3 cells by 35.4% (P<0.01) in
COMT knockdown cells compared to scrambled control. COMT silencing in DU-145 resulted in a non-
statistically significant increase in NF-κB phosphorylation by 13% (Fig. 5 D).
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3.9. Effect of COMT silencing on the gene expression of aromatase (CYP19A1)
COMT silencing significantly increased the expression of CYP19A1 in MCF-7, MDA-MB231 and PC-3
cancer cell lines by about 4, 2.9-fold (P<0.001) and 1.37-fold (P<0.05) compared to scrambled control
(Fig. 4C).
3.10. Effect of COMT substrate 2-OHE2 on docetaxel (DOC) response
This is the first study to examine the effect of catechol E2 metabolite 2-OHE2 on DOC potency in a cell
system. Exposure of the cells to low concentration 0.01 µM of 2-OHE2 resulted in a significant decrease
in DOC potency. The IC50 of DOC alone in MCF-7 cells was 2.63± 0.19 nM, while upon concomitant
exposure to 2-OHE2 the IC50 increased to 5.8±1.45 nM which is significantly higher by 2.2 folds
(p<0.05). The IC50 of DOC alone in MDA-MB231 cells was 3.49± 0.29nM, while in the presence of 2-
OHE2 it was increased to 8±0.86 nM which is 2.3 folds(p<0.01) higher. Similar findings were shown in
PC cells where the IC50 of DOC alone in PC-3 cells was 2.33±0.72 nM, while DOC IC50 under conditions
of concurrent exposure to 2-OHE2 it was increased to 6.6±0.72 nM which is 2.8 folds(P<0.001) higher.
The IC50 of DOC alone in DU-145 cells was 5.7±0.68 nM, while in the presence of 2-OHE2 it was
increased by 1.9 folds (p<0.01) to be 11.35±1.8 nM (Fig. 3C).
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4. Discussion
COMT has a pivotal role in E2 metabolism. It helps in the detoxification of the deleterious
catechol E2 metabolites and converts them to either inactive metabolites as 4-methoxyestradiol or a
metabolite with an anticancer activity as 2-methoxyestradiol [19]. Thus, COMT seems to play a critical
role in modulating the cancer progression. Our long-term goal is to reduce death from BC and PC and to
improve the well-being of patients experiencing the impact of these tumors. Toward achieving this goal,
we investigated the relationship between compromised COMT expression and cancer cell proliferation.
COMT knockdown (COMT-si) cells were established through lipofection using a validated COMT
specific siRNA. Two cell lines of different genotypes were selected to be studied from each cancer type.
For BC the ER-α positive cell line MCF7 was used along with the triple negative cell line MDA-MB231
that lacks ER-α expression. For PC we used stage-IV androgen-independent cell line PC-3 which
expresses ER-α and DU-145 cell line which is deficient in ER-α expression. The cell proliferation of
COMT-si cells that express low COMT levels was assessed and compared to scrambled control groups.
Interestingly, MCF-7 cell proliferation was significantly boosted by 28% (P<0.001) 24h after
COMT silencing. Furthermore, the cell proliferation was significantly enhanced by 54 and 41.5%
(P<0.01) in MCF-7 and PC-3 COMT-si cells 48h post-silencing compared to the scrambled control
counterparts. The proliferative response in MCF-7 breast cancer cells after COMT knockdown was
evident at an earlier time point (24h) compared to PC-3 prostate cancer cells. In the next step, we wanted
to assess if the enhanced cell proliferation is related to the presence of E2 in the medium or is it solely due
to reduced COMT expression. Therefore, the cell proliferation was assessed and compared for cells
cultured in the presence of E2 (0.01µM) and cells cultured in E2-free conditions using phenol red-free
medium supplemented with charcoal-stripped FBS. The data showed a significant increase in cell
proliferation of COMT-si MCF-7 and PC-3 cells by 62% and 44.8% (P<0.001) compared to scrambled
controls cultured in the same E2 exposure conditions. However, there was no significant difference in the
proliferation between COMT-si cells cultured in the presence of E2 and cells cultured in the absence of
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E2. Collectively, these results indicated that the proliferative effect of COMT silencing in the
aforementioned cell types is independent on the presence of E2 in the medium and is triggered by the
reduction in COMT gene expression. These data highlight a possible role of COMT as a tumor suppressor
enzyme in BC and PC. The proliferative effect of COMT gene knockdown was previously reported for
leiomyoma and colon cancer cells [20,32]. In the current study, we also tested the effect of COMT
inhibitor entacapone (ENT) on cell proliferation. The proliferation of PC-3 cells was significantly
enhanced by ENT alone or combined with 2OHE2, while MCF-7 cells were not affected. This differential
effect of ENT can be justified based on the difference in the basal levels of COMT in these cell lines,
where it is abundant at significantly higher levels in MCF-7 compared to PC-3 cells. Therefore, the same
concentration of ENT used in PC-3 might be not sufficient for the enzyme inhibition in MCF-7.
On the other hand, the proliferation of MDA-MB231 cells was slightly reduced (9%) 48 h after COMT
knockdown compared to scrambled control. No change in cell proliferation was observed in DU-145 cells
after COMT silencing. The baseline protein expression of S-COMT in MCF-7 and PC-3 cells was noticed
to be higher compared to MDA-MB231 and DU-145 cells. This can partly explain the differential
proliferative response after COMT silencing in these cell lines. The high level of S-COMT in MCF-7
breast cancer cells was previously reported [33]. Additionally, previous reports showed higher level of S-
COMT expression in endometrial cancer tissues compared to the normal surrounding endometrium [34].
Moreover, the levels of both S-COMT and MB-COMT were shown to be significantly higher in breast
cancer tissues compared to the adjacent nonmalignant mammary tissues [35,36]. Treatment of hamsters
with estradiol resulted in increased translocation of S-COMT to the nuclei, where catechol estrogens-
mediated DNA damage occurs [37]. Collectively, these studies support the notion that COMT expression
and nuclear translocation might be a compensatory mechanism to minimize the oxidative DNA damage
by the catechol estrogen metabolites [9].
Our data indicated that the proliferative effect of COMT knockdown is exclusive to cells
expressing ER-α. Therefore, we evaluated the effect of COMT silencing on ER-α activation in ER-α
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positive cells MCF-7 and PC-3 via assessing ER-α S167 phosphorylation (ER-α p-S167). Silencing
COMT significantly boosted ER-α p-S167 compared to scrambled control. ER-α phosphorylation is
known to increase the receptor transcriptional activity and hence increasing ER-α-mediated effects such
as induction of cell proliferation [38]. Our results partly explain the controversial results of
epidemiological studies linking COMT polymorphisms to PC risk [23-28], where the subgroup analyses
in most of PC studies did not include the ER-α status of PC. We also investigated the effect of the COMT
product 2-methoxyestradiol (2ME) on the cell viability. 2ME was cytotoxic to all the tested cell lines and
was more potent in BC cells. This result is not mirroring the differential effect of COMT-silencing in ER-
α positive and ER-α negative cell lines. This is because the anti-proliferative effects of 2ME are
independent of ER-α [39].
Cell proliferation is negatively regulated by cyclin-dependent kinase inhibitors such as p27kip1 and
p21cip1[40,41]. Therefore, we evaluated the effect of COMT silencing on the expression of p27kip1and
p21cip1genes. For PC cells, the expression of both p27kip1 and p21cip1 genes were significantly
downregulated in PC-3 COMT-si cells compared to scrambled control. Such changes explain the
enhanced cell proliferation of PC-3 COMT-si cells and gains support from previous data indicating that
reduced expression of both p27kip1 and p21cip1genes is linked to aggressive PC phenotype [42]. For BC
cells, our data showed that MCF-7 and MDA-MB231 cells expressing low COMT-levels showed
downregulation of p27kip1 mRNA transcripts. The reduction of p27kip1 levels was more prominent in MCF-
7 cells versus MDA-MB231 (5-fold vs 1.6-fold) compared to the scrambled control counterparts.
Moreover, the levels of p27kip1 transcripts were significantly diminished in MCF-7 by 3 folds compared to
MDA-MB231 COMT-si cells. The evidenced proliferative effect of COMT silencing in MCF-7 cells is
partly due to the marked decline of p27kip1 levels. Reduced p27kip1 expression was reported as an
independent factor for poor prognosis in patients suffering from localized BC or PC[43,44].
Activation of the pro-inflammatory transcription factor NF-κB is implicated in the progression
and resistance to therapy in both BC and PC due to its ability to regulate several anti-apoptotic,
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proliferation, metastasis and angiogenesis genes [45-48]. Herein we evaluated the basal NF-κB(p65) S536
phosphorylation (p-NF-κB) status compared to COMT expression level in wild-type (WT) BC and PC
cells. The basal p-NF-κB level in MDA-MB231 was significantly higher than MCF-7. This is in line with
previous studies indicating higher levels of NF-κB activity in triple negative BC cells [49]. Regarding
COMT expression, MCF-7 cells showed a significantly higher level compared to MDA-MB231 cells. An
inverse relationship was observed between COMT expression and p-NF-κB in BC cells. Both PC-3 and
DU-145 cells showed comparable basal levels of p-NF-κB. The constitutive activation of NF-κB was
reported in androgen-independent PC cell lines PC-3 and DU145 [48]. There was no significant
difference in the basal COMT levels in both PC cells. In the subsequent step, we wanted to study the
effect of COMT silencing on NF-κB activation. For BC cells, COMT silencing in MCF-7 cells
significantly increased p-NF-κB compared to scrambled control, while no change was noticed in MDA-
MB231 cells. For PC cells, p-NF-κB was significantly boosted in PC-3 cells but not DU-145 cells after
COMT silencing. These findings supported that the proliferative effect of COMT silencing in MCF-7 and
PC-3 cells is partly due to increased p-NF-κB. The lack of changes in p-NF-κB in MDA-MB231 and DU-
145 is consistent with the absence of a proliferative response after COMT silencing in these cells and
support that COMT expression in these cell lines does not affect p-NF-κB.
Aromatase(CYP19A1) enzyme is responsible for the in situ synthesis of E2 in the breast and
prostate cells[2,50]. The expression of CYP19A1 is higher in BC and PC tissues and was associated with
poor response to docetaxel (DOC) in PC patients [51]. Inhibition of CYP19A1 enzyme is an important
strategy for the management of hormone receptor-positive BC [52]. Our data showed that CYP19A1
expression was significantly up-regulated in COMT knockdown cells compared to scrambled control. The
level of CYP19A1 is down-regulated in response to the increase in the level of 2-methoxyestradiol
(COMT product) as a negative feedback [53]. Therefore, it is expected that COMT silencing will
subsequently reduce the level of this COMT product and hence decrease the negative feedback on
CYP19A1 expression. It is noteworthy that the induction of CYP19A1 expression by COMT silencing in
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PC cells was less prominent compared to that observed in BC cell lines. This is the first study to show the
relationship between COMT expression and CYP19A1 expression in BC and PC. The increased CYP19A1
expression in COMT-si cells is consistent with the evidenced proliferative response of COMT silencing
in the E2 responsive cell lines MCF-7 and PC-3. It indicates that COMT silencing can increase the in situ
E2 production by the virtue of boosting CYP19A1 expression in these cells which will consequently
activate E2-mediated proliferation.
Catechol E2 substrates such as 2-OHE2 and 4-OHE2 if not detoxified by COMT they have the
ability to form quinine intermediates that were reported to form adducts with DOC and interfere with
DOC binding to its main cellular target tubulin [54]. Herein, we investigated the effect of the catechol
substrate 2-OHE2 on DOC IC50. There was a marked reduction in DOC potency in all the tested cell lines
as evidenced from the significant increase in the IC50 by 1.9-2.3 folds compared to DOC-alone treatments.
The effect of COMT substrates on the responsiveness of the cells to DOC is independent on ER-α status
of the cells because it is mainly due to the chemical interaction between 2-OHE2 and DOC and
interference with DOC-tubulin interaction [54].
In conclusion, the data generated from our study provides a better understanding of the effect of
COMT on critical signaling pathways involved in the development and progression of BC and PC (Fig. 6)
This would ultimately contribute to the development of effective treatment modalities for the disease. Our
data showed that COMT silencing enhances the cell proliferation of ER-α positive cancer cells as MCF-7
and PC-3. This finding suggests that COMT gene plays a tumor suppressor role in such tumors which
opens the door for future studies to validate COMT expression as a novel biomarker for the prediction of
cancer aggressiveness and treatment efficacy.
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Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgments
MFT is supported by Fulbright scholarship (FY2015/2016) and L’Oréal-UNESCO for Women in Science
Levant and Egypt fellowship-2016. ACFN, received support from Brazil Science Without Borders
Program. The authors would like to thank Dr. Sherif Z. Abdel-Rahman, The University of Texas Medical
Branch (UTMB), Galveston, Texas, USA, for his guidance and support.
Authors’ contributions
MFT and HAO generated the work idea, designed and performed the experiments, data analyses,
contributed with materials and analysis tools and wrote the manuscript. FH contributed in experiments
and data analysis. ACFN, helped in some preliminary Western blot experiments. AMN contributed with
materials, analysis tools and bench space. All authors approved the final manuscript.
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Legends to Figures
Fig. 1. The effect of COMT silencing on cancer cell proliferation in BC and PC cell lines.
(A) Western blot analysis of the differential expression levels of COMT in control siRNA and
COMT siRNA. Cell lysates were collected and processed for western blotting 48h after siRNA
transfection. (B) Bar charts for the ratio of COMT expression normalized to β-actin. (C) The
effect of COMT knockdown on cancer cell proliferation of MCF-7, MDA-MB231, PC-3 and
DU-145. Cell viability was assessed by MTT assays at 24 and 48h after siRNA transfection. All
data are depicted as mean ± S.D(n=6) of three independent experiments. * indicates significant
difference vs the corresponding scrambled control at P<0.05, ** at P<0.01, *** at P<0.001.
Fig.2. Estrogen receptor-positive cancer cells and COMT silencing or COMT inhibition.
(A) The proliferative effect of COMT silencing in MCF-7 and PC-3 cells in the presence of E2
in the culture medium. (B)Entacapone effect on cancer cell proliferation. Cell viability was
assessed via MTT assay at 48h post-silencing or ENT exposure. (C) Western blot analysis for
ERα and ERα (PS167). (D) Bar charts for the ratio of ERα (PS167)/ERα. All data are depicted as
mean ± S.D(n=6) of three independent experiments. ** indicates significant difference vs the
corresponding scrambled control at P<0.01, *** at P<0.001.
Fig.3. The effect of COMT product 2ME2 on cancer cell proliferation. (A) The anti-
proliferative effects of 2ME2 in MCF-7 and MDA-MB231 cell lines. (B)The anti-proliferative
effects of 2ME2 in PC-3 and DU-145 cell lines. (C) The effect of COMT substrate 2-OHE2 on
DOC potency in the tested cell lines. Cell viability was assessed by MTT assays at 48h of
exposure. All data are depicted as mean ± S.D (n=6) of three independent experiments. *
indicates significant difference vs the corresponding control at P<0.05, ** at P<0.01, *** at
P<0.001.
Fig.4. Effect of COMT silencing on the gene expression of cyclin-dependent kinase
inhibitors p27kip1(A), p21cip1(B) and aromatase (CYP19A1) (C)in BC (i)and PC(ii) cells. All
data are depicted as mean ± S.D (n=4) of three independent experiments. The fold changes
displayed are normalized to β-actin expression. * indicates significant difference vs the
corresponding scrambled control at P<0.05, ** at P<0.01, *** at P<0.001.
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Fig.5. The relationship between COMT expression and NF-κB phosphorylation. (A)
Differential COMT protein levels in wild-type (WT) PC-3, DU-145, MCF-7 and MDA-MB231
cell lines. (B) Basal levels of NF-κB phosphorylation in the different tested wild-type cell lines.
(C) The effect of COMT silencing on NF-κB phosphorylation in MCF-7 and MDA-MB231
cells. (D)The effect of COMT silencing on NF-κB phosphorylation in PC-3 and DU-145 cells.
All data are depicted as mean ± S.D (n=4) of three independent experiments. $indicates
significant difference vs WT PC-3at P<0.05; #indicates significant difference vs WT DU-145at
P<0.05; @indicates significant difference vs WT MCF-7 at P<0.05; * indicates significant
difference vs the corresponding scrambled control at P<0.05, ** at P<0.01, *** at P<0.001.
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Highlights
● COMT silencing enhanced the cell proliferation of ER-α positive cancer cells
● The decreased activity of COMT enhanced ER-α and NF-kB signaling pathways
● The expression of p27kip1
and aromatase were COMT-dependent
● COMT enzyme plays a tumor suppressor role in hormone receptor-positive tumors
● Cancer aggressiveness can be predicted through COMT expression levels