Original Article Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

Received: February 18, 2014 Accepted after revision: May 3, 2014 Published online: June 18, 2014

Green Tea Extract Inhibition of Human Leiomyoma Cell Proliferation Is Mediated via Catechol-O-Methyltransferase Dong Zhang a Veera Rajaratnam a Omar Al-Hendy a Sunil Halder a, b Ayman Al-Hendy a, b a

Center for Women’s Health Research and b Department of Obstetrics and Gynecology, Meharry Medical College, Nashville, Tenn., USA

Key Words Epigallocatechin gallate · Green tea · Leiomyoma · Uterine fibroids · Proliferation inhibition · Catechol-o-methyltransferase

Abstract Background/Aims: To investigate the inhibitory effect of green tea extract, epigallocatechin gallate (EGCG), on wildtype human leiomyoma (WT-HuLM) cells and its potential action via catechol-o-methyltransferase (COMT) activity. Methods: Cell proliferation of WT-HuLM and COMT gene-silenced HuLM (COMT-shRNA-HuLM) cells treated with 0 or 100 μM EGCG for 7 days was measured using the MTT method. Total RNA and protein were extracted from cells treated with 0 or 100 μM of EGCG for 48 h. Gene expression profiling was performed using Human Signal Transduction PathwayFinder. Proliferation cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (Cdk4) and COMT protein levels were detected by Western blot analyses. COMT enzyme activity was evaluated by HPLC. Results: EGCG-treated WT-HuLM cells showed significantly decreased COMT expression (p < 0.001) and enzyme activity (p < 0.05) compared to untreated WTHuLM cells, while COMT-shRNA-HuLM cells showed no significant change. At 100 μM of EGCG, survival of WT-HuLM cells was significantly lower (p < 0.05) compared to COMT-

© 2014 S. Karger AG, Basel 0378–7346/14/0782–0109$39.50/0 E-Mail [email protected] www.karger.com/goi

shRNA-HuLM cells. EGCG treatment modulated multiple signaling pathways in WT-HuLM compared to untreated control, while changes were minimal or reversed in COMTshRNA-HuLM cells. EGCG significantly decreased PCNA, Cdk4 and soluble COMT protein levels (p < 0.001) in WTHuLM, but not in COMT-shRNA-HuLM cells. Conclusions: The antiproliferative and gene-modulating effects of EGCG on HuLM cells are mediated, at least partially, via its effect on COMT expression and enzyme activity. © 2014 S. Karger AG, Basel

Introduction

Uterine fibroids (leiomyomas), benign tumors of the uterine wall, are a major women’s health problem. Leiomyomas are clinically symptomatic in at least 25% of women of reproductive age [1]. We and others have reported on the higher incidence of symptomatic leiomyomas in African American women compared to white women [2–5]. The basis of this marked health disparity is not very clear. Potential etiologies of uterine fibroids including correlation to metabolic syndrome and genetic factors have been investigated [6–8]. We have recently identified two genetic polymorphisms in estrogen receptor-α and catechol-o-methyltransferase (COMT) Ayman Al-Hendy, MD, PhD Department of Obstetrics and Gynecology, Center for Women’s Health Research Meharry Medical College, Hubbard Hospital, Room 5131, 1005 Dr. D.B. Todd Boulevard, Nashville, TN 37208 (USA) E-Mail ahendy @ mmc.edu

genes that are associated with uterine fibroids and are more prevalent in African Americans [9, 10]. We have demonstrated a higher COMT expression in leiomyoma compared to adjacent normal myometrium and presented a mechanistic model in which COMT is a key factor in fibroid tumorigenesis [10]. Available treatment options for fibroids including surgery and medication are effective, but with limitations [11]. Novel gene therapy using modified adenovirus delivery systems is being developed and evaluated [12]. We have recently reported a remarkable decrease in fibroid volume utilizing adenovirus-mediated gene therapy on a rat model [13]. Women of color who are known to be high users of alternative medicine approaches will benefit from safe and simple nonsurgical strategies for the treatment of uterine fibroids. Although medications that manipulate concentrations of steroid hormones are effective, side effects limit long-term use. An orally administered medication that can effectively and safely reduce or eliminate fibroid-associated symptoms will certainly be a welcome addition and will have a major favorable impact on women’s health. Tea is the most popular beverage in the world, next to water, and consists of three major commercial types: green, black and oolong tea. Green tea is processed in a manner to prevent the oxidation of green tea-leaf polyphenols, which contain five major catechins, including catechin, epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate (EGCG) [14]. These compounds show diverse chemical and biological activities and regulate the expression of a number of cyclins, oncogenes and tumor suppressor genes [15]. EGCG comprises >40% of the total polyphenolic mixture of green tea catechins and possesses antioxidant, anti-inflammation and antitumor capacity [16]. Previous studies have shown that EGCG inhibited the growth of various human cancer cells such as epidermoid carcinoma cells [17], hepatoma cells [15], prostate carcinoma cells [18] and breast cancer cells [19] by inhibiting proliferation or inducing apoptosis of tumor cells [20–22]. Notably, several reports demonstrate the ability of EGCG to selectively inhibit the enzyme activity of human or rodent liver cytosolic COMT [23–26]. Recent studies from our laboratory reported that the COMT gene is highly expressed in human leiomyoma (HuLM) tissue compared to adjacent normal myometrium and is a central step in leiomyoma tumorigenesis [2, 10]. We have found that EGCG could effectively inhibit the proliferation of HuLM cells in vitro [27] and in a nude mouse model of fibroids [28]. In this study, we wanted to 110

Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

evaluate the potential mechanism of EGCG as an inhibitory agent on uterine fibroid tumorigenesis and, specifically, whether any effect of EGCG is mediated via COMT. Materials and Methods Chemicals, Reagents and DNA Smooth muscle cell basal medium was purchased from Lonza (Walkersville, Md., USA). (–)- EGCG, shRNA plasmid for human COMT, HPLC grade methanol and water were purchased from Sigma (St. Louis, Mo., USA). An RNease kit was purchased from Qiagen (Valencia, Calif., USA). An RT First Strand kit, SuperArray RT qPCR master mix and human signal PathwayFinder kit were purchased from SuperArray Bioscience Corporation (Frederick, Md., USA). All other chemicals and biochemicals were available from commercial resources. Cell Culture The human uterine leiomyoma (HuLM) cell line was a gift from Dr. D. Dixon (National Institute of Environmental Health Sciences, Research Triangle Park, N.C., USA). To specifically and selectively downregulate the expression of the COMT gene, HuLM cells were transferred with the plasmid containing the shRNA of COMT using the electroporation method as described earlier [29]. Colonies of stable transferred cells were selected using puromycin (2.5 ug/ml; Sigma). shRNA-COMT-HuLM cells demonstrate a marked decrease in COMT protein expression and the stable clone with the lowest expression of soluble COMT (S-COMT) was used [30]. The wild-type HuLM cells (WT-HuLM) and the shRNACOMT-HuLM cells were cultured [30] in SmBM medium supplied with 5% FBS, 0.1% insulin, 0.2% hFGF-b, 0.1% GA-1000 and 0.1% hEGF (Lonza). The cells were maintained at 37 ° C in a humidified atmosphere of 5% CO2 and 95% air.  

 

Cell Proliferation Assay The effect of EGCG on the proliferation of both WT-HuLM and shRNA-COMT-HuLM cells was measured using the MTT method as we have described previously [31]. Briefly, HuLM cells (2 × 103 cells/well) were plated in 96-well plates. The cells were allowed to attach overnight at 37 ° C and were then treated with the indicated concentrations of EGCG (0 or 100 μM). The survival rate of both WT-HuLM and shRNA-COMT-HuLM cells was measured on days 1, 3, 5 and 7 after EGCG treatment. The effect of EGCG on growth inhibition was assessed as the percentage of optical density value in which cells with no EGCG treatment were taken as 100% viable.  

 

RNA Isolation and Assay of Leiomyoma Tumor Signal Transduction Pathways To explore the expression of representative genes, both WTHuLM and shRNA-COMT-HuLM cells were treated with or without 100 μM EGCG for 48 h. Total RNA was isolated and cDNA produced by reverse transcription PCR using the RT First Strand kit. Gene expression profiling was performed using signal transduction PathwayFinder PCR arrays (PAHS-014; SuperArray Bioscience Corp.) which contain 84 key genes representing 18 different signal transduction pathways. The PCR reactions were carried out in a final volume of 50 μl. The standard cycling condition was

Zhang/Rajaratnam/Al-Hendy/Halder/ Al-Hendy

50 ° C for 2 min and 90 ° C for 10 min, followed by 40 cycles of 95 ° C for 15 s and 60 ° C for 1 min. Raw gene array expression data was normalized against five reference genes included in each array through Excel-based analysis (SuperArray Bioscience Corp.). Fold units were calculated using the comparative threshold cycle method, dividing the expression fold changes of the candidate genes by the expression fold changes of the reference genes.  

 

 

 

 

 

1.2

 

Western Blot Analysis Equivalent amounts of protein extracts from both WT-HuLM and shRNA-COMT-HuLM cells, treated with or without 100 μM EGCG for 48 h, were separated by NuPAGE Novex 10% Bis-Tris Gel (Invitrogen Life Technologies, Carlsbad, Calif., USA) under a reducing condition using 200 V for 50 min. The proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Billerica, Mass., USA) using the XCell II Blot Module (Invitrogen). After blocking nonspecific binding sites by incubation for 1 h with PBS containing 5% fat-free milk and 0.1% Tween 20, the membranes were incubated with the primary antibodies overnight at 4 ° C. Immunological detection was performed using the following primary antibodies: human proliferation cell nuclear antigen (PCNA, 1:500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), cyclin-dependent kinase 4 (Cdk4, 1: 1000 dilution; Sigma) and COMT (1: 100,000 dilution; Chemicon, Temecula, Calif., USA). The membranes were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (diluted 1: 5,000 with blocking buffer). The antigen-antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Bioscience, Piscataway, N.J., USA). The membranes were reprobed with a monoclonal antibody raised against β-actin (diluted 1:5,000; Sigma) as an internal control for protein loading and normalization between samples. Films exposed to blots were scanned and optical densities of the band signals were quantified.  

1.0

* 0.6

 

 

*

0.4 0.2 0

Day 1

Day 3

Day 5

Day 7

EGCG treatment

 

Effects of EGCG on COMT Enzyme Activity To evaluate COMT enzyme activity, both WT-HuLM and shRNA-COMT-HuLM cells were treated with or without 100 μM EGCG for 48 h. Protein was extracted using lysis buffer as we have described previously [32]. Protein concentration was measured using Pierce BCA Protein Assay kit (Pierce Biotechnology, Rockford, Ill., USA). The preparation of COMT enzyme HPLC assay consists of 100 μl cell lysate, 40 μl sodium phosphate (0.1 M, pH 7.8), 10 μl magnesium chloride (40 mM), 10 μl S-adenosyl-L-methionine (2 mM), 30 μl HPLC grade water and 10 μl 3,4 DOBA (500 μM) to give a final volume of 200 μl. The enzyme reaction was incubated for 60 min at 37 ° C. The reaction was stopped by adding 20 μl HCl (2 M). After centrifuging at 14,000 rpm for 10 min, the supernatants were analyzed by HPLC using Waters Scanning Fluorescence Detector (Waters Corp., Milford, Mass., USA). A standard curve was generated from the average of 4-hydroxy-3-methoxybenzoic acid (4H3MBA) measured during the assay. The final COMT enzyme activity was expressed on a pictogram of 4H3MBA per minute per milligram protein basis [33].

Untreated WT-HuLM shRNA WT-HuLM

0.8 Survival (%)

 

Fig. 1. Reduced sensitivity to EGCG-induced inhibition of HuLM cell proliferation in shRNA-COMT-HuLM cells. The normalized survival percent of WT-HuLM cells at 100 μM EGCG was significantly decreased compared to shRNA-COMT-HuLM cells at day 5 and 7 (p < 0.05). Data represent means ± SD of the results of 3 experiments.

Results

Antiproliferative Effect of EGCG on HuLM Cells Is Abolished in shRNA-COMT-HuLM Cells WT-HuLM and shRNA-COMT-HuLM cells displayed fibroblast-like morphology and there were no distinct morphological changes between them under lowdose EGCG treatment. To evaluate the antiproliferative effect of EGCG on WT-HuLM versus shRNA-COMTHuLM cells, the cells were treated with or without 100 μM EGCG for 7 days. Cell proliferation was evaluated using an MTT assay. A gradual decrease in survival rate was observed in both WT-HuLM and shRNA-COMT-HuLM cells throughout the 7-day culture period compared to their untreated control. The inhibitory effect of 100 μM EGCG on WT-HuLM was significantly greater compared to its effect on shRNA-COMT-HuLM cells or untreated WT-HuLM cells at day 5 and 7 (p < 0.05; fig. 1).

Statistical Analysis Data were expressed as the mean ± SD of the values obtained from three replicates. Statistical significance was determined using one-way ANOVA. A difference with p < 0.05 was considered statistically significant.

EGCG-Induced Gene Expression Modulation Affecting Multiple Signaling Pathways Is Diminished in shRNA-COMT-HuLM Cells For the gene expression analysis, total mRNA was isolated from WT-HuLM and shRNA-COMT-HuLM cells treated with a dose of 100 μM EGCG for 48 h. The basis

EGCG Inhibits COMT in Human Leiomyoma Cells

Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

111

20

WT-HuLM COMT-shRNA-HuLM

15

5 0 –5

for the choice of this concentration was cell growth studies. Figure 2 shows genes exhibiting at least a 2-fold change (up- or downregulation) in expression after EGCG treatment. The analysis of the signal transduction pathway revealed that 100 μM EGCG treatment significantly inhibited the expression of BCL2, BCL2A1 and CCND1, while enhancing the expression of p21 WAF1, p15, p53 binding protein (MDM2) and tumor protein p53 inducible protein 3 (TP53I3; fig. 2; table 1) in WTHuLM cells. EGCG dramatically enhanced expression of bone morphogenetic protein 2 (BMP2; fig. 2, gene No. 4) and heat shock factor 1 (HSF1; fig. 2, gene No. 15) at 14.1and 16.2-fold higher than control, respectively. Similar trends were observed in TGF-β (CDKN2B) and p53 (BAX, CDKN1A, MDM2 and TP53I3) pathways (fig. 2; table  1). Interestingly, shRNA-COMT-HuLM cells showed reversal of most of EGCG-induced modulation of gene expression observed with WT-HuLM cells (fig. 2). EGCG treatment of shRNA-COMT-HuLM cells showed significant upregulation of BAX, BCL2 (fig. 2, gene No. 1 and 2) and Wnt pathway downstream genes (CCND1 and WISP1; fig. 2, genes No. 7 and 25), while stress pathway genes HSF1 and HSPB1 were downregulated (fig. 2, gene No. 15 and 16). It is apparent that EGCG treatment of WT-HuLM cells has affected the expression of at least 25 genes that belong to multiple signal pathways (table 1). 112

Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

WISP1

VEGFA

VCAM1

PTGS2

TP5313

PRKCA

LEF1

MDM2

ICAM1

HSF1

HSPB1

GYS1

GREB1

FOS

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 GADD45A

8

CDKN2B

7

CDK2

6

CDKN1A

5

CCL20

4

CCND1

3

BMP2

2

BRCA1

1

BCL2A1

–15

BAX

–10

BCL2

Fig. 2. EGCG-induced gene expression modulation observed in HuLM cells is diminished or reversed in shRNA-COMTHuLM cells. The fold changes in the expression of representative genes are shown for WT-HuLM and shRNA-COMTHuLM cells after treatment with 100 μM EGCG for 48 h. Genes exhibiting at least a 2-fold change in expression (up- or downregulation) after EGCG treatment are shown. The shRNA-COMT-HuLM cells show reversal of most of EGCG-induced modulation of gene expression seen with WT-HuLM cells.

Fold changes

10

Gene symbol

Additionally, COMT-silenced shRNA-COMT-HuLM cells show diminished or reversed EGCG-induced changes observed with WT-HuLM cells in all these 25 genes (fig. 2). Inhibition of Proliferation-Associated Proteins in HuLM Cells by EGCG Treatment Is Abolished in shRNA-COMT-HuLM Cells To further confirm the effect of EGCG on signal transduction in HuLM cells, WT-HuLM and shRNA-COMTHuLM cells cultured in the presence or absence 100 μM of EGCG for 48 h were assessed by Western blotting for PCNA and Cdk4 expression. As shown in figure 3, EGCG treatment significantly decreased the expression of PCNA and Cdk4 in WT-HuLM cells compared to untreated control cells (p  < 0.001). Additionally, shRNA-COMTHuLM cells treated with EGCG demonstrated no significant reduction in PCNA and CDK4 expression. The expression levels in EGCG-treated cells remained similar to those of untreated shRNA-COMT-HuLM cells (fig. 3). EGCG Inhibits COMT Enzyme Activity and Protein Expression in HuLM Cells To explore the potential effect of EGCG on COMT enzyme activity and protein expression levels in leiomyoma cells, WT-HuLM and shRNA-COMT-HuLM cells were Zhang/Rajaratnam/Al-Hendy/Halder/ Al-Hendy

Table 1. EGCG-responsive genes (symbols and description) in HuLM cells sensitive to COMT protein expression and activity status

Gene number (as in fig. 2)

Symbol

Description

Related signal transduction pathway

BAX BCL2

BCL2-associated X protein B-cell CLL/lymphoma 2

3 4 5 6 7 8 9 10

BCL2A1 BMP2 BRCA1 CCL20 CCND1 CDK2 CDKN1A CDKN2B

11

FOS

12 13 14 15 16 17

GADD45A GREB1 GYS1 HSF1 HSPB1 ICAM1

18 19

LEF1 MDM2

20 21

PRKCA PTGS2

22 23 24 25

TP53I3 VCAM1 VEGFA WISP1

BCL2-related protein A1 bone morphogenetic protein 2 breast cancer 1, early onset chemokine (C-C motif) ligand 20 cyclin D1 cyclin-dependent kinase 2 cyclin-dependent kinase inhibitor 1A (p21, Cip1) cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) V-fos FBJ murine osteosarcoma viral oncogene homolog growth arrest and DNA damage-inducible, alpha GREB1 protein glycogen synthase 1 (muscle) heat shock transcription factor 1 heat shock 27 kDa protein 1 intercellular adhesion molecule 1 (CD54), human rhinovirus receptor lymphoid enhancer-binding factor 1 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) protein kinase C, alpha prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) tumor protein p53 inducible protein 3 vascular cell adhesion molecule 1 vascular endothelial growth factor A WNT1 inducible signaling pathway protein 1

p53 pathway survival (PI3 Kinase/AKT, Jak/Src), estrogen, phospholipase C pathway survival (NFκB) pathway hedgehog pathway estrogen pathway NFκB pathway Wnt, survival (PI3 Kinase/AKT) pathway androgen pathway p53, TGF-β, androgen pathway TGF-β pathway

1 2

CREB, stress, mitogenic, calcium and protein kinase C, phospholipase C pathway p53 pathway estrogen pathway insulin pathway stress pathway stress pathway NFκB, phospholipase C pathway Wnt pathway p53 pathway calcium and protein kinase C pathway phospholipase C pathway p53 pathway NFκB, phospholipase C, LDL pathway Wnt pathway Wnt pathway

compared to expression levels in WT-HuLM cells (p  < 0.001). Treatment with 100 μM EGCG did not decrease S-COMT expression in shRNA-COMT-HuLM cells (fig. 4b).

treated with or without 100 μM EGCG for 48 h followed by cell lysis and protein extraction for COMT enzyme activity or Western blot analysis. The methylation of the standard substrate 4H3MBA was determined by HPLC. As expected, the COMT enzyme activities were 1,054.60 ± 414.16 and 139.75 ± 12.40 pg/min/mg in untreated WTHuLM and shRNA-COMT-HuLM cells, respectively (p < 0.05). EGCG treatment significantly decreased COMT enzyme activity (the yield of 4H3MBA) in WT-HuLM cells (p < 0.05) while there were no significant effects on shRNA-COMT-HuLM cells (fig. 4a). The inhibitory effect of EGCG on COMT enzyme expression levels in WTHuLM was further confirmed by Western blot analysis. Expression of S-COMT was significantly decreased in WT-HuLM cells treated with 100 μM EGCG compared to untreated control (p < 0.001). As expected, S-COMT expression was significantly low in shRNA-COMT-HuLM

Several studies have shown the beneficial effects of green tea against various diseases including many types of cancers and diabetes [33–39]. In a randomized controlled clinical pilot study, we have recently demonstrated the effectiveness of EGCG treatment on symptomatic uterine fibroids [40]. We have also shown previously that EGCG could effectively inhibit the proliferation of HuLM cells in vitro [27] and in vivo in a nude mouse model of uterine fibroids [28]. In this study, we have

EGCG Inhibits COMT in Human Leiomyoma Cells

Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

Discussion

113

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0.2 0

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1,400

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p < 0.05

WT-HuLM PCNA

CDK4

:7+X/0

PCNA

CDK4

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3 Fig. 3. Inhibition of HuLM cell proliferation-associated proteins by EGCG is abolished in shRNA-COMT-HuLM cells. a Effects of

shown that the inhibitory effect of EGCG on uterine fibroid tumorigenesis is mediated, at least in part, via COMT. The results from the current study confirm the reduced expression and low enzyme activity of COMT in shRNA-COMT-HuLM cells, which evidently render them less sensitive to EGCG treatment compared to WTHuLM cells with normal COMT activity. 114

Gynecol Obstet Invest 2014;78:109–118 DOI: 10.1159/000363410

DŽ$FWLQ 0

EGCG (μM) 1.4 &207SURWHLQOHYHOV QRUPDOL]HGWRDŽDFWLQ

EGCG on the PCNA and CDK4 protein levels in WT-HuLM and shRNA-COMT-HuLM cells were detected by Western blot. b β-Actin reprobing was performed as a control for protein (abundance) loading. The protein levels of PCNA and CDK4 were significantly downregulated in WT-HuLM cells treated with 100 μM of EGCG for 48 h compared to those of shRNA-COMT-HuLM cells (p < 0.001). Representative Western blots of PCNA, CDK4 and β-actin as detected by chemiluminescence are shown. Data represent means ± SD of the results of 3 experiments. Fig. 4. EGCG treatment effect on COMT enzyme activity and expression levels in WT-HuLM and shRNA-COMT-HuLM cells. Treatment of WT-HuLM cells with 100 μM EGCG for 48 h significantly inhibited COMT activity (the yield of 4H3MBA) as detected by HPLC analysis (p < 0.05; a) and also decreased the expression levels of S-COMT protein (p  < 0.001; b) compared to untreated control, while no similar effects were observed in shRNA-COMT-HuLM cells. In shRNA-COMT-HuLM cells, the COMT activity levels (a) and the expression levels of S-COMT protein (b) were significantly lower than in WT-HuLM cells. Data represent means ± SD of the results of 3 experiments.

0

p < 0.001

1.2

p < 0.001

100 EGCG 0 μM 100 μM

1.0 0.8 0.6 0.4 0.2 0

b

100

MB-COMT

S-COMT

WT-HuLM

MB-COMT

S-COMT

&207VK51$+X/0 (*&*WUHDWPHQW

4

Effects of COMT Expression Modulation COMT is an ubiquitous intracellular enzyme encoded by a single gene with six exons, which has been mapped to chromosome 22q11.21 [41]. The COMT protein exists in two isoforms, resulting from two mRNA transcripts from two promoters (P1 and P2): a soluble form found in the cell cytoplasm (S-COMT; 221 aa) and a longer, membrane-bound form (MB-COMT; 271 aa) [42]. The MBZhang/Rajaratnam/Al-Hendy/Halder/ Al-Hendy

COMT is predominantly expressed in the brain, while SCOMT is predominantly expressed in blood and in other tissues such as the liver and kidney [43]. We have also demonstrated COMT expression in normal myometrium [44], leiomyoma [45], endometrium [46], fetal membranes [47], ovary [48] and endometrial cancer cells [49]. A common Val/Met substitution at codons 108 and 158 in the S-COMT and MB-COMT transcripts, respectively, has been reported to account for >95% of the variations of this enzyme activity in humans [50]. The general function of COMT is to eliminate the potentially active or toxic catechol structures of endogenous and exogenous compounds such as dopamine, norepinephrine or flavonoid. It also converts 2-hydroxyestradiol (an antiestrogen) to 2-methoxyestradiol (proestrogen) and creates a high estrogenic environment by the accumulation of 2-methoxyestrodiol [32]. A recent study has found that COMTdeficient mice displayed a preeclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol [51]. Our previous studies have revealed that high-activity COMT Val/Val genotype is associated with increased risk of uterine leiomyoma and is more common in African American women compared to Caucasian women, which provides a possible explanation for the higher prevalence of uterine leiomyoma among African American women [2, 9]. Thus, the formation of uterine leiomyoma, an estradiol-dependent benign tumor, may be modulated indirectly by COMT activity which in turn affects estrogen bioavailability. shRNA is a powerful tool to investigate the effects of specific genes in mammalian cells. In this study, we were able to render the HuLM cells nonresponsive or more resilient to EGCG treatment by silencing the expression of COMT genes and thus reducing the COMT activity in shRNA-COMT-HuLM cells.

In this study, we have confirmed by HPLC analysis the ability of EGCG to inhibit COMT activity in HuLM cells. The shRNA-COMT-HuLM cells in which COMT is silenced showed a reduced or opposite response compared to WT-HuLM cells when treated with EGCG, indicating that the effects of EGCG might be mediated in part by COMT. In WT-HuLM cells, EGCG treatment dramatically altered the expression of genes related to the survival pathway, stress pathway, NFκB pathway and phospholipase C pathway. These changes of multisignal pathways indicate that EGCG has substantial effects on cellular behavior and characteristics, which have been well documented [26, 55, 56]. Cell cycle regulation plays an essential role in cellular homeostasis and contributes to the determination of the fate of cells. The activation of complexes of cyclin D with CDKs is the first step leading to cell cycle entry. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis [57]. EGCG significantly inhibited the levels of PCNA and CDK4 protein expression in WT-HuLM cells, while mildly decreasing these proteins in shRNA-COMT-HuLM cells. These results are consistent with cell proliferation assay, which show that EGCG inhibited proliferation of WT-HuLM cells remarkably at day 5 and 7, while having virtually no effect on shRNACOMT-HuLM cells observed at the same time points and concentration.

Effects of EGCG Treatment Recent studies have found that EGCG, having a catechol structure like other compounds such as catechol estrogens and catechol-containing flavonoids, is both a substrate and a potent inhibitor of COMT [52]. An epidemiological study in which the ability of tea catechins to reduce the risk of breast cancer was strongest among persons who had low-activity COMT alleles [53] suggests that these individuals were less efficient in eliminating tea catechins and have derived the most benefit from these compounds. EGCG acts as a noncompetitive inhibitor by a tightly binding interaction with COMT [54]. These findings are consistent with our results which demonstrate the ability of EGCG to exert its effects on HuLM cells via inhibiting COMT activity.

EGCG-Responsive Genes in HuLM Cells Sensitive to COMT Expression and Activity The results of the current study confirm the ability of EGCG to inhibit proliferation, induce apoptosis and regulate the expression of multiple genes in HuLM cells [27, 28]. Notably, the shRNA-COMT-HuLM cells in which COMT expression and activity is minimal resulted in an opposite effect or no response of key pathway genes that responded to EGCG treatment of HuLM cells. EGCG treatment caused a 14.1-fold increase in the expression of the BMP2 gene in WT-HuLM cells but not in shRNACOMT-HuLM cells. The BMPs are members of the transforming growth factor-β (TGF-β) superfamily, which regulates cell differentiation, proliferation and apoptosis [58]. Similar to TGF-β, BMP2 exert its function as a tumor suppressor by inhibiting proliferation and inducing apoptosis. Our gene array data indicate that BMP2 may play a critical role in EGCG-induced inhibition of leiomyoma cells. We also found that HSF1 gene expression was significantly higher (16.2-fold) in EGCG-treated HuLM cells. However, COMT-silenced shRNA-COMTHuLM cells showed a decrease in HSF1 gene expression

EGCG Inhibits COMT in Human Leiomyoma Cells

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in response to EGCG treatment. The function of HSF1 is to regulate the transcription of heat-shock genes induced by stress. It has been demonstrated that overexpression of constitutively active HSF1 sensitizes HeLa cells to Fasmediated killing [59]. Therefore, the notion is that the induced expression of HSF1 could sensitize leiomyoma cells to the suppression effects of EGCG that may function through the Fas receptor. Thus, the inhibitory effect of EGCG through the induction of HSF1 may also possibly contribute to the therapeutic application of EGCG in the treatment of uterine leiomyoma. The BCL2, BCL2A1, CCND1 (cyclin D1) and other cell cycle proliferation pathway genes are known to be overexpressed in several cancers [60, 61]. The downregulation of BCL2 and BCL2A1 gene expression by EGCG treatment suggests that the altered expression of BCL2 family members might play a key role in the EGCG-mediated induction of apoptosis in HuLM cells. Also, positive expression of CCND1 – a downstream WNT/wingless signaling pathway gene – usually has a poor prognosis compared to negative expression in lung cancer, bladder cancer and head and neck squamous cell carcinoma [60]. CCND1 is also known to be regulated by estrogen and progesterone in breast cancer cells and overexpression of CCDN1 mimicked the estrogen effect [61, 62]. CCND1 downregulation by EGCG through COMT inhibition in this study is in line with estrogen regulation of leiomyoma cell proliferation. In addition to CCND1, the expression of WISP1 (WNT-1-induced secreted protein 1), another WNT pathway gene expression, was decreased by EGCG in WT-HuLM cells. Notably, EGCG treatment showed no inhibition of WISP1 expression in shRNA-COMTHuLM cells. WISP1 is also a member of the CCN family of growth factors and is a β-catenin-regulated gene that can contribute to tumorigenesis [63]. WISP1 is known to have oncogenic activities, and overexpression of WISP1 in normal rat kidney fibroblast cells has been shown to induce morphological transformation and accelerated cell proliferation [63, 64]. WISP1 (or CCN4) has been shown to be upregulated in colon cancer [64, 65]. Inhibition of WNT pathway genes by EGCG has also been observed in cancer cells [66]. WNT proteins are secreted glycoproteins that activate signal transduction pathways to control a wide variety of cellular processes such as determination of cell fate, proliferation, migration and apoptosis. Recently, several studies have demonstrated that the mediator complex subunit 12 (MED12) gene is frequently mutated in exon 2 in at least 70% of cases of uterine fibroids in a Finnish 116

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population, a North American population and in South African populations, while no mutation was found in adjacent normal myometrium [67, 68]. The MED12 gene is directly involved in the gene-specific transcription process and it plays an important role in the activation of WNT/β-catenin signaling. Uterine fibroids with MED12 gene mutations expressed significantly higher levels of the gene-encoding wingless-type MMTV integration site family member 4 (WNT4) [69]. Mutations in the MED12 gene are known to activate the WNT signaling pathway, which in turn activates β-catenin to further stimulate the downstream mammalian target of rapamycin (mTOR, a crucial master regulator of cell function), and are involved in tumor cell proliferation [70]. It would be interesting to elucidate in future work the role of COMT in the WNT/β-catenin pathway and downstream signaling targets in leiomyoma cell proliferation and fibroid pathogenesis. In this study, nonresponsiveness to EGCG or reversed response of the COMT-silenced shRNA-COMT cells compared to WT-HuLM cells clearly show that EGCG effects are mediated, at least in part, via modulating COMT expression and enzyme activity. Further preclinical and clinical studies are warranted to evaluate the key target pathway networks and the utility of EGCG as a potential simple nonsurgical alternative treatment for uterine leiomyomas. Acknowledgments We thank Dr. S.A. Salama, Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Tex., USA, for his generous gift of the COMT-shRNA construct utilized in this study. This work was supported by NIH/NICHD R01 HD046228, U54 HD044315 and RCMI grant G12 RR 03032.

Disclosure Statement The authors have no conflicts of interest to disclose.

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