Resveratrol inhibits proliferation of myometrial and leiomyoma cells and decreases extracellular matrix-associated protein expression

Journal of Functional Foods 23 (2016) 241–252

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Resveratrol inhibits proliferation of myometrial and leiomyoma cells and decreases extracellular matrix-associated protein expression Chi-Hao Wu a,1, Tzong-Ming Shieh b,1, Lin-Hung Wei c,d, Ting-Fang Cheng a, Hsin-Yuan Chen a, Tsui-Chin Huang e, Kai-Lee Wang a, Shih-Min Hsia a,* a

School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan Department of Dental Hygiene, College of Health Care, China Medical University, Taichung, Taiwan c Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan d Department of Obstetrics & Gynecology, National Taiwan University College of Medicine, Taipei, Taiwan e PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Uterine fibroids (leiomyomas) are the most common benign tumours in women during their

Received 30 November 2015

reproductive age. Hyperplasia of uterine smooth muscle cells and abnormal deposition of

Received in revised form 19

extracellular matrix (ECM) are responsible for the development of uterine fibroids. Studies

February 2016

have shown that food rich in phytochemicals can prevent or treat leiomyoma. Therefore,

Accepted 22 February 2016

the purpose of this study is to demonstrate the inhibitory effects of resveratrol on uterine

Available online

fibroid cell growth and ECM-associated protein expression. We found that resveratrol inhibited the proliferation of leiomyoma cell line (ELT3) and uterine smooth muscle cell line

Keywords:

(UtSMC) and affected the cell morphology of both cell lines, induced cell cycle arrest at S

Resveratrol

and G2/M phase, regulated cell apoptosis associated protein expression and induced cell

Uterine fibroids

apoptosis, and affected ECM-associated mRNA and protein expression. Our results suggest

Proliferation

that resveratrol is potentially effective in preventing the hyperplasia of leiomyoma and uterine

Cell cycle

smooth muscle cells.

Apoptosis

© 2016 Elsevier Ltd. All rights reserved.

Extracellular matrix

1.

Introduction

Uterine leiomyoma is a significant health concern to women of reproductive age as it is the most common benign tumour

of the female reproductive tract. It has been reported that the cumulative incidence of uterine leiomyoma among women 50 years of age is approximately 70–80% in the United States (Bulun, 2013). According to Taiwan’s Ministry of Health and Welfare, leiomyomas affect one in four women of reproductive

* Corresponding author. School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan. Tel.: +886 2 7361661 6558; fax: +886 2 27373112. E-mail address: [email protected] (S.-M. Hsia). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jff.2016.02.038 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

242

Journal of Functional Foods 23 (2016) 241–252

age in Taiwan. Women with leiomyomas usually suffer from a reduced quality of life with symptoms such as heavy uterine bleeding, pelvic pain, and infertility (Islam et al., 2013). Thus, understanding the molecular background with regard to disease progression is crucial in the development of effective therapeutic strategy for uterine leiomyoma. Leiomyomas are believed to arise from the transformation of myometrial cells (Bulun, 2013). While the aetiology of the formation is not well understood, genetic, cytokines, growth factors, extracellular matrix (ECM) components, and steroid hormones such as progesterone and oestrogen are factors that contribute to the growth and formation of leiomyomas (Islam et al., 2013). Chronic inflammation is also associated with leiomyoma growth as it induces the release of oestrogen, causing hyperplasia of smooth muscle cells and formation of fibrous tissue (Wegienka, 2012). The deposition of ECM-associated proteins is an important characteristic feature of leiomyoma. Under normal physiological conditions, degradation of ECM is precisely regulated. However, when the cells do not respond correctly to the usual signals, this will lead to the development of several diseases such as cancer and arthritis. ECMassociated proteins that are overexpressed include collagen type 1, fibronectin and proteoglycans (biglycan, fibromodulin). The matrix metalloproteinase (MMP) families, such as zincdependent endoproteinases, are responsible for the degradation of ECM. Among the previously reported human MMPs, MMP-2 (gelatinase-A/72 kDa type IV collagenase) and MMP-9 (gelatinase-B/92 kDa type IV collagenase) are abnormally expressed in leiomyomas, and are thought to be the key enzymes that contribute to cancer invasion and metastasis because these enzymes cleave native type IV collagen, one of the major components of basement membrane (Lu et al., 2015). One of the hallmarks of cancer is that cancer cells escape the process of apoptosis (Hanahan & Weinberg, 2000). Apoptosis is controlled by two different pathways: the membrane death receptor-mediated pathway (Fumarola & Guidotti, 2004) and the mitochondria-mediated pathway (Karunagaran, Joseph, & Kumar, 2007). The death receptor pathway is initiated when the ligand binds to its receptor and the process leads to apoptosis. Alternatively, the mitochondrial pathway is mediated by the change in mitochondrial membrane permeability, which promotes the release of cytochrome c from the mitochondria and consequently results in apoptotic cell death. Therefore, it is important to develop an effective strategy that promotes apoptosis in all types of cancer cells. Resveratrol is present in red wine and grape skin and its content in red grapes ranges from 1.5 to 7.3 µg/g (Bertelli & Das, 2009; Burns, Yokota, Ashihara, Lean, & Crozier, 2002; Kroon, Iyer, Chunduri, Chan, & Brown, 2010). Research has discovered the many health benefits of resveratrol. It is a potent antioxidant with anti-inflammatory and anti-proliferative actions on cancer cells (e.g., breast, ovary and prostate cancer), and has the ability to improve dysmenorrhoea (Hsia, Wang, & Wang, 2011; Hudson et al., 2007; Lin et al., 2013; Opipari et al., 2004; Riha et al., 2014; Shahidi & Ambigaipalan, 2015). However, its action on leiomyoma growth and the underlying mechanisms remain unclear. The aim of the present study was to investigate the direct effect of resveratrol on myometrial and leiomyoma cell proliferation and ECM-associated protein expression in vitro.

2.

Materials and methods

2.1.

Chemicals and antibodies

Materials used for cell culture (DMEM/F12 medium, foetal calf serum, foetal bovine serum (FBS), epidermal growth factor, fibroblast growth factor, insulin, penicillin and streptomycin) were purchased from Gibco (Grand Island, NY, USA). Resveratrol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue solution and other chemicals were purchased from Sigma Chemical (St Louis, MO, USA). Coomassie Brilliant Blue R-250 was purchased from Bio-Rad (Hercules, CA, USA). Primary antibodies against Bcl-2, Bax, Caspase3, cleaved caspase-3, Cytochrome c, Cyclin D1, CDK2, CDK4 and PARP, were obtained from Cell Signaling Technology (Danvers, MA, USA). The following 3 primary antibodies, Fibromodulin, Collagen Type I and Biglycan, were obtained from Abcam (Cambridge, UK). Primary antibodies against GAPDH, TIMP2 and MMP9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2.

Cell lines and culture conditions

Eker uterine leiomyoma cell line (ELT-3) that expresses oestrogen and progesterone receptors was kindly provided by Dr. Lin-Hung Wei (Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan). ELT-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum and maintained at 37 °C in 95% humidified air–5% CO2 atmosphere (Wei et al., 2011). Uterine smooth muscle cells (UtSMC) were purchased from PromoCell Co. (PromoCell, Heidelberg, Germany). UtSMC were cultured in DMEM/F12 medium supplemented with 5% (v/v) foetal calf serum, 0.5 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor and 5 µg/mL insulin, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C.

2.3.

Cell viability assay

Cell viability of ELT-3 and UtSMC cells were assessed using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (methyl thiazolyltetrazolium [MTT]) assay. Cells were treated with either vehicle alone (0.1% dimethyl sulphoxide, DMSO) or different concentrations of resveratrol (1–100 µM) in 10% FBS culture medium for 24 and 48 h. The MTT assay was conducted as previously described (Wang et al., 2013).

2.4.

Cell morphological evaluation

ELT-3 and UtSMC cells were seeded in 6-well plates and allowed to grow at 37 °C under humidified atmosphere containing 5% CO2. When the cells reached 50–60% confluence, the cells were treated with different concentrations of resveratrol. Before and after 24 h or 48 h of incubation, the cells were visualized and photographed using EVOS® FL cell imaging system microscope (Invitrogen, Carlsbad, CA, USA).

2.5.

Trypan blue dye exclusion assay

For trypan blue dye exclusion assay, ELT-3 and UtSMC were seeded at the density of 5 × 103 cells/well in 6-well plates, and

Journal of Functional Foods 23 (2016) 241–252

allowed to attach overnight. The medium was replaced with fresh complete medium containing the desired concentrations of resveratrol and the plates were incubated for 24 and 48 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Both floating and adherent cells were collected and centrifuged at 700 × g for 5 min. The cells were re-suspended in 25 µL phosphate buffered saline (PBS), mixed with 5 µL of 0.2% trypan blue solution and counted using a haemocytometer.

CFX Connect™ Real-Time PCR Detection System (Bio-Rad). Amplification of all genes was performed under the following cycling conditions: denaturation at 95 °C for 3 min followed by 45 cycles for 15 s at 95 °C and 30 s at 60 °C. Synthesis of DNA product of the expected size was confirmed by melting curve analysis. The comparative threshold cycle (Ct) values of each gene were normalized to Ct values of GAPDH (internal control).

2.9. 2.6.

Cell cycle distribution and apoptosis analysis

To analyse cell cycle distribution, 2 × 10 ELT-3 and UtSMC cells were plated in 6 cm culture dish and cultured for 24 h at 37 °C in 95% humidified air/5% CO2 atmosphere. Cells were treated with vehicle alone, 10 µM or 50 µM resveratrol in 10% FBS culture media for 48 h. For cell cycle distribution assay, the cells were fixed in 70% alcohol at −20 °C and stained with a propidium iodide solution (PI, 0.02 mg/mL) containing RNase A (0.2 mg/ mL) for 30 min at room temperature. For apoptosis analysis, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and PI from the commercial annexin V-FITC apoptosis detection kit (BD Biosciences, Bedford, MA, USA). Cell cycle distribution and apoptosis were analysed using BD FACSCanto, and the results were analysed by ModFitLT V3.0 software program. 5

2.8.

Zymography

Western blotting analysis

Cellular proteins were prepared by separating the proteins (20 µg) in the samples using 8–15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 50 V for 30 min and 90 V for 90 min in running buffer. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products, Inc., Boston, MA, USA) using a Trans-Blot SD semidry transfer cell (170–3940, Bio-Rad, Hercules, CA, USA) at 50 mA (for 8 mm × 10 mm membrane) for 60 min in blotting solution. After blocking for 1 hour in phosphate buffered saline containing 5% skim milk, blots were incubated with primary antibodies at 4 °C overnight, and probed with secondary antibodies for 2 h. ECL detection reagent (PerkinElmer Life Sciences Boston, MA, USA) was used to visualize the immunoreactive proteins on PVDF membranes after transfer. Multi-Gauge V3.0 software was used for protein quantification.

2.7.

243

Real-time polymerase chain reaction analysis

Real-time PCR analysis was performed to determine MMP2, TIMP1 and TIMP2 mRNA expression using total RNA from resveratrol-treated cells as described above. Primer sequences were as follows: MMP2, sense 5′-TCTACTCAGCCAGCACCCTGGA3′ and anti-sense 5′-TGCAGGTCCACGACGGCATCCA-3′; TIMP1, sense 5′-CTTCTGGCATCCTGTTGTTG-3′ and anti-sense 5′AGAAGGCCGTCTGTG GGT-3′; TIMP2, sense 5′-CGACATTT ATGGCAACCCTATCA-3′ and anti-sense 5′-CAGGCCCTTTGAA CATCTTTATCT-3′; GAPDH, sense 5′-AACATCATCCCTGCCTCTAC3′ and anti-sense 5′-CTGCTTCACCACCTTCTTG-3′. Amplification reactions were performed using iQ™ SYBR® Green Supermix (Bio-Rad). In total, 500 ng of each cDNA was added to the mix containing appropriate primer sets (200 nM each) and SYBR green in a 10 µL reaction volume. All samples were analysed in triplicate. Real-time PCR analyses were performed using a

In the zymography assay, 2 × 105 cells were seeded in a 24well plate and cultured for 24 h in an incubator. Cells were treated with vehicle alone, 10 µM or 50 µM resveratrol (1 mL serum free medium) for 48 h. The cells were calculated, and the conditional medium was collected and stored in −80 °C. Electrophoresis was carried out on the unboiled samples using the 8% SDS-polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gel was washed twice in 2.5% Triton X-100 for 30 min at room temperature and incubated in developing buffer (10 mM CaCl2, 0.01% NaN3 and 40 mM Tris–HCl, pH 8.0) for 12 h at 37 °C. Bands corresponding to activity were visualized by negative staining using 0.5% Coomassie Brilliant Blue R-250, and subsequently, bands of gelatinolytic activity were analysed as previously described (Shih et al., 2015).

2.10.

Immunofluorescence analysis

Cells were cultured on sterile glass coverslips and treated with 10 or 50 µM resveratrol for 48 h. After treatment, cells were fixed with 4% formaldehyde solution at room temperature for 15 min. The cells were then washed in 1 X PBS, permeabilized using 0.1% triton X-100/PBS for 15 min, and blocked in blocking solution (5% (w/v) BSA in 1X TBST). Cells were incubated with primary antibodies overnight at 4 °C. Cells were washed for 15 min (3 times × 5 min each) with 1X PBS, and then incubated with secondary antibodies (Alexa Fluor® 488 dye and Alexa Fluor® 546 dye, Life Technologies, Gaithersburg, MD, USA) for 1 hour at room temperature followed by washing with 1 X PBS. Cells were then added with ProLong® Gold Antifade Mountant. Fluorescent images were taken using EVOS® microscope.

2.11.

Statistical analysis

Data are expressed as mean ± SEM and were obtained from three independent experiments performed in triplicate. Statistical analyses were performed using unpaired t-test. Differences between the variants were considered statistically significant when p < 0.05.

3.

Results and discussion

3.1. Effect of resveratrol on the growth of ELT-3 cells and UtSMCs We first determined the antiproliferative effect of resveratrol on ELT-3 cells and UtSMCs. The effects of resveratrol on the growth of ELT-3 cells and UtSMCs were assessed by morphological examination, MTT assay and trypan blue dye exclusion

244

Journal of Functional Foods 23 (2016) 241–252

assay. We found that the number of attached ELT-3 cells and UtSMCs decreased after the cells were treated with 25 to 100 µM resveratrol for 24 and 48 h. Changes in the morphology of ELT-3 cells and UtSMCs were also observed after resveratrol treatment for 24 and 48 h. The cells became shrunken and rounded, and a fraction of the cells floated in the medium, whereas the untreated control cells were well attached to the surface (Fig. 1A). After ELT-3 cells and UtSMCs were exposed to resveratrol for 24–96 h, cell viability was assessed by MTT assay. Resveratrol at 10 to 100 µM concentrations significantly inhibited the viability of ELT-3 cells in a dose- and time-dependent manner. UtSMC proliferation was also significantly inhibited at 25 to 100 µM concentrations of resveratrol in a dose- and time-dependent manner (p < 0.05) (Fig. 1B). Resveratrol at 10 µM did not influence cell viability. However, resveratrol at 25 to 100 µM significantly decreased the number of ELT-3 cells following incubation for 48 h and longer (p < 0.01) (Fig. 1C). Previous studies have demonstrated that resveratrol can inhibit the proliferation of various cancer cells (Benitez, Pozo-Guisado, Alvarez-Barrientos, Fernandez-Salguero, & Castellon, 2007). Prostate cancer cells, as well as LNCaP (androgen dependent), PC-3 (androgen independent) and PZ-HPV-7 (normal cells) cells, were treated with resveratrol. Resveratrol inhibited prostate cancer cell viability. At 24 h, cell viability significantly decreased in LANCaP (from 50 µM), PC-3 (from 10 µM) and PZ-HPV-7 (from 100 µM) cells. In addition, cell viability significantly decreased in LANCaP (from 10 µM), PC-3 (from 10 µM) and PZHPV-7 (from 1 µM) cells at 48 h. One study found that resveratrol could inhibit cell proliferation in lung cancer cells (Yuan et al., 2015). Resveratrol at concentrations above 50 µM significantly inhibited cell proliferation in A548 cells at 24 and 48 h. In our current study, resveratrol also affected cell morphology; cells became shrunken, distorted and rounded after exposure to resveratrol. This study also found that resveratrol could inhibit cell proliferation and alter cell morphology in ELT3 cells and UtSMCs. In the current study, the bioavailability of resveratrol, particularly the tissue distribution concentration in the uterus, must be a major issue. However, there are limited studies on resveratrol distribution concentration in the uterus. Previous studies have shown that resveratrol in systemic organs and tissue (kidneys, livers, lung, cerebellum, heart, brain, aorta tissue, ovaries, uterus, urinary bladder tissue, pancreas, spleen, lymph node, perineal fat, mesenteric fat, subcutaneous fat, longissimus dorsi and semimembranous muscle) was very small and amount up to 0.5% of the initial dose of resveratrol (472 mg) administered. The distribution concentration in the uterus is near 9 µg/g tissue weight (Azorín-Ortuño et al., 2011). This study showed that resveratrol at 50 µM (11.41 µg/mL of concentration) had more physiological effect in ELT-3 cells and UtSMCs. This result indicated that the dosage of resveratrol used in this study was 50 µM (11.41 µg/mL of concentration), which was very close to the physiologically reasonable concentration as shown above.

3.2. Resveratrol activated the caspase pathway and enhanced the expression of apoptosis-related proteins in ELT-3 cells and UtSMCs We next evaluated whether the reduced proliferation of ELT-3 cells and UtSMCs caused by resveratrol treatment is due to

decreased survival of ELT-3 cells and UtSMCs. We evaluated the percentage of apoptotic cells using PI and annexin V staining. Treatment with resveratrol (10 and 50 µM) enhanced the percentage of apoptotic cells in a dose-dependent manner in ELT-3 cells (p < 0.05). However only at the 50 µM concentration, resveratrol increased the percentage of apoptotic UtSMCs (p < 0.05) (Fig. 2A–C). Effects of resveratrol (10 and 50 µM) on caspase and cytochrome c protein expression were examined in ELT-3 cells and UtSMCs (Fig. 2D). Activation of caspase cascades leads to a reduction of the proenzyme forms. Caspase 3 is capable of specific cleavage. In ELT-3 cells, resveratrol treatment reduced the amounts of procaspase 3 and cytochrome c after 48 h. Simultaneously, active caspase cleaves cellular target proteins, including PARP, thus leading to cell death. In this study, we also determined the effect of resveratrol on PARP activation. ELT-3 cells treated with resveratrol (10 and 50 µM) for 48 h resulted in a dose-dependent increase in PARP cleavage (p < 0.05). Reduction in the expression of procaspase is a key point in apoptotic signal transduction. Caspases are involved in the pathways that induce apoptosis and are subdivided into two subfamilies: initiator caspases (including caspases 8 and 9) and effector caspases (including caspases 3, 6, and 7) (Kumar & Harvey, 1995; Stegh & Peter, 2001; Thorburn, 2004). Here, we showed that procaspase 3 protein levels in ELT3 cells and UtSMCs were reduced after resveratrol treatments, indicating caspase cascade activation. Furthermore, resveratrol treatments resulted in PARP cleavage in ELT3 cells and UtSMCs. In this study, we found that 50 µM resveratrol significantly induced apoptosis in ELT3 cells. Resveratrol treatment also increased the protein levels of cleaved caspase 3 (active form) and cleaved PARP (inactive form). Previous studies have shown that resveratrol induces apoptosis in various cancer cells, such as breast cancer cells (Lin et al., 2013; Riha et al., 2014), liver cancer cells (Bishayee & Dhir, 2009), colorectal cancer cells (Juan, Wenzel, Daniel, & Planas, 2008; Weng, Liao, Li, Chang, & Chiou, 2010), lung cancer cells (Weng, Yang, Ho, & Yen, 2009; Yu et al., 2013) and gastric cancer cells (Jing, Cheng, Wang, Li, & He, 2016; Yang et al., 2013). A previous study has also indicated that resveratrol induces cell apoptosis via an endogenous apoptosis pathway (Han et al., 2015). The cells treated with resveratrol proceed to apoptosis, cytochrome c is released from mitochondria and the inactive form of caspase is cleaved to its active form. Then, cleaved caspase converts PARP to cleaved PARP (inactive form). Therefore, DNA in the cells cannot be repaired, and apoptosis is induced.

3.3. Resveratrol induced cell cycle arrest in ELT-3 cells and UtSMCs Cell proliferation can be inhibited via cell cycle arrest; hence, in this study, the effect of resveratrol on the cell cycle was investigated. Previous studies have demonstrated that resveratrol can induce cell cycle arrest (Benitez et al., 2007; Sakamoto, Horiguchi, Oguma, & Kayama, 2010). In the present study, we found that 50 µM resveratrol promoted cell cycle arrest in S and G2/M phases in ELT3 cells and S phase in UtSMCs (Fig. 3A–B). The cell cycle is regulated by CDK and cyclin. In this study, we found that 50 µM resveratrol inhibited protein expression of CDK4, cyclin D1 and CDK2 in ELT3 cells and UtSMCs (Fig. 3C). In contrast to the present study, resveratrol was found to induce

Journal of Functional Foods 23 (2016) 241–252

245

Fig. 1 – Effect of resveratrol on proliferation and viability of UtSMC and ELT-3 cell lines. (A) Morphology. UtSMC and ELT-3 cells were treated with vehicle alone or 10–100 µM resveratrol for 48 h. (B) Cell viability was assessed by MTT assay. (C) Cell number was assessed by trypan blue dye exclusion assay. Data represent mean ± SEM from six independent experiments. *p < 0.05, **p < 0.01 when compared with the vehicle group.

246

Journal of Functional Foods 23 (2016) 241–252

Fig. 2 – Effect of resveratrol on apoptosis of UtSMC and ELT-3 cell lines. (A) Cell apoptosis. UtSMC and ELT-3 cells were treated with vehicle alone, resveratrol (10 and 50 µM) for 48 h, fixed, stained with PI and annexin V, and then subjected to flow cytometry. (B) Apoptotic cell death was shown as percentage to the total cells counted after UtSMC and ELT-3 cells were treated with vehicle alone, resveratrol (10 and 50 µM) for 48 h. (C) Late and early apoptotic cell death was shown as percentage to the total cells counted. (D) Resveratrol significantly increased cytochrome c, cleavage caspase 3 and PARP in UtSMC and ELT-3 cell lines. Data represent mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 when compared with the vehicle group.

Journal of Functional Foods 23 (2016) 241–252

247

Fig. 3 – Inhibitory actions of resveratrol on cell cycle of UtSMC and ELT-3 cell lines. (A) Cell cycle. UtSMC and ELT-3 cells were treated with vehicle alone, resveratrol (10 and 50 µM) for 48 h, fixed, stained with PI, and then subjected to flow cytometry. (B) Different phases of cell cycle were shown as percentage to the total cells counted after UtSMC and ELT-3 cells were treated with vehicle alone, resveratrol (10 and 50 µM) for 48 h. (C) Resveratrol significantly increased CDK4, cyclin D1 and CDK2 in UtSMC and ELT-3 cell lines. Data represent mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 when compared with the vehicle group.

248

Journal of Functional Foods 23 (2016) 241–252

Fig. 4 – Effect of resveratrol on protein expressions of ECM-associated proteoglycans in UtSMC and ELT-3 cells. Equal amounts of each lysate from ELT-3 cells (A) and UtSMC cells (B) treated with different concentrations of resveratrol (0, 10 and 50 µM) were analysed by western blotting using anti-fibronectin, anti-collage type 1 (collagen), anti-fibromodulin, antibiglycan antibodies. Western blot with anti-GAPDH antibody was used as a loading control. Data represent one of three independent experiments.

cycle arrest in G0/G1 phase in LANCaP cells (100 µM) and PC-3 cells (50 µM) (Benitez et al., 2007). However, it has also been stated that resveratrol induced cell cycle arrest in S and G2/M phases in MCF-7 cells (Sakamoto et al., 2010).

3.4. Resveratrol treatment regulated ECM-associated mRNA and protein expressions and the proteolytic activation of MMPs in ELT-3 cells and UtSMCs Leiomyomas are characterized by the deposition of ECMassociated proteins and hyperplasia of smooth muscle cells

(Halder, Osteen, & Al-Hendy, 2013a, 2013b). To verify the effect of resveratrol on the ECM in ELT-3 cells and UtSMCs, we performed western blot analysis to investigate the effect of resveratrol on ECM-associated protein expression. Resveratrol treatment significantly decreased fibronectin, collagen I, fibromodulin and biglycan levels in ELT-3 cells and UtSMCs (Fig. 4A–B). Moreover, fluorescence images revealed a reduction in fibronectin staining in ELT3 cells and UtSMCs (Fig. 5). Since leiomyoma cells show high MMP-2 and MMP-9 expression and low TIMP2 expression (Halder et al., 2013b), we then examined whether resveratrol could attenuate specific mRNA

Journal of Functional Foods 23 (2016) 241–252

249

Fig. 5 – Effect of resveratrol on protein expressions of fibronectin in UtSMC and ELT-3 cells. Immunofluorescence analyses of fibronectin were performed using UtSMC and ELT-3 after treatment with different concentrations of resveratrol (0, 10 and 50 µM) for 48 h. Data represent one of three independent experiments.

250

Journal of Functional Foods 23 (2016) 241–252

Fig. 6 – Effect of resveratrol on MMP-related mRNA and protein expressions in UtSMC and ELT-3 cells. (A) Real-time PCR analyses for mRNA expressions of MMP2 and (B) TIMP-2 were performed using gene-specific sense and anti-sense primers as described in the Materials and methods section. The mRNA expression levels of MMP2 and TIMP-2 were normalized with GAPDH and the normalized values were used to generate the graphs. (C) Resveratrol significantly increased TIMP-2 and decreased MMP9 protein expression in UtSMC and ELT-3 cell lines. (D) Effect of resveratrol on gelatinolytic activities of MMP 2 and 9 in cultured human uterine fibroid cells. Data represent mean ± SEM from three independent experiments.

Journal of Functional Foods 23 (2016) 241–252

251

Resveratrol

Cell growth Cell cycle arrest

Cell proliferation

Apoptosis

Leiomyoma MMPs TIMPs

Abnormal deposition of ECM

ECM Fig. 7 – A hypothetical sketch of proposed mechanism by which resveratrol inhibits growth retardation in UtSMC and ELT-3 cell lines. The proliferation of UtSMC and ELT-3 cells were inhibited by resveratrol through cell cycle arrest and apoptosis pathway; resveratrol reduced the ECM-related protein expression; resveratrol inhibited MMP-2 and MMP-9 expression.

and protein expressions, as well as the secretion and proteolytic activation of MMP-2 and MMP-9 in ELT-3 cells and UtSMCs. We found that MMP-2 mRNA expression in ELT3 and UtSMC cells was not affected (Fig. 6A). In contrast, resveratrol at 50 µM concentration significantly increased TIMP2 mRNA expression in ELT3 cells and UtSMCs (Fig. 6B). We also observed that resveratrol reduced MMP-9 protein expression and increased TIMP2 protein expression (Fig. 6C). Our results also indicated that a gelatinolytic band corresponding to MMP-2 was markedly decreased by resveratrol treatment in ELT3 cells and UtSMCs (Fig. 6D). Previous studies have indicated that ECM accumulation promotes the growth of uterine fibroids (Walker & Stewart, 2005). This study aimed to investigate the effect of resveratrol on the expression of ECM-associated mRNAs and proteins. A previous study found that vitamin D3 reduced MMP-2 and MMP-9 mRNA and protein expressions and increased TIMP2 mRNA and protein expressions but did not affect TIMP1 mRNA and protein expressions (Halder et al., 2013a, 2013b). Therefore, vitamin D3 can suppress the growth of uterine fibroids. In the present study, we found that resveratrol reduced MMP-9 protein expression, increased TIMP1 mRNA expression and increased TIMP2 mRNA and protein expressions; however, resveratrol did not regulate MMP-2 mRNA expression. Gweon and Kim (2014) performed western blot and immunofluorescence analyses using chondrosarcoma cells (HTB94) treated with increasing concentrations above 10 µM resveratrol, and the results showed that MMP-2 and MMP-9 protein expression levels were reduced. The results were similar to the results of this study. In addition, vitamin D3 can suppress uterine fibroid proliferation by reducing ECM-associated proteins, such as proteoglycans (biglycan, fibromodulin, and versican), fibronectin and collagen I (Halder et al., 2013a, 2013b). In this study, we observed that resveratrol reduced the protein levels of fibronectin, collagen I, fibromodulin and biglycan in ELT3 cells and UtSMCs. In conclusion, this study demonstrates for the first time that resveratrol inhibits leiomyoma cell proliferation, induces cell

apoptosis, promotes cell cycle arrest and regulates ECMassociated mRNA and protein expression (Fig. 7). Thus, resveratrol is potentially effective in preventing the hyperplasia of leiomyoma and uterine smooth muscle cells.

Acknowledgement This study was supported by the grants [MOST103-2313-B038-003-MY3, MOST103-2629-B-038-002-, MOST103-2313-B038-001-MY3, NSC102-2313-B-038-001-] from the Ministry of Science and Technology, Taiwan, Republic of China. REFERENCES

Azorín-Ortuño, M., Yáñez-Gascón, M., Vallejo, F., Pallarés, F. J., Larrosa, M., Lucas, R., Morales, J. C., Tomás-Barberán, F. A., García-Conesa, M. T., & Espín, J. C. (2011). Metabolites and tissue distribution of resveratrol in the pig. Molecular Nutrition & Food Research, 55(8), 1154–1168. Benitez, D. A., Pozo-Guisado, E., Alvarez-Barrientos, A., Fernandez-Salguero, P. M., & Castellon, E. A. (2007). Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest in prostate cancer-derived cell lines. Journal of Andrology, 28(2), 282–293. Bertelli, A. A., & Das, D. K. (2009). Grapes, wines, resveratrol, and heart health. Journal of Cardiovascular Pharmacology, 54(6), 468– 476. Bishayee, A., & Dhir, N. (2009). Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: Inhibition of cell proliferation and induction of apoptosis. Chemico-Biological Interactions, 179(2–3), 131–144. Bulun, S. E. (2013). Uterine fibroids. The New England Journal of Medicine, 369(14), 1344–1355. Burns, J., Yokota, T., Ashihara, H., Lean, M. E., & Crozier, A. (2002). Plant foods and herbal sources of resveratrol. Journal of Agricultural and Food Chemistry, 50(11), 3337–3340.

252

Journal of Functional Foods 23 (2016) 241–252

Fumarola, C., & Guidotti, G. G. (2004). Stress-induced apoptosis: Toward a symmetry with receptor-mediated cell death. Apoptosis: An International Journal on Programmed Cell Death, 9(1), 77–82. Gweon, E. J., & Kim, S. J. (2014). Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways. Oncology Reports, 32(1), 71–78. Halder, S. K., Osteen, K. G., & Al-Hendy, A. (2013a). 1,25Dihydroxyvitamin d3 reduces extracellular matrix-associated protein expression in human uterine fibroid cells. Biology of Reproduction, 89(6), 150. Halder, S. K., Osteen, K. G., & Al-Hendy, A. (2013b). Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells. Human Reproduction, 28(9), 2407–2416. Han, G., Xia, J., Gao, J., Inagaki, Y., Tang, W., & Kokudo, N. (2015). Anti-tumor effects and cellular mechanisms of resveratrol. Drug Discoveries & Therapeutics, 9(1), 1–12. Hanahan, D., & Weinberg, R. A. (2000). The hallmarks of cancer. Cell, 100(1), 57–70. Hsia, S. M., Wang, K. L., & Wang, P. S. (2011). Effects of resveratrol, a grape polyphenol, on uterine contraction and Ca2+ mobilization in rats in vivo and in vitro. Endocrinology, 152(5), 2090–2099. Hudson, T. S., Hartle, D. K., Hursting, S. D., Nunez, N. P., Wang, T. T., Young, H. A., Arany, P., & Green, J. E. (2007). Inhibition of prostate cancer growth by muscadine grape skin extract and resveratrol through distinct mechanisms. Cancer Research, 67(17), 8396–8405. Islam, M. S., Protic, O., Giannubilo, S. R., Toti, P., Tranquilli, A. L., Petraglia, F., Castellucci, M., & Ciarmela, P. (2013). Uterine leiomyoma: Available medical treatments and new possible therapeutic options. The Journal of Clinical Endocrinology and Metabolism, 98(3), 921–934. Jing, X., Cheng, W., Wang, S., Li, P., & He, L. (2016). Resveratrol induces cell cycle arrest in human gastric cancer MGC803 cells via the PTENregulated PI3K/Akt signaling pathway. Oncology Reports, 35(1), 472–478. Juan, M. E., Wenzel, U., Daniel, H., & Planas, J. M. (2008). Resveratrol induces apoptosis through ROS-dependent mitochondria pathway in HT-29 human colorectal carcinoma cells. Journal of Agricultural and Food Chemistry, 56(12), 4813– 4818. Karunagaran, D., Joseph, J., & Kumar, T. R. (2007). Cell growth regulation. Advances in Experimental Medicine and Biology, 595, 245–268. Kroon, P. A., Iyer, A., Chunduri, P., Chan, V., & Brown, L. (2010). The cardiovascular nutrapharmacology of resveratrol: Pharmacokinetics, molecular mechanisms and therapeutic potential. Current Medicinal Chemistry, 17(23), 2442–2455. Kumar, S., & Harvey, N. L. (1995). Role of multiple cellular proteases in the execution of programmed cell death. FEBS Letters, 375(3), 169–173. Lin, C. Y., Hsiao, W. C., Wright, D. E., Hsu, C. L., Lo, Y. C., Wang Hsu, G. S., & Kao, C. F. (2013). Resveratrol activates the histone H2B ubiquitin ligase, RNF20, in MDA-MB-231 breast cancer cells. Journal of Functional Foods, 5(2), 790–800. Lu, S., Zhu, Q., Zhang, Y., Song, W., Wilson, M. J., & Liu, P. (2015). Dual-functions of miR-373 and miR-520c by differently regulating the activities of MMP2 and MMP9. Journal of Cellular Physiology, 230(8), 1862–1870. Opipari, A. W., Jr., Tan, L., Boitano, A. E., Sorenson, D. R., Aurora, A., & Liu, J. R. (2004). Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Research, 64(2), 696–703.

Riha, J., Brenner, S., Bohmdorfer, M., Giessrigl, B., Pignitter, M., Schueller, K., Thalhammer, T., Stieger, B., Somoza, V., Szekeres, T., & Jäger, W. (2014). Resveratrol and its major sulfated conjugates are substrates of organic anion transporting polypeptides (OATPs): Impact on growth of ZR75-1 breast cancer cells. Molecular Nutrition & Food Research, 58(9), 1830–1842. Sakamoto, T., Horiguchi, H., Oguma, E., & Kayama, F. (2010). Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells. The Journal of Nutritional Biochemistry, 21(9), 856– 864. Shahidi, F., & Ambigaipalan, P. (2015). Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects – A review. Journal of Functional Foods, 18, 820–897. Shih, Y. H., Chang, K. W., Yu, C. C., Kao, M. C., Chen, M. Y., Wang, T. H., Chih, T. Y., Chenh, Y. L., & Shieh, T. M. (2015). Hinokitiol suppressed pan-histone expression and cell growth in oral squamous cell carcinoma cells. Journal of Functional Foods, 15, 452–463. Stegh, A. H., & Peter, M. E. (2001). Apoptosis and caspases. Cardiology Clinics, 19(1), 13–29. Thorburn, A. (2004). Death receptor-induced cell killing. Cellular Signalling, 16(2), 139–144. Walker, C. L., & Stewart, E. A. (2005). Uterine fibroids: The elephant in the room. Science, 308(5728), 1589–1592. Wang, K. L., Hsia, S. M., Chan, C. J., Chang, F. Y., Huang, C. Y., Bau, D. T., & Wang, P. S. (2013). Inhibitory effects of isoliquiritigenin on the migration and invasion of human breast cancer cells. Expert Opinion on Therapeutic Targets, 17(4), 337–349. Wegienka, G. (2012). Are uterine leiomyoma a consequence of a chronically inflammatory immune system? Medical Hypotheses, 79(2), 226–231. Wei, L. H., Torng, P. L., Hsiao, S. M., Jeng, Y. M., Chen, M. W., & Chen, C. A. (2011). Histone deacetylase 6 regulates estrogen receptor alpha in uterine leiomyoma. Reproductive Sciences, 18(8), 755–762. Weng, C. J., Yang, Y. T., Ho, C. T., & Yen, G. C. (2009). Mechanisms of apoptotic effects induced by resveratrol, dibenzoylmethane, and their analogues on human lung carcinoma cells. Journal of Agricultural and Food Chemistry, 57(12), 5235–5243. Weng, Y. L., Liao, H. F., Li, A. F., Chang, J. C., & Chiou, R. Y. (2010). Oral administration of resveratrol in suppression of pulmonary metastasis of BALB/c mice challenged with CT26 colorectal adenocarcinoma cells. Molecular Nutrition & Food Research, 54(2), 259–267. Yang, Q., Wang, B., Zang, W., Wang, X., Liu, Z., Li, W., & Jia, J. (2013). Resveratrol inhibits the growth of gastric cancer by inducing G1 phase arrest and senescence in a Sirt1-dependent manner. PLoS ONE, 8(11), e70627. Yu, Y. H., Chen, H. A., Chen, P. S., Cheng, Y. J., Hsu, W. H., Chang, Y. W., Chen, Y. H., Jan, Y., Hsiao, M., Chang, T. Y., Liu, Y. H., Jeng, Y. M., Wu, C. H., Huang, M. T., Su, Y. H., Hung, M. C., Chien, M. H., Chen, C. Y., Kuo, M. L., & Su, J. L. (2013). MiR520h-mediated FOXC2 regulation is critical for inhibition of lung cancer progression by resveratrol. Oncogene, 32(4), 431– 443. Yuan, L., Zhang, Y., Xia, J., Liu, B., Zhang, Q., Liu, J., Luo, L., Peng, Z., Song, Z., & Zhu, R. (2015). Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells. Molecular Medicine Reports, 11(4), 2459–2464.