Tranilast, an orally active antiallergic compound, inhibits extracellular matrix production in human uterine leiomyoma and myometrial cells Md Soriful Islam, Ph.D.,a,b Olga Protic, M.Sc.,a Andrea Ciavattini, M.D.,c Stefano Raffaele Giannubilo, M.D.,c Andrea Luigi Tranquilli, M.D.,c William H. Catherino, M.D., Ph.D.,d Mario Castellucci, M.D., Ph.D.,a and Pasquapina Ciarmela, Ph.D.a,e a
Department of Experimental and Clinical Medicine, Faculty of Medicine, c Department of Clinical Science, and Department of Information Engineering, Polytechnic University of Marche, Ancona, Italy; b Biotechnology and Microbiology Laboratory, Department of Botany, University of Rajshahi, Rajshahi, Bangladesh; and d Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, Maryland e
Objective: To determine the effect of tranilast (an antiallergic drug known to suppress fibrosis or to stabilize mast cells) on extracellular matrix production in human leiomyoma and myometrial cells. Design: Laboratory study. Setting: University-affiliated laboratory. Patient(s): Seven premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Intervention(s): Cells were treated with tranilast (300 mM) for 48 hours to measure extracellular matrix and activin-A expression by real-time reverse-transcription polymerase chain reaction and/or immunocytochemistry. Main Outcome Measure(s): The expression of fibronectin, collagen1A1, versican, and activin-A in myometrial and leiomyoma cells. Result(s): Tranilast decreased fibronectin, collagen 1A1, and versican messenger RNA (mRNA) expression in human primary leiomyoma cell culture. Similar results were found in an immortalized human leiomyoma cell line. Tranilast also decreased the mRNA expression of fibronectin, collagen 1A1, and versican in human primary myometrial cells. The reduced expression of fibronectin and collagen 1 were observed by immunocytochemistry as well. Tranilast also reduced profibrotic growth factor, activin-A mRNA expression in primary myometrial and leiomyoma cells. Conclusion(s): Our results indicate that tranilast reduced fibronectin, collagen 1A1, versican, and activin-A expression in leiomyoma and myometrial cells, demonstrating its potential as an antifibrotic therapy for human Use your smartphone leiomyomas. (Fertil SterilÒ 2014;102:597–606. Ó2014 by American Society for Reproductive to scan this QR code Medicine.) and connect to the Key Words: Fibroid, tranilast, fibronectin, collagen 1A1, activin-A Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/islammd-tranilast-extracellular-matrix-uterine-leiomyomamyometrial/
Received January 11, 2014; revised and accepted May 7, 2014; published online June 14, 2014. M.S.I. has nothing to disclose. O.P. has nothing to disclose. A.C. has nothing to disclose. S.R.G. has nothing to disclose. A.L.T. has nothing to disclose. W.H.C. has nothing to disclose. M.C. has nothing to disclose. P.C. has nothing to disclose. This work was supported by the ‘‘Fondazione Cassa di Risparmio di Fabriano e Cupramontana’’ (to M.C. and P.C.) and by Italian Ministry of the University and Research (PRIN 2010-2011, no. 20102CHST5_007, to S.R.G.). M.S.I. was recipient of a fellowship from Polytechnic University of Marche, reserved for a Ph.D. student from a non–European Union country. O.P. is recipient of a fellowship from Polytechnic University of Marche, reserved for a Ph.D. student coming from universities of the UNIADRION network. Reprint requests: Pasquapina Ciarmela, Ph.D., Department of Experimental and Clinical Medicine, Polytechnic University of Marche, Faculty of Medicine, Via Tronto 10/a, 60020 Ancona, Italy (E-mail:
[email protected]). Fertility and Sterility® Vol. 102, No. 2, August 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.013 VOL. 102 NO. 2 / AUGUST 2014
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terine fibroids or leiomyomas are the most common female reproductive tract tumors (1, 2). They are highly prevalent, with 70%–80% of women burdened by the end of their reproductive years (3). Beside their high prevalence leiomyomas are associated with a variety of problems, such as menorrhagia, pelvic pain, and pelvic pressure, as well as infertility and
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ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE pregnancy complications (4). Hysterectomy is definitive treatment for this tumor, but loss of reproductive potential and significant morbidity and mortality are major limitations of this surgical intervention. Furthermore, surgical intervention for leiomyomas is associated with a substantial economic impact on health care systems that amounts to approximately $2.2 billion per year in the United States alone (5). To avoid the risks of surgical intervention, many novel therapies are currently under investigation. The GnRH agonist leuprolide acetate is the only medical intervention approved by the US Food and Drug Administration for presurgical treatment of uterine leiomyomas (6). Unfortunately, because of the hypoestrogenic side effects associated with such therapy, long-term intervention is contraindicated. As a result, medical therapeutic options are quite limited for leiomyoma treatment. Fibroids originate from the smooth muscle layer of the uterus and probably develop from a single transformed myometrial smooth muscle cell (7, 8). Tumor bulk results from a disorder of fibrosis (9–12). Leiomyomas produce large amounts of extracellular matrix (ECM) proteins, such as collagens, fibronectin, and proteoglycans (1, 11, 13–18). The majority of the tumor is made up of this disrupted matrix (19), and recent findings suggested that alterations in ECM can modify mechanical stress on cells, which leads to activation of internal mechanical signaling that may contribute to leiomyoma growth (20–22). Considering that leiomyoma has tumor and fibrotic characteristics, any effective therapy should regulate both leiomyoma cell proliferation and ECM production. Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) is a synthetic drug of low toxicity that has been widely used clinically in Japan since the 1980s (23). This drug is taken orally and is effective against allergic diseases such as bronchial asthma, allergic rhinitis, atopic dermatitis, and allergic conjunctivitis (23, 24). Tranilast exhibits its therapeutic effect in these conditions by inhibiting the release of chemical mediators from mast cells and basophils (25, 26). Tranilast is also used to prevent keloid tumor (a fibrotic disorder that shares similar molecular and epidemiologic features with leiomyomas) (10, 27, 28) formation after skin injury by reducing collagen synthesis in keloid fibroblasts through interference with transforming growth factor (TGF)-b effects (29). Tranilast has been reported to inhibit the TGF-b–induced transformation of fibroblasts to myofibroblasts and their contraction in vitro (30) and vascular endothelial growh factor–induced angiogenesis (31). Tranilast also inhibits the release of inflammatory and fibrotic mediators such as TGF-b1, interleukin (IL)-1b, prostaglandin E2, IL-2, IL-8, and leukotriene C4 from human monocytes and macrophages (24, 32, 33). Furthermore, tranilast antagonizes angiotensin II (34), restores cytokineinduced nitric oxide production against platelet-derived growth factor (35), and inhibits calcium entry in smooth muscle cells (36). Subsequent studies have confirmed the ability of tranilast to inhibit cancer cell growth and proliferation in various tumor models, including breast, pancreatic, gastric, and prostate cancer, glioma, and other tumors (37). 598
Although tranilast exhibits multiple therapeutic effects in diverse pathologic conditions, limited work has been reported in uterine fibroid biology. In 2002, Shime et al. (38) reported that tranilast arrested the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of cyclindependent kinase 2 activity via an induction of p21waf1and p53. Similar to a previous study, our group also found that tranilast inhibited the proliferation of human primary uterine leiomyoma cells (39). We also noted that tranilast inhibited the proliferation of normal myometrial cells (39). However, no study has addressed the effect of tranilast on ECM production in leiomyoma cells. Therefore, in the present study we hypothesized that tranilast could regulate ECM production in leiomyoma and myometrial cells.
MATERIALS AND METHODS Drugs and Chemicals Tranilast was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) at 30 mM. Further, it was diluted with medium to reach 1, 10, 30, 100, 300, and 1,000 mM before treatment.
Ethics Statement This study was conducted according to the principles of the Declaration of Helsinki. It was approved by the internal institutional review and ethical board of the Department of Obstetrics and Gynaecology. All patients provided written, informed consent for the collection of samples and subsequent analysis.
Tissue Collection Seven premenopausal Caucasian women aged 41–49 years were included in this study, and they were not receiving any type of hormonal therapy. Fibroid and myometrial tissues were obtained by hysterectomy or laparotomic myomectomy from patients with symptomatic fibroid. The diagnosis of leiomyoma was confirmed by histologic examination of the specimens removed. After surgically removing fibroid and adjacent myometrial tissues, fresh tissue specimens 1.5 cm 1.5 cm 1.5 cm were collected from both submucosal and intramural leiomyomas. The size range was 3–10 cm in diameter.
Primary Cell Cultures Myometrial and leiomyoma samples were collected in Hanks' balanced salt solution (Euroclone) at the time of surgery. Samples were cut into small pieces with 0.1% collagenase type 8 (Serva Electrophoresis) solutions (serum-free Dulbecco's modified Eagle medium [DMEM; Sigma-Aldrich] containing 1% penicillin–streptomycin [EuroClone], 50 mg/L gentamicin [Lonza], and 1% amphotericin B [Lonza]). Tissues were incubated at 37 C for 5 to 6 hours in a water bath and shaken manually until complete digestion. Digested cell suspensions were then centrifuged at 1,200 rpm for 10 minutes and washed with regular media (DMEM containing 10% fetal bovine serum [Sigma-Aldrich], 1% penicillin–streptomycin [EuroClone], 50 mg/L gentamicin [Lonza], and 1% amphotericin B [Lonza]). Cells were plated in T75 plastic dishes and VOL. 102 NO. 2 / AUGUST 2014
Fertility and Sterility® incubated at 37 C in 95% air–5% CO2. The growth medium was changed after 24 hours or 48 hours to remove unattached cells and then subsequently twice weekly. The purity of cells was assessed by immunocytochemical staining with a specific smooth muscle cell marker, monoclonal mouse anti-asmooth muscle actin (a-SMA) (Sigma-Aldrich). All cells were strongly positive for a-SMA (Fig. 1A). The lower passage number (up to four) of cells was used for experiments to avoid changes in phenotype and gene expression.
Cell Lines Culture The leiomyoma cell line was provided by William H. Catherino, M.D., Ph.D. (Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, Maryland). The primary myometrial and leiomyoma cells were immortalized following the modified protocol of Rhim (40) using human papillomavirus type 16 as previously described by Malik et al. (41). Cells were cultured in fresh DMEM-F12 supplemented with 10% fetal bovine serum, 1% antibiotic (penicillin–streptomycin; EuroClone), 1% fungizone (amphotericin B; Lonza), and 1% glutamine (Gibco, Life Technologies) at 37 C in 95% air–5% CO2.
RNA Isolation and Real-time Polymerase Chain Reaction Analysis All cell lines were treated with tranilast (300 mM) and negative control (only respective solvent, 1% DMSO as we used for tranilast) for 48 hours, lysed using TRIzol reagent (Ambion, Life Technologies), and stored at 80 C. Total RNA (colorless upper aqueous phase) was separated using chloroform according to the manufacturer's instructions. After that, RNA was purified and concentrated using the ReliaPrep RNA Cell Miniprep System (Promega Italia), and the levels of fibronectin, collagen 1A1, and versican messenger RNA (mRNA) were determined using real-time polymerase chain reaction (PCR) performed on the StepOnePlus detection system (Applied Biosystems, Life Technologies). Briefly, complementary DNA (cDNA) was generated from 1 mg of RNA using a high-capacity cDNA reverse transcriptase (RT) kit (Applied Biosystems, Life Technologies), and newly synthesized cDNA was used for real-time PCR. Real-time PCR was performed in 96-well optical reaction plates with 50 ng cDNA in a final volume of 15 mL, containing 1 Taqman Universal Master Mix, optimized concentrations of 6-carboxyfluorescein (FAM)-labelled probe, and specific forward and reverse primers for fibronectin (Hs00365052_m1), collagen 1A1 (Hs00164004_m1), versican (Hs00171642_m1), activin-A (Hs00170103_m1), and HPRT (Hs99999909_m1) (housekeeping gene) (Applied Biosystems, Life Technologies). Controls, consisting of RNA subjected to RT-PCR without reverse transcriptase, were included.
Immunocytochemistry Analysis Immunocytochemical staining was performed to detect fibronectin and collagen expression in human myometrial and leiomyoma cells, as previously described (42). Primary myometrial and leiomyoma cells were cultured in eight-well chamber tissue culture slides and treated with tranilast (300 mM) and negative VOL. 102 NO. 2 / AUGUST 2014
control (only respective solvent, 1% DMSO as we used for tranilast) for 48 hours. At indicated times, slides were washed three times with phosphate-buffered saline (PBS), fixed in cold methanol (20 C) for 20 minutes, and again washed with PBS three times. Next, cells were treated with 0.2% Triton X-100 in PBS for 5 minutes and washed three times with PBS. Hydrogen peroxide was applied for 10 minutes to block peroxidase activity. After washing three times, cells were incubated with normal horse serum diluted 1:75 in 1% bovine serum albumin in PBS for 20 minutes at room temperature to block nonspecific background staining. Serum was removed, and cells were incubated with primary antibody (fibronectin [dilution of 1:600; SigmaAldrich] and type I collagen [1 mg/mL; Immunological Sciences]) for 1 hour at room temperature and then washed three times with PBS. Cells were incubated with biotinylated horse anti-mouse IgG at a dilution of 1:200 (Vector Laboratories). The peroxidase avidin-biotin-peroxidase complex method (Vector Laboratories) was performed for 1 hour at room temperature using 30 , 30 diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as chromogen. Slides were counterstained in Mayer's hematoxylin, dehydrated, and mounted with Eukitt solution (Kindler). The fibronectin and collagen expression was quantified by measuring the intensity of staining using a Nikon H600 L Microscope and an image analysis system (Adobe Photoshop 7.0).
Data Analysis Wilcoxon signed rank test was used for data analysis using GraphPad Prism version 4.01 for Windows. Differences were considered significant at P< .05.
RESULTS Effect of Tranilast on ECM in Myometrial and Leiomyoma Cells Considering that uterine leiomyoma is a fibrotic disorder, our aim was to check whether tranilast is able to alter the expression of ECM proteins overexpressed in leiomyoma (fibronectin, collagen, and versican) (17, 18, 43). The established dose of tranilast treatment was 300 mM, as reported by an earlier study (38). To verify the effective concentration of tranilast, in our preliminary experiments we also tested several concentrations (1, 10, 30, 100, 300, and 1,000 mM) to check fibronectin, collagen, and versican expression by real-time PCR in immortalized human leiomyoma cells. We found that lower concentrations (1, 10, 30, and 100 mM) of tranilast were not able to significantly reduce fibronectin, collagen, versican, and activin-A mRNA expression, whereas a high concentration (1,000 mM) of tranilast was toxic for the cells (data not shown). Therefore, we treated human primary myometrial and leioymoma cells and immortalized leiomyoma cells with tranilast at 300 mM for 48 hours. Real-time PCR showed that tranilast significantly reduced fibronectin, collagen 1A1, and versican mRNA expression in primary leiomyoma cells compared with untreated leiomyoma cells (Fig. 1B–D). Tranilast also significantly reduced fibronectin, collagen 1A1, and versican mRNA expression in primary myometrial cells (Fig. 1B–D). Immortalized human 599
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FIGURE 1
(A) Immunocytochemical staining of a-SMA in human primary myometrial and leiomyoma cells. (B–D) Real-time PCR of fibronectin, collagen 1A1, and versican in human primary myometrial and leiomyoma cells. Results are the mean SEM of three experiments performed using independent cell cultures from different tissues. Each experiment was done in triplicate. *P
