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Romidepsin reduces histone deacetylase activity, induces acetylation of histones, inhibits proliferation, and activates apoptosis in immortalized epithelial endometriotic cells

2010; Elsevier BV; Volume: 94; Issue: 7 Linguagem: Inglês

10.1016/j.fertnstert.2010.04.052

1556-5653

Patrick Imesch, Daniel Fink, André Fedier,

Reproductive System and Pregnancy

Romidepsin inhibited HDAC activity, produced acetylation of the histone proteins, up-regulated p21, and down-regulated cyclins B1 and D1, resulting in proliferation inhibition and apoptosis activation in 11z immortalized epithelial endometriotic cells. Our findings provide evidence that endometriotic cells are sensitive to the epigenetic effects of romidepsin and suggest that endometriosis may be therapeutically targeted by romidepsin. Romidepsin inhibited HDAC activity, produced acetylation of the histone proteins, up-regulated p21, and down-regulated cyclins B1 and D1, resulting in proliferation inhibition and apoptosis activation in 11z immortalized epithelial endometriotic cells. Our findings provide evidence that endometriotic cells are sensitive to the epigenetic effects of romidepsin and suggest that endometriosis may be therapeutically targeted by romidepsin. Histone deacetylase inhibitors (HDAC-i) are epigenetically acting agents which are usually well tolerated and show promising results in phase I/II clinical trials for the treatment of both hematologic malignancies and solid tumors. They inhibit the enzymatic activity of HDAC and cause the acetylation of histones, and therefore they are thought to facilitate gene expression. They also demonstrate significant anticancer activity, including the inhibition of proliferation, stimulation of differentiation, and activation of apoptosis (1Glaser K.B. HDAC inhibitors: clinical update and mechanism-based potential.Biochem Pharmacol. 2007; 74: 659-671Crossref PubMed Scopus (319) Google Scholar, 2Bolden J.E. Peart M.J. Johnstone R.W. Anticancer activities of histone deacetylase inhibitors.Nat Rev Drug Discov. 2006; 5: 769-784Crossref PubMed Scopus (2465) Google Scholar, 3Rasheed W.K. Johnstone R.W. Prince H.M. 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Gong Z. et al.Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish.Dev Biol. 2008; 317: 336-353Crossref PubMed Scopus (103) Google Scholar) and may have a role in the development of spina bifida when taken during pregnancy (11Gurvich N. Berman M.G. Wittner B.S. Gentleman R.C. Klein P.S. Green J.B. Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo.FASEB J. 2005; 19: 1166-1168PubMed Google Scholar). Romidepsin (also known as FK-228 and depsipeptide), which was originally isolated from a broth culture of Chromobacterium violaceum, is a natural bicyclic tetrapeptide-type member among the HDAC-i and shows anticancer activity (12Konstantinopoulos P.A. Vandoros G.P. Papavassiliou A.G. FK228 (depsipeptide): a HDAC inhibitor with pleiotropic antitumor activities.Cancer Chemother Pharmacol. 2006; 58: 711-715Crossref PubMed Scopus (65) Google Scholar). Endometriosis is a benign disease affecting 10%–15% women of reproductive age and is frequently associated with pelvic pain and infertility. It is characterized by the ectopic proliferation of endometrial tissue outside the uterine cavity (13Giudice L.C. Kao L.C. Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2437) Google Scholar, 14Farquhar C. Endometriosis.BMJ. 2007; 334: 249-253Crossref PubMed Scopus (129) Google Scholar, 15Rodgers A.K. Falcone T. Treatment strategies for endometriosis.Expert Opin Pharmacother. 2008; 9: 243-255Crossref PubMed Scopus (54) Google Scholar, 16Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1415) Google Scholar). Its pathophysiology is still not fully understood, but epigenetic alterations such as chromatin modification (17Wu Y. Guo S.W. Inhibition of proliferation of endometrial stromal cells by trichostatin A, RU486, CDB-2914, N-acetylcysteine, and ICI 182780.Gynecol Obstet Invest. 2006; 62: 193-205Crossref PubMed Scopus (53) Google Scholar, 18Wu Y. Starzinski-Powitz A. Guo S.W. Trichostatin A, a histone deacetylase inhibitor, attenuates invasiveness and reactivates E-cadherin expression in immortalized endometriotic epithelial cells.Reprod Sci. 2007; 14: 374-382Crossref PubMed Scopus (83) Google Scholar) and DNA methylation (19Wu Y. Halverson G. Basir Z. Strawn E. Yan P. Guo S.W. Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis.Am J Obstet Gynecol. 2005; 193: 371-380Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 20Wu Y. Strawn E. Basir Z. Halverson G. Guo S.W. Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis.Fertil Steril. 2007; 87: 24-32Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 21Wu Y. Strawn E. Basir Z. Halverson G. Guo S.W. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis.Epigenetics. 2006; 1: 106-111Crossref PubMed Scopus (236) Google Scholar) are thought to contribute to the pathogenesis of endometriosis. Recent in vitro studies suggest HDAC-i as a potential medical option in the treatment of endometriosis (16Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1415) Google Scholar, 22Wu Y. Guo S.W. Histone deacetylase inhibitors trichostatin A and valproic acid induce cell cycle arrest and p21 expression in immortalized human endometrial stromal cells.Eur J Obstet Gynecol Reprod Biol. 2008; 137: 198-203Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Wu Y. Guo S.W. Reconstructing cellular lineages in endometrial cells.Fertil Steril. 2008; 89: 481-484Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Because increased proliferation and decreased apoptosis are characteristics of endometriosis, we hypothesized that romidepsin, by reducing HDAC activity and producing histone acetylation, inhibits proliferation and activates apoptosis in an immortalized epithelial endometriotic cell line (11z). Primary peritoneal epithelial endometriotic cells were immortalized by in situ electroporation with SV-40 T-antigen. The characteristics of this cell line have been described elsewhere (24Zeitvogel A. Baumann R. Starzinski-Powitz A. Identification of an invasive, N-cadherin-expressing epithelial cell type in endometriosis using a new cell culture model.Am J Pathol. 2001; 159: 1839-1852Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 25Banu S.K. Lee J. Starzinski-Powitz A. Arosh J.A. Gene expression profiles and functional characterization of human immortalized endometriotic epithelial and stromal cells.Fertil Steril. 2008; 90: 972-987Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The 11z cells were cultured in Dulbecco modified Eagle medium (31053; Invitrogen, Basel, Switzerland) containing 10% fetal calf serum (Oxoid, Basel, Switzerland), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in an atmosphere with 10% CO2 and 95% humidity. Romidepsin was kindly provided by Gloucester Pharmaceuticals (Cambridge, MA), and stock solutions were prepared in dimethylsulfoxide (DMSO) and stored at −20°C. The methylthiazolyl tetrazolium (MTT) assay was used to determine the romidepsin-induced inhibition of 11z cell proliferation. Cells (30,000 in 200 μL medium) seeded into 96-well plates were incubated without or with romidepsin (10 nmol/L, 30 nmol/L, or 100 nmol/L) for 48 hours, 72 hours, 96 hours, 120 hours, or 144 hours 3 days after seeding. MTT dye (dissolved in phosphate-buffered saline solution [PBS]) was added to a final concentration of 500 μg/mL. Four hours later, the medium was removed, the crystals were dissolved in 200 μL DMSO, and optical density (OD, absorbance at 540 nm) was measured spectrophotometrically (SpectraFluor Plus Reader; Tecan, Hombrechtikon, Switzerland). Data are presented as relative proliferation (calculated from OD data) as a function of romidepsin concentration and duration of treatment. Statistical analysis was performed using the two-tailed Student t test. P values of <.05 were considered to be statistically significant. Cell lysates to determine the HDAC enzymatic activity in 11z cells and to perform immunoblot analyses were produced from untreated and romidepsin-treated 11z cultures following standard laboratory protocols (26Sambrook J. Fritsh E. Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York1989Google Scholar). These cultures were subconfluent at the time of analysis (to avoid undesired effects due to factors generated after contact inhibition). To determine HDAC enzymatic activity in lysates from untreated 11z cultures, a colorimetric HDAC activity assay kit (ab1432; Abcam, Cambridge, U.K.) was used. Briefly, these lysates were assayed for HDAC enzymatic activity in absence (untreated control) or presence of various concentrations of romidepsin (as indicated) according to the manufacturer's instructions (including all standard assays). This assay was performed under conditions where neither the sample enzymatic activity, the substrate, nor the assay incubation time was rate limiting. The OD (absorbance at 405 nm) was measured spectrophotometrically (SpectraFluor Plus Reader). Enzymatic activity was normalized per amount of protein and expressed as OD. Romidepsin-mediated reduction of HDAC activity is presented as the relative values of the enzymatic activities (percentage of untreated control). To determine romidepsin-induced histone acetylation and alterations in protein expression, cells grown to 60% confluence were treated with various concentrations of romidepsin for various periods of time, washed in PBS, and lysed. Protein concentration of cell lysates was determined by the BCA Protein Assay Kit (23227; Pierce, Perbio Science, Lausanne, Switzerland). Immunoblot analysis was performed according to standard laboratory protocols (26Sambrook J. Fritsh E. Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York1989Google Scholar). Twenty micrograms of cell lysate protein was separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by blotting onto a polyvinylidene difluoride membrane (Amersham Biosciences, Otelfingen, Switzerland). Proteins were detected by the specific primary antibodies and the respective secondary horseradish peroxidase (HRPO)–conjugated antimouse (M15345; Transduction Laboratories, Lexington, KY) or HRPO-conjugated antirabbit (7074, Cell Signaling, BioConcept, Allschwil, Switzerland) antibodies. The following primary antibodies were used: HDAC-1 (2062; Cell Signaling), HDAC-2 (05–814; Lake Placid, NY), HDAC-3 (2632; Cell Signaling), HDAC-4 (2072; Cell Signaling), HDAC-5 (2082; Cell Signaling), HDAC-6 (2162; Cell Signaling), acetyl-H2A (2576; Cell Signaling), acetyl-H2B (2575; Cell Signaling), acetyl-H3 (9671; Cell Signaling), acetyl-H4 (2594; Cell Signaling), caspase-3 (9662; Cell Signaling; recognizing both the 37-kD full-length caspase-3 and the cleaved 17-kD fragment), caspase-9 (9502; Cell Signaling; recognizing both the 47-kD full-length caspase-9 and the cleaved 37-kD fragment), and poly(ADP-ribose) polymerase (PARP) 1 (9542; Cell Signaling; recognizing both the 116-kD full-length PARP-1 and the cleaved 89-kD fragment), p21 (2946; Cell Signaling), p27 (2552; Cell Signaling), cyclin B1 (4135; Cell Signaling), cyclin D1 (2926; Cell Signaling), multi-drug resistance (MDR) (sc-13131; Santa Cruz Biotechnology, Santa Cruz, CA), and multidrug resistance–associated protein (MRP) 1 (sc-18835; Santa Cruz Biotechnology). Mouse β-actin (A5441; Sigma, Buchs, Switzerland) was used as sample loading control. Complexes were visualized by enhanced chemiluminescence (Amersham Biosciences) and autoradiography. We determined the effects of romidepsin on the HDAC enzymatic activity and on histone acetylation. Our results showed that 11z cells expressed class I HDAC-1, HDAC-2, HDAC-3, and HDAC6 and class II HDAC-4 and HDAC-5 (Fig. 1A). Romidepsin reduced HDAC enzymatic activity (IC50 = 6.5 ± 0.6 nmol/L) in a concentration-dependent manner (Fig. 1B) and produced a time- and concentration-dependent accumulation of the acetylated histones H2A, H2B, H3, and H4 (Fig. 1C). Detectable levels of histone acetylation were observed at 24 hours in response to 25 nmol/L or 100 nmol/L romidepsin. With lower romidepsin concentrations (10 nmol/L), histone acetylation was detectable after 48 hours. The 11z cells thus responded to nanomolar concentrations of romidepsin in a way typically seen with HDAC-i in tumor cells, indicating that 11z cells are sensitive to the epigenetic effect of romidepsin. The observed responses were closely associated with and functionally related to proliferation inhibition and apoptosis activation. This relationship is one of the key findings, because it was demonstrated for the first time in epithelial endometriotic cells. Trichostatin A and valproic acid have been shown to negatively affect proliferation of these cells (17Wu Y. Guo S.W. Inhibition of proliferation of endometrial stromal cells by trichostatin A, RU486, CDB-2914, N-acetylcysteine, and ICI 182780.Gynecol Obstet Invest. 2006; 62: 193-205Crossref PubMed Scopus (53) Google Scholar, 22Wu Y. Guo S.W. Histone deacetylase inhibitors trichostatin A and valproic acid induce cell cycle arrest and p21 expression in immortalized human endometrial stromal cells.Eur J Obstet Gynecol Reprod Biol. 2008; 137: 198-203Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), but neither the effects of these agents on HDAC activity and histone acetylation nor those on apoptosis have been studied. We determined the effects of romidepsin on the expression of some cell cycle regulatory proteins and on proliferation. P21 and p27 are members of the family of endogenous cyclin-dependent kinase inhibitors that negatively regulate cell cycle progression and are therefore relevant for proliferation inhibition in tumors (27Sandor V. Senderowicz A. Mertins S. Sackett D. Sausville E. Blagosklonny M.V. et al.P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228.Br J Cancer. 2000; 83: 817-825Crossref PubMed Scopus (276) Google Scholar, 28Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.Proc Natl Acad Sci U S A. 2000; 97: 10014-10019Crossref PubMed Scopus (1002) Google Scholar). The induction of p21 gene expression, the concomitant acetylation of histones, and the subsequent cell cycle arrest and proliferation inhibition are well known responses to HDAC-i in tumor cells (28Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.Proc Natl Acad Sci U S A. 2000; 97: 10014-10019Crossref PubMed Scopus (1002) Google Scholar). P27 can also be induced by HDAC-i in some tumor cells (29Dedes K.J. Dedes I. Imesch P. von Bueren A.O. Fink D. Fedier A. Acquired vorinostat resistance shows partial cross-resistance to “second-generation” HDAC inhibitors and correlates with loss of histone acetylation and apoptosis but not with altered HDAC and HAT activities.Anticancer Drugs. 2009; 20: 321-333Crossref PubMed Google Scholar). P21 and p27 may also have a role in endometriosis. The HDAC-i trichostatin A and valproic acid up-regulated p21 in epithelial endometriotic cells (22Wu Y. Guo S.W. Histone deacetylase inhibitors trichostatin A and valproic acid induce cell cycle arrest and p21 expression in immortalized human endometrial stromal cells.Eur J Obstet Gynecol Reprod Biol. 2008; 137: 198-203Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and p27 seems to be down-regulated in the endometrium of women with endometriosis (30Schor E. da Silva I.D. Sato H. Baracat E.C. Girão M.J. de Freitas V. P27Kip1 is down-regulated in the endometrium of women with endometriosis.Fertil Steril. 2009; 91: 682-686Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is possible that p27 gene expression may be epigenetically silenced in endometriotic cells and therefore might be reactivated by romidepsin. Our results showed that low nanomolar concentrations (2–5 nmol/L) of romidepsin rapidly (within 6 h) upregulated p21 in a time- and concentration-dependent manner (Fig. 1D) and thus preceded histone acetylation. P21 up-regulation is perhaps a more sensitive marker of the effect of romidepsin in 11z cells. Measurable accumulation of acetylation may take time, but the pleotropic effects of HDAC-i appear to occur more quickly. P27 expression was not found in these cells and neither was an induction of p27 expression by romidepsin (Fig. 1D). Romidepsin also down-regulated the expression of the cyclins B1 and D1, two positive regulators of cell cycle progression (Fig. 1E). Romidepsin thus reduced proliferation of these cells in a concentration-dependent manner (Fig. 1F). Untreated control cultures continued proliferating, but cultures treated with romidepsin ceased to proliferate after 72 hours of treatment (P<.05 vs. control). The observed antiproliferative effect of romidepsin is important because increased proliferation is one characteristic of endometriosis. Another characteristic of endometriosis is reduced susceptibility to apoptosis. Our results demonstrated that romidepsin induces apoptosis in 11z cells. Romidepsin produced proteolytic cleavage of the 47-kD initiator procaspase-9 into its 37-kD fragment, the 37-kD effector procaspase-3 into its 17-kD fragment (Fig. 1G), and the 116-kD PARP-1 precursor into its 89-kD fragment in a concentration- and time-dependent manner (Fig. 1H). PARP-1 cleavage, an established measure for ongoing apoptosis, was detectable after 24 hours and 48 hours in cells treated with 20 nmol/L and with 10 nmol/L romidepsin, respectively. Romidepsin activated apoptosis in 11z cells in a close time and concentration correlation with the acetylation of histones, suggesting that histone acetylation is a prerequisite of apoptosis activation. This apoptosis was clearly caspase dependent. Caspases are a family of proteases that are one of the main executors of the apoptotic process. They exist as inactive zymogens and are cleaved to form active enzymes after the induction of apoptosis (31Danial N.N. Korsmeyer S.J. Cell death: critical control points.Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (3959) Google Scholar). The involvement of caspase-9 indicates that romidepsin activated the intrinsic apoptotic pathway in the 11z epithelial endometriotic cells. Caspase-9 is the initiator caspase of the intrinsic apoptotic pathway. It is activated upon release of cytochrome c from the mitochondria and can activate effector caspases, such as caspase-3. This leads to the cleavage of key cellular proteins (e.g., PARP-1 and cytoskeletal proteins) to drive forward the biochemical events that culminate in death and the dismantling of the cell. By activating intrinsic apoptosis, romidepsin might overcome the apoptosis resistance brought about by the occasionally altered Fas–Fas ligand system of the extrinsic apoptotic pathway in endometriotic cells (32Cakmak H. Guzeloglu-Kayisli O. Kayisli U.A. Arici A. Immune-endocrine interactions in endometriosis.Front Biosci. 2009; 1: 429-443Google Scholar). The expression of MDR and MRP-1, two multidrug resistance transporters, confers a multidrug apoptosis resistance phenotype. Romidepsin can induce the expression of MDR and MRP-1 in tumor cells and is a substrate for these transporters (33Okada T. Tanaka K. Nakatani F. Sakimura R. Matsunobu T. Li X. et al.Involvement of P-glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamily of histone deacetylase inhibitors in the drug-resistant osteosarcoma and Ewing's sarcoma cells.Int J Cancer. 2006; 118: 90-97Crossref PubMed Scopus (48) Google Scholar, 34Xiao J.J. Huang Y. Dai Z. Sadée W. Chen J. Liu S. et al.Chemoresistance to depsipeptide FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone] is mediated by reversible MDR1 induction in human cancer cell lines.J Pharmacol Exp Ther. 2005; 314: 467-475Crossref PubMed Scopus (67) Google Scholar, 35Robey R.W. Zhan Z. Piekarz R.L. Kayastha G.L. Fojo T. Bates S.E. Increased MDR1 expression in normal and malignant peripheral blood mononuclear cells obtained from patients receiving depsipeptide (FR901228, FK228, NSC630176).Clin Cancer Res. 2006; 12: 1547-1555Crossref PubMed Scopus (87) Google Scholar). However, MDR and MRP-1 were neither expressed in 11z cells nor induced by romidepsin (data not shown). Taken together, the present study demonstated that 11z epithelial endometriotic cells are responsive to romidepsin in a manner typically seen with HDAC-i in tumor cells. Romidepsin is well studied in cancer patients and seems to be relatively well tolerated. In line with the putative contribution of epigenetic alterations to endometriosis, epigenetically acting romidepsin may be a candidate to therapeutically encounter endometriosis. Its therapeutic efficiency may not be compromised by resistance mechanisms arising from expression of multidrug resistance transporters. The authors thank Dr. Anna Starzinski-Powitz for kindly providing the 11z cell line and Gloucester Pharmaceuticals for kindly providing romidepsin.