{"paper_id":"9dd90049-2f18-4488-b6a2-3ba974951f61","body_text":"R E S E A R C H Open Access\nArcyriaflavin a, a cyclin D1 –cyclin-\ndependent kinase4 inhibitor, induces\napoptosis and inhibits proliferation of\nhuman endometriotic stromal cells: a\npotential therapeutic agent in\nendometriosis\nTomoko Hirakawa 1* , Kaei Nasu 1,2, Yoko Aoyagi 1, Kanetoshi Takebayashi 1 and Hisashi Narahara 1\nAbstract\nBackground: We previously showed that microRNA-503 (miR-503) transfection into endometriotic cyst stromal cells\n(ECSCs) induced cell cycle arrest at the G0/G1 phase by suppressing cyclin D1. This finding prompted us to evaluate the\npotential therapeutic effects of cyclin D1 inhibitors in endometriotic cells. This study aimed to determine whether arcyriaflavin\nA, a representative inhibitor of cyclin D1–cyclin-dependent kinase 4 (CDK4), is beneficial in the treatment of endometriosis.\nMethods:ECSCs were isolated from the ovarian endometriotic tissues of 32 women. The effects of arcyriaflavin A on cell\nviability and proliferation, vascular endothelial growth factor A expression, apoptosis, and cell cycle progression were\nevaluated using a modified methylthiazoletetrazolium assay,enzyme-linked immunosorbent assay (ELISA), Caspase-Glo®\n3/7 assay, and flow cytometry.\nResults: Arcyriaflavin A significantly inhibited cell viability, proliferation, and angiogenesis of ECSCs as assessed using the\n5-bromo-2-deoxyuridine (BrdU) and methylthiazoletetrazolium bromide (MTT) assays, and vascular endothelial growth\nfactor (VEGF) ELISA. Arcyriaflavin A induced apoptosis as shown in the Caspase-Glo® 3/7 assay and cell death detection\nELISA whilethe cell cycle was arrested at the G0/G1 phase.\nConclusion: The findings indicate that cyclin D1–CDK4 inhibitors may be promising candidates for the treatment of\nendometriosis. This is the first study to demonstrate the potential usefulness of arcyriaflavin A as a therapeutic agent\nfor endometriosis. Further studies of the effects of cyclin D1 –CDK4 inhibitors on endometriosis may provide useful\ninformation on pathogenesis and treatment.\nKeywords: Endometriosis, Cyclin D1 inhibitor, Cell cycle, Apoptosis, Cell proliferation\n* Correspondence: tomokoh@oita-u.ac.jp\n1Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University,\nIdaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan\nFull list of author information is available at the end of the article\n© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nHirakawa et al. Reproductive Biology and Endocrinology  (2017) 15:53 \nDOI 10.1186/s12958-017-0272-3\n\nBackground\nEndometriosis is an estrogen-dependent condition char-\nacterized by the benign ectopic growth of proliferative\nendometrial tissue. It most frequently occurs in women\nof reproductive age and usually involves the peritoneum,\novaries, and rectovaginal septum [1]. Its main symptoms\nare dysmenorrhea, chronic pelvic pain, subfertility, and\ndyspareunia, which often greatly decrease the quality of\nlife of the patients [1].\nAlthough endometriotic tissue shares many histological\ncharacteristics with normal proliferative endometrial tis-\nsues [1], there are several interesting molecular differences\nsuch as in gene expression and protein production, syn-\nthesis, and responsiveness to steroids and cytokines.\nEndometriotic cells exhibit proliferative, antiapoptotic,\nangiogenic, and fibrogenic features [2– 5].\nTo identify the mechanisms underlying the pathogenesis\nof endometriosis, our research has focused on the dysregu-\nlation of the expression of several microRNAs (miRNAs)\ninvolved in endometriosis [4 – 6]. miRNAs, which regulate\nthe translation of specific targeted protein-coding genes,\na r es h o r tn o n c o d i n gR N A s .I nap r e v i o u ss t u d y ,w eo b -\nserved the anti-proliferative, pro-apoptotic, angiostatic, and\nanti-fibrogenic functions of miR-503 and identified its pos-\nsible downstream targets using miR-503-transfected endo-\nmetriotic cyst stromal cells (ECSCs) [5]. The transfection\nof miR-503 into ECSCs induces apoptosis by B-cell\nlymphoma-2 (Bcl-2) suppression, inhibition of vascular\nendothelial growth factor A (VEGF-A) production and cell\nproliferation, and induction of cell cycle arrest at the G0/\nG1 phase by cyclin D1 suppression [5]. These findings\nprompted us to evaluate the therapeutic effects of cyclin\nD1 inhibitors on endometriotic cells.\nNumerous studies have reported the functions of cyc-\nlin D1 and cyclin-dependent kinase (CDK). Cyclin D1\ninteracts with the enzymes CDK 4/6 to regulate cell\ncycle progression from the G1 phase to the S phase [7].\nThere are few reports on the effects of cyclin D1 – CDK4\ninhibitors on endometriosis.\nWe hypothesized that cyclin D1 and CDK inhibitors\nmight alleviat endometriosis. Arcyriaflavin A, a represen-\ntative cyclin D1 – CDK4 inhibitor, exhibited potent inhib-\nition of cyclin D1 – CDK4 [8]. Furthermore, it was\npreviously described as a novel antiviral compound [9]\nand was reported to induce cancer cells apoptosis [10].\nTherefore, we designed the present study to evaluate the\nefficacy of arcyriaflavin A in treating endometriosis.\nMethods\nIsolation of ECSCs and cell culture conditions\nOvarian endometriosis tissue_samples were obtained from\npatients with regular menstrual cycles who had undergone\nsalpingo-oophorectomy or cystectomy for the treatment of\novarian endometriotic cysts (27 patients, aged 26– 45 years).\nNone of the patients had received any hormonal treatments\nfor at least 6 months prior to surgery. All specimens were\nconfirmed to_be in the mid- to late-proliferative phases\nbased on pathological observation, the menstrual cycles, or\nboth. The ECSCs were isolated from the ovarian\nendometriotic tissues using enzymatic digestion with\ncollagenase, as previously described [11]. Isolated\nECSCs were cultured in Dulbecco ’ s modified Eagle ’ s\nmedium (DMEM) supplemented with 100 IU/mL\npenicillin, 50 mg/mL streptomycin, and 10% heat-\ninactivated fetal bovine serum (FBS, all obtained from\nGibco-BRL, Gaithersburg, MD, USA) at 37 °C in air\ncontaining 5% CO 2. The purity of ECSCs in the\nmonolayer culture after the third passage was >99%,\ndetermined using immunocytochemical staining with\nantibodies for vimentin, CD10, cytokeratin, factor\nVIII, and leukocyte common antigen [11]. Each ex-\nperiment was performed in triplicate and repeated\nwith at least three replicates with cells from at least\nthree different patients.\nAssessment of viability of arcyriaflavin A-treated cells\nThe viability of ECSCs after arcyriaflavin A treatment\nwas determined using a modified methylthiazoletetrazo-\nlium (MTT) assay using the cell proliferation kit I\n(Roche Diagnostics GmbH, Basel, Switzerland). Briefly,\n5×1 0 3 ECSCs were placed into each well of 96-well\nflat-bottomed microplates (Corning, New York, NY,\nUSA) and incubated with arcyriaflavin A (0.1 – 10 μM, di-\nluted with culture medium, Santa Cruz Biotechnology,\nDallas, TX, USA) for 48 h. Thereafter, 20 μL of the MTT\ndye was added to each well, the cells were incubated for\n4 h, and then, the cell viability was determined by meas-\nuring the absorbance at 570 nm. The data were obtained\nfrom triplicate samples, and the values obtained from\nthe arcyriaflavin A-treated ECSCs are presented as a per-\ncentage of the values of untreated ECSCs.\nAssessment of proliferation of arcyriaflavin A-treated ECSCs\nThe proliferation of ECSCs following arcyriaflavin A\ntreatment was determined based on the 5-bromo-2 ′-\ndeoxyuridine (BrdU) incorporation using an enzyme-\nlinked immunosorbent assay (ELISA) kit (cell proliferation\nELISA, Roche Diagnostics) [6]. We seeded 5 × 10 3 ECSCs\ninto each well of a 96-well flat-bottomed microplate\n(Corning), incubated them with arcyriaflavin A (0.1 –\n10 μM) for 48 h, and then 10 μL BrdU (10 mM) was\nadded to each well, followed by incubation for 2 h. The\nincorporation of BrdU was determined by measuring the\nabsorbance of the resultant solution at 450 nm. The data\nwere obtained from triplicate samples, and the values of\nthe arcyriaflavin A-treated ECSCs are presented as a per-\ncentage of values of untreated ECSCs.\nHirakawa et al. Reproductive Biology and Endocrinology  (2017) 15:53 Page 2 of 5\n\nAssessment of VEGF-A protein levels of arcyriaflavin A-treated\nECSC culture supernatant\nThe protein levels of VEGF-A in the supernatant of\nECSCs following arcyriaflavin A treatment were deter-\nmined using an ELISA, as described previously [4]. Briefly,\nsubconfluent ECSCs treated without and with arcyriaflavin\nA( 0 . 1– 10 μM) were cultured for 48 h in 24-well culture\nplates (Corning), and the supernatants were collected. The\nconcentration of VEGF-A was determined using commer-\ncially available ELISA kits (Human VEGF immunoassay,\nR&D Systems, Minneapolis, MN, USA). The sensitivity of\nthe VEGF-A assay was 9.0 pg/mL.\nAssessment of apoptosis of arcyriaflavin A-treated ECSCs\nWe determined the apoptosis levels of ECSCs following\narcyriaflavin A treatment by direct determination of nu-\ncleosomal DNA fragmentation using an ELISA (cell death\ndetection ELISA, Roche Diagnostics) [6]. The ECSCs\n(5 × 10 3 cells/well) were placed in 96-well flat-bottomed\nmicroplates (Corning). After incubation with arcyriaflavin\nA( 0 . 1– 10 μM) for 48 h, the cells were lysed, centrifuged at\n200×g for 10 min, and the mono- and oligo-nucleosomes\nin the supernatants were quantified using an anti-histone-\nbiotin antibody. The concentration of the nucleosome-\nantibody complex was determined by measuring the\nabsorbance at 405 nm using 2,2 ′-azino-di(3-ethylbenzthia-\nzolinesulfonate) as the substrate. The data analyzed were\nfrom triplicate samples, and values of the arcyriaflavin A-\ntreated ECSCs are presented as a percentage of those from\nuntreated ECSCs.\nAssessment of caspase-3 and caspase-7 activities in\narcyriaflavin a treated ECSC\nThe caspase-3 and caspase-7 activities of ECSCs follow-\ning incubation with arcyriaflavin A were evaluated using\nthe Caspase-Glo® 3/7 assay (Promega, Madison, WI,\nUSA), as described previously [6]. The ECSCs (5 × 10 3\ncells/well) were plated in 96-well flat-bottomed micro-\nplates (Promega). After a 48-h incubation with arcyria-\nflavin A (0.1 – 10 μM), the Caspase-Glo® 3/7 reagent was\nadded to each well, the plates were shaken gently for\n120 min at 20 – 25 °C, and then the luminescence was\nmeasured using a plate-reading luminometer. The data\nanalyzed were of triplicate samples, and the values of\nECSCs treated with arcyriaflavin A are presented as a\npercentage of those of the untreated ECSCs.\nAssessment of cell cycle of arcyriaflavin A-treated ECSCs\nThe cell cycle of ECSCs following treatment with arcyr-\niaflavin A was analyzed using flow cytometry, as previ-\nously described [5, 12]. Briefly, 72 h after arcyriaflavin A\ntreatment (10 μM), the ECSCs were trypsinized, rinsed\nin phosphate-buffered saline, fixed in 70% ethanol, and\nthen incubated for 30 min at 4 °C in the dark with a\nsolution containing 5 μg/mL propidium iodide and\n1 mg/mL RNase (Sigma-Aldrich, St. Louis, MO, USA).\nFlow cytometric analysis of the cell cycle was performed\nafter propidium iodide staining using the CellFIT pro-\ngram (Becton-Dickinson, Franklin Lakes, NJ, USA),\nwhich analyzed the S-phase using a ModFit model.\nStatistical analysis\nThe data analyzed were of triplicate samples and are\npresented as a percentage relative to the corresponding\ncontrol values as the mean ± standard deviation. The\ndata were appropriately analyzed using the Bonferroni\nmethod and Student ’ s t-test using the SigmaPlot 11.2\n(Systat Software, Chicago, IL, USA) while a p < 0.05 was\nconsidered significant.\nResults\nSuppression of ECSC viability and proliferation by arcyriaflavin\nat r e a t m e n t\nThe effects of arcyriaflavin A on the viability and prolif-\neration of ECSCs were evaluated using modified MTT\nand BrdU incorporation assays, respectively. As shown\nin Fig. 1a, the number of viable cells decreased signifi-\ncantly after treatment with arcyriaflavin A at 1 and\n10 μM. Furthermore, arcyriaflavin A treatment signifi-\ncantly inhibited BrdU incorporation in ECSCs at 1 and\n10 μM (Fig. 1b).\nDownregulation of VEGF-A expression in ECSCs by\narcyriaflavin a treatment\nVEGF-A protein expression in ECSCs was suppressed by\narcyriaflavin A at 1 and 10 μM (Fig. 1c).\nInduction of ECSC apoptosis by arcyriaflavin a treatment\nThe effects of arcyriaflavin A on apoptosis in ECSCs\nwere determined using an ELISA kit. As shown in Fig. 1d,\narcyriaflavin A induced apoptosis at 10 μM. The pro-\napoptotic effects of arcyriaflavin A on ECSCs were also\nassessed by evaluating caspase-3 and caspase-7 activities,\nwhich were significantly at 10 μM (Fig. 1e).\nInduction of cell cycle arrest in ECSCs by arcyriaflavin a\ntreatment\nThe effects of arcyriaflavin A on the cell cycle were\ndetermined using flow cytometry. As shown in Fig. 1f,\narcyriaflavin A induced the accumulation of ECSCs in\nthe G0/G1 phase ( p = 0.000, Bonferroni method), with a\nconcomitant decrease in the proportion of cells in the S\nand G2/M phases ( p = 0.001 and p = 0.000, respectively;\nBonferroni method).\nDiscussion\nIn our previous study, we investigated the expression of\nmiR-503 in ECSCs and normal endometrial stromal cells\nHirakawa et al. Reproductive Biology and Endocrinology  (2017) 15:53 Page 3 of 5\n\nisolated from eutopic endometrial tissues. We evaluated\nthe effects of miR-503 on the cellular functions of\nECSCs and the mechanisms underlying the suppression\nof miR-503 expression in ECSCs. Transfection of ECSCs\nwith miR-503 inhibited cell proliferation and VEGF-A\nproduction and induced apoptosis and G0/G1 cell cycle\narrest in these cells [5]. Furthermore, we previously\ndemonstrated that Bcl-2 and VEGF-A expression was\nupregulated in ECSCs, and this upregulation was re-\nsponsible for the anti-apoptotic and angiogenic features\nof endometriosis [4, 13].\nBased on these findings, we designed the present study\nto investigate the potential of arcyriaflavin A, a cyclin\nD1-CDK4 inhibitor, for treating endometriosis. Similar\nto the effects of miR-503, arcyriaflavin A inhibited cell\nproliferation and VEGF-A production, and induced\napoptosis and G0/G1 cell cycle arrest in these cells\nmainly at 1 and 10 μM.\nCyclin-CDK complexes regulate the progression of\ncells through the cell cycle. Strong lines of evidence sug-\ngest the involvement of D-type cyclins in the G1 phase\nthrough an association with CDK4 and CDK6. Aberra-\ntions in CDKs and their regulators have been found in a\nlarge percentage of human tumors including melanoma,\nlymphoma, and carcinomas of the breast, lung, ovary,\noropharynx, and colon [8]. Ovarian endometriotic cyst is\nan ovarian tumor and endometriosis is a well-established\novarian cancer risk factor. Thus, inhibitors of cyclin-\nCDK complexes may offer a broad range of therapeutic\napplications in endometriosis.\nOver the last decade, many small molecules have\nbeen reported as CDK inhibitors. In particular, arcyria-\nflavin A has shown strong inhibitory activity (half-max-\nimal inhibitory concentration [IC 50]=1 4 0n M )a g a i n s t\ncyclin D1-CDK4. Interestingly, it also demonstrated sig-\nnificant selectivity towards several other kinases inclu-\nding cyclin B-CDK1, cyclin E-CDK2, protein kinase A\n(PKA), and protein kinase C (PKC) [8]. Furthermore,\narcyriaflavin A has been described as a novel antiviral\ncompound that selectively inhibits the replication of\nthe human cytomegalovirus [9].\nmiR-503 has been demonstrated to downregulate cyc-\nlin D1 expression and induce G0/G1 phase cell cycle ar-\nrest in several cell types [5, 7, 14]. We confirmed that\narcyriaflavin A induced cell cycle arrest in the G1 phase\nof ECSCs. Induction of apoptosis and inhibition of cell\nproliferation and angiogenesis follow cell cycle arrest.\nArcyriaflavin A exhibited a variety of therapeutic effects\non ECSCs that are similar to those induced by miR-503\ntransfection. A number of CDK 4/6 inhibitors have been\n25\n0\n75\n100\n50\nArcyriaflavin A (µµM)\n0.1 1 10\nBrdU incorporation (%) *\n**\n25\n0\n75\n100\n50\nArcyriaflavin A (µM)\nCell viability (%)\n**\n0.1 1 10\n**\nArcyriaflavin A (µM)\n0.1 1 10\n50\n0\n150\n200\n100\n0.1 1 10\nCaspase 3/7 activity (%)\n*\n0\n75\n100\n50\nControl\nCells (%)\n25\nG0/G1\nS\nG2/M\n**\n** **\nArcyriaflavin A (µM)\n(a)\n(d)\n(b) (c)\n(f)(e)\nArcyriaflavin A\n(10 µM)\n50\n0\n150\n100\n****\nVEGF-A protein levels (%)\n50\n0\n150\n200\n100\nArcyriaflavin A (µM)\n0.1 1 10\nApoptotic cells (%)\n**\nFig. 1 Therapeutic effects of arcyriaflavin A on endometriotic cyst stromal cells (ECSCs).a Cell viability; b 5-bromo-2-deoxyuridine (BrdU) incorporation;\nc vascular endothelial growth factor (VEGF)-A protein level;d apoptotic activity; e caspase-3/7 activity;f cell cycle progression. a–e ECSCs were analyzed\nfollowing 48-h incubation with arcyriaflavin A. f ECSCs were analyzed using flow cytometry following a 72-h incubation with arcyriaflavin A. *p < 0.05\nand **p < 0.005, Bonferroni method\nHirakawa et al. Reproductive Biology and Endocrinology  (2017) 15:53 Page 4 of 5\n\nevaluated for clinical use [15 – 19]. With the development\nof more effective and less toxic agents, cyclin D1 inhibi-\ntors could be used clinically for treating endometriosis\nin the future.\nThere are some limitations to the present study. First,\nthe effects of arcyriaflavin A were evaluated only in\nECSCs, but not in normal endometrial stromal cells.\nAnother limitation is the study design. There is currently\nno established protocol for assessing the effects of\narcyriaflavin A on endometriotic lesions in vivo.\nConclusion\nWe demonstrated that the cyclin D1-CDK4 inhibitor,\narcyriaflavin A, exerted therapeutic effects on ECSCs that\nare similar to those of miR-503, which is considered a\npromising candidate for the treatment of endometriosis.\nFuture studies on the effects of cyclin D1-CDK4 inhibitors\non endometriosis may provide useful information on the\npathogenesis of this condition.\nAbbreviations\nCDK:Cyclin-dependent kinase; DSBs: DNA double-strand breaks; ECSCs: Endometriotic\nc y s ts t r o m a lc e l l s ;m i R N A s :m i c r o R N A s ;P K A :P r o t e i nk i n a s eA ;V E G F - A :V a s c u l a r\nendothelial growth factor A\nAcknowledgements\nNot applicable.\nFunding\nThis work was supported in part by Grants-in-Aid for Scientific Research from\nthe Japan Society for the Promotion of Science (no. 13237327 to K. Nasu, and\nno. 23592407 to H. Narahara).\nAvailability of data and materials\nThe data of this study are available from the corresponding author.\nAuthors’ contributions\nTH, KN, and HN participated in the study design, analysis, and manuscript\ndrafting. YA, KT, and TH executed the study. All autho\nrs read and approved the final manuscript.\nEthics approval and consent to participate\nThis study was approved by the Institutional Ethics Committee of the Faculty\nof Medicine, Oita University (registration number: P-16-01). Written informed\nconsent was obtained from all patients.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interest.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in published\nmaps and institutional affiliations.\nAuthor details\n1Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University,\nIdaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan.2Division of\nObstetrics and Gynecology, Support System for Community Medicine, Faculty\nof Medicine, Oita University, Oita Prefecture, Oita 879-5593, Japan.\nReceived: 19 April 2017 Accepted: 28 June 2017\nReferences\n1. Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364:1789 –99.\n2. Nasu K, Yuge A, Tsuno A, Narah ara H. 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PD 0332991,\na selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of\nluminal estrogen receptor-positive human breast cancer cell lines in vitro.\nBreast Cancer Res. 2009;11:R77.\n19. Michel L, Ley J, Wildes TM, Schaffer A, Robinson A, Chun SE, et al. Phase I\ntrial of palbociclib, a selective cyclin-dependent kinase 4/6 inhibitor, in\ncombination with cetuximab in patients with recurrent/metastatic head and\nneck squamous cell carcinoma. Oral Oncol. 2016;58:41 –8.\nHirakawa et al. Reproductive Biology and Endocrinology  (2017) 15:53 Page 5 of 5","source_license":"CC0","license_restricted":false}