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Histone deacetylase inhibitor, Romidepsin (FK228) inhibits endometrial cancer cell growth through augmentation of p53-p21 pathway
Biomedicine & Pharmacotherapy 82 (2016) 161–166
Available online at
ScienceDirect www.sciencedirect.com
Original article
Histone deacetylase inhibitor, Romidepsin (FK228) inhibits endometrial cancer cell growth through augmentation of p53-p21 pathway Lu-Hong Lia,* , Pei-Ru Zhanga , Pei-Ya Caia , Zhi-Chao Lib a b
Department of Obstetrics and Gynecology, Second Affiliated Hospital of Fujian Medical College, China Department of Radiology, Second Affiliated Hospital of Fujian Medical College, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 March 2016 Received in revised form 25 April 2016 Accepted 25 April 2016
Objective: Romidepsin (FK228), a Histone Deacetylase (HDAC) inhibitor, has been used for anti-cancer therapies. However, the anti-cancer effect of FK228 and its underlying mechanism in endometrial carcinoma (EC) have not been studied. The aime of this study was to investigate the anti-cancer effects of FK228 and the associated mechanism(s) in EC. Methods: Ishikawa and HEC-1-A endometrial cancer cells were treated with 8 nM concentration of FK228 and cell growth was measured by XTT assay. The cell cycle distribution and cell death were measured by flow cytometry, immunofluorescence, respectively. The mNRA and protein expressions were analyzed by quantitative RT-PCR and western blot, respectively. Results: Based on assays carried out in EC cell lines, it was observed that FK228 inhibited EC cell proliferation in a dose and time-dependent manner. Furthermore, following treatment with FK228 for 48 h, there were significant induction of apoptosis and cell cycle arrest at G0/G1 phase in EC cells. Moreover, FK228 treatment significantly increased the mRNA and protein expressions of p53, p21, cleaved caspases such as 3, 7 and 8 and PARP. Further, FK228 treatment increased the levels of acetylated histone H3 and H4 that confirms the HDAC inhibition. Conclusion: In conclusion, FK228 inhibits EC tumor cell proliferation and induces apoptosis by activation caspase/PARP via the induction of p53/p21 signaling cascades, suggesting that FK228 is a potential therapeutic agent for EC. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Endometrial cancer FK228 Apoptosis Cell-cycle and p53
1. Introduction Endometrial cancer (EC) is the most common malignancy diagnosed in women and the incidence of EC is increasing in the world wide. In the United States, EC is now the fourth common cancer among women, with an estimated 54, 870 new cases and 10, 170 deaths in 2015 [1]. The most EC cases are diagnosed after menopause, however; 14% of EC are diagnosed before menopause and the age of 40 [2,3]. The standard treatment for EC are chemotherapy with cytotoxic agents and surgical staging such as hysterectomy, bilateral salpingo-oophorectomy and sampling of lymph nodes[4]. However, EC patients with surgery and long-term exposure of chemotherapeutic drugs that leads to have significant medical comorbidities. Therefore, finding new targets for therapy
* Corresponding author at: Department of Obstetrics and Gynecology, Second Affiliated Hospital of Fujian Medical College, No. 34, Zhongshan North Road, Licheng District, Quangzhou, Fujian 362000, China. E-mail address: [email protected] (L.-H. Li). http://dx.doi.org/10.1016/j.biopha.2016.04.053 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
and therapeutic options are necessary to decrease the morbidity and mortality observed with advanced stage EC. Histone deacetylase (HDAC) and DNA methyltransferases (DNMTs) are important factors for the dysregulation of epigenetic process which leads to altered gene expression and play important role in a multiple cancers [5,6]. The differential activities of HDAC are associated with certain types of human cancer, including EC human cancer thereby affecting signal transduction pathways and the expression of tumor suppressor genes such as p53 and p21/ WAF, [7–9]. The drugs including HDAC inhibitors are a promising class of anti-cancer drugs because of their ability to inhibit/activate cancer cell proliferation, cell cycle arrest, apoptosis, metastasis and angiogenesis [10–12]. The HDAC inhibitors such as such as valproic acid, suberoylanilide hydroxamic acid (SAHA), LBH-589 and romidepsin (FK228) have been shown to have anti-neoplastic effects in a variety of tumor [10]. The advantage of HDAC inhibitors are less toxic alternative to standard therapy due its selective targeting effect of cancer cells versus normal cells [13]. Recently, FK228 has been approved by FDA for the treatment of T cell
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cutaneous lymphoma and now it is in phase II clinical trial in solid tumors and hematopoietic malignancies [14]. To best of our knowledge, the role(s) and mechanism(s) of action of FK228 in EC cells has not been examined. Hence, in the present study, we intended to determine the effect of FK228 on the suppression of EC cell growth and its mechanism(s) of action. We show here that FK228 inhibits EC cell growth, induces cell death and arrest cells at G0/G1 phase by augmenting p53-p21 signaling pathway. 2. Materials and methods 2.1. Reagents Romidepsin (FK228), penicillin/streptomycin and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide were as purchased from Sigma (St. Louis, MO). All cell culture media were obtained from Invitrogen Life Technologies (Grand Island, NY). Antibodies including p53, p21, cleaved caspase-7, cleaved PARP, acetylated H3 and H4 and b-actin were procured from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). 2.2. Cell culture
Fig. 1. FK228 reduces endometrial cell proliferation in a dose-dependent manner. (A) Ishikawa and (B) HEC-1-A cells were treated with different concentrations of FK228 (0, 4, 8, 16, 32 and 64 nM) for 48 h and their survival rate was then measured using a XTT assay as described in materials and methods. Data are reported as the means standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
Ishikawa and HEC-1-A, the human endometrial cell lines were procured from Chinese Academy of Sciences Committee Type Culture Collection cell bank (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 10% FBS, 1X penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 C. 2.3. Cell proliferation assay Ishikawa and HEC-1-A cells were plated in 96-well plate at 50,000 cells/ml and treated with different concentrations (0, 4, 8 and 16 nM) of FK228 for 48 h. After 72 h of treatment, 50 ml of XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide) was added and incubated at 37 C for 2 h and then read absorbance at 450. 2.4. Annexin V staining Cell death were determined using apoptosis cell quantitation kit (Biotium, Hayward, USA). Ishikawa and HEC-1-A cells were seeded in 2-well chamber coverglass slides. After 24 h of cells attached to the slides, cells were treated either vehicle (DMSO) or 8 nM of FK228 for 48 h. After 48 h of treatment, cells were stained with 5 ml of Annexin V and Hoechst 33342 dye to 100 ml of 1 binding buffer and incubate for 15 min at room temperature in the dark. The cells were washed with 1 binding buffer and observe under fluorescence microscope at 20 objective. 2.5. Cell death analysis Cell death were analyzed by using FITC Annexin V kit according to the manufacturer’s instruction. FK228 treated cells were pelleted by centrifugation at 1000 rpm for 5 min. The pellets were suspended in binding buffer and add Annexin V-FITC (5 ml) and propidium iodide (50 mg/ml) and incubated at room temperature for 5 min in the dark. FITC Annexin V stained cells were analyzed in a FACScan.
Fig. 2. FK228 reduces endometrial cell proliferation in a time-dependent manner. (A) Ishikawa and (B) HEC-1-A cells were treated with 8 nM of FK228 for 24, 48 and 72 h and their survival rate was then measured using a XTT assay as described in materials and methods. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
2.6. Cell cycle analysis Ishikawa and HEC-1-A cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 48 h and then fixed with 75% ethanol at 20 C. The ethanol fixed cells were resuspended in PBS with
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Fig. 3. FK228 induces apoptosis in EC cells. (A, B) Ishikawa and (C, D) HEC-1-A cells were treated with 8 nM of FK228 for 48 h and their apoptotic index was measured using Annexin V staining by immunofluorescence and FACS as described in materials and methods. Green fluorescent staining shows apoptotic cells. All nuclei are stained with blue fluorescence. Bar graph show the quantitation of apoptotic cells as expressed relative green fluorescence. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
addition of RNase A (1 mg/ml) and incubated for 1 h at 37 C. Propidium iodide (50 mg/ml) was used for staining the fixed cells for 30 min at room temperature. The DNA contents of the stained cells were analyzed by using the CELL Quest Software with a FACScan flowcytometer.
2.7. Quantitative RT-PCR (qRT-PCR) Ishikawa cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 24 h and total RNA was isolated by using AxyPre TM Multi-source RNA miniprep kit (Axygen, USA). Two mg of total RNA was converted into complementary DNA (cDNA) with PrimeScript
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RT reagent kit (Takara, Japan). P53, p21, caspase-3, 7 and 8 and PARP mRNA expressions were measured by using specific TaqMan Probes and TaqMan assay kit (Life Technologies) according to manufacturer’s protocol with real-time PCR machine. GAPDH was used as a control to determine relative mRNA expression. 2.8. Immunoblot analysis Ishikawa cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 24 h and cells were lysed with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 1 mM PMSF. The protein concentrations were measured using the Bio-Rad assays. An equal amount proteins were separated in 10% SDS-PAGE, transferred to a nitrocellulose membrane. The membranes were incubated with indicated primary antibodies (p53, p21, cleaved caspase 3, 7, and 8 cleaved PARP, acetylated histone H3 and H4 and b-actin) for overnight at 4 C. After 5, 10 and 15 min washes with TBST, the membranes were incubated with corresponding secondary IgG antibodies for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescence reagent (ECL). 2.9. Statistical analysis Statistical analysis was performed by using SPSs 16.0 software. The data are expressed as mean standard deviation (SD). Oneway ANOVA followed by post hoc test was used to analysis significant differences between each sample. p < 0.05 values were considered statistically significant. 3. Results 3.1. FK228 inhibits EC proliferation Fist, to examine the different concentrations of FK228 on cell survival of two different endometrial cells, we treated Ishikawa and HEC-1-A cells with 0, 4, 8, 16, 32 and 64 nM concentrations of FK228 for 48 h and measured cell proliferation by XTT assay. As shown in Fig. 1A and B, the percentage of cell survival rate was decreased in a dose-dependent manner and 8 nM of FK228 was inhibited 50% of cell growth in both Ishikawa and HEC-1-A. Next we determine the time dependent cell growth inhibition of FK228. The cells were treated with 8 nM of FK228 for 24, 48 and 72 h and measured cell proliferation. The cell survival rate were significantly decreased in a time-dependent manner and 50% of inhibition was observed at 48 h (Fig. 2A and B). 3.2. FK228 induces EC cell death To investigate the effect of FK228 on apoptosis, we measured cell death by measuring apoptotic cells by immunofluorescence and FACS. Ishikawa and HEC-1-A cells were treated with 8 nM of FK228 for 48 h and measured apoptotic cells. There were significant increases of apoptotic cells as seen green fluorescence (Fig. 3A and C) and percentage of apoptotic cells measured by FACS (Fig. 3B and D) in FK228 treated both cells when compared to vehicle treated (Control) cells, indicating that FK228 induces cell death in both endometrial cells. 3.3. FK228 arrests cells at G0/G1 phase Fig. 4. FK228 regulates cell-cycle in EC cells. (A) Ishikawa and (B) HEC-1-A cells were treated with 8 nM of FK228 for 48 h. After 48 h, cells were harvested and fixed with 75% ethanol as described in materials and methods. Cells were stained with propidium iodide. Cell cycle distribution was assessed by flow cytometry. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control.
To confirm the cell death effect of FK228, we performed cellcycle to determine the positions of cells in the cell cycle were determined by flow cytometry. Consistent with apoptosis data (Fig. 3A and B), treatment of cells with FK228 for 48 h significantly increased the percentage of cells in G0/G1 from 40% to 80% (Fig. 4A
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increased in FK228 treated cells (Fig. 6A), indicating that activation of p53-p21 pathway due to FK228 treatment may be responsible for the induction of apoptosis by FK228 treatment. Further, treatment of FK228 significantly increased the acetylated histone H3 and H4 (Fig. 6B), suggesting that FK228 inhibits deacetylase activity that increases acetylation of histone H3 and H4. 4. Discussion
Fig. 5. FK228 increases the mRNA expression of p53, p21, caspases 3, 7 and 8 and PARP in EC cells. Ishikawa cells were treated with 8 nM of FK228 for 24 h and then subjected to qPCR analysis as described in materials and methods. GAPDH was used as endogenous control to analysis mRNA expression and expressed as relative mRNA expression. Values represent the mean standard deviation of three independent experiments. *P < 0.05 compared with control.
and B), showing that FK228 arrests Ishikawa and HEC-1-A cells at G0/G1 and consistent with cell death as shown in Fig. 3A and B. 3.4. FK228 increases the expression of p53, p21, caspase-7, PARP, Achistone H3 and H4 in EC cells To further study the effect of FK228 on the expression of genes involved in apoptosis, we measured the mRNA and protein levels of p53, p21, caspase 3, 7 and 8 and PARP. Ishikawa cells were treated with 8 nM of FK228 for 24 h and measured mRNA and protein expressions as described in materials and methods. The mRNA expressions of p53, p21, caspase 3, 7 and 8 and PARP were significantly increased in FK228 treated cells as compared to control cells (Fig. 5). Further, the protein levels of p53, p21, cleaved caspase 3, 7, 8 and cleaved PARP as the indicator for apoptosis were also
EC is the most common gynecological malignancy with increasing incidence in developed countries[15]. Over the past 20 years, the EC incidence has also increased in China with an increase of over 100% in the overall mortality rate [16]. The current chemotherapy and radiotherapy has been the strength for cancer treatment but has severe side effects and drug resistance that limit its clinical effectiveness. Therefore, identification of novel drugs and the mechanisms underlying the invasion of EC is likely to assist in the development of novel therapeutic approaches. HDAC inhibitors, including FK228 enhance histone acetylation and this response contributes to the anticancer activities against numerous tumors in vitro and in vivo [10,14]. However, to date there are no studies into the anti-cancer effect of FK228 on EC. In the present study, FK228 inhibited the proliferation of the Ishikawa and HEC-1-A EC cell lines in a dose and time-dependent manner and induced cell death. Further, we investigated the molecular mechanisms underlying the anti-cancer of effect of FK228 on the inhibition of endometrial cell growth. FK228 treatment markedly increased the number of cells at G0/G0 phase and consequently induces apoptosis. Further, FK228-increased G0/G0 arrest and apoptotic responses are appeared to be the regulation of p53-p21 pathway which leads to activation of PARP and caspase-7. Apoptosis, called programmed cell death, including the intrinsic mitochondrial pathway and the extrinsic cell death pathway and the activation of the p53-p21 is also related to
Fig. 6. FK228 increases the protein levels of p53, p21, cleaved caspases 3, 7 and 8 and PARP, acetylated H3 and H4 in EC cells. Ishikawa cells were treated with 8 nM of FK228 for 24 h and then subjected to western blotting to analysis the protein levels (A) p53, p21, cleaved caspase-7 and PARP (B) acetylated histone H3 and H4 as described in materials and methods. B-actin was used as loading control.
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apoptosis [17,18]. Here, FK228 treatment resulted in the cleavages of caspase 3, 7 and 8 and PARP and upregulation of p53 and p21 as apoptotic protein in Ishikawa cells, demonstrating the apoptotic effect of FK228 via p53-p21 pathway. Studies reported that FK228 increases cell cycle arrest and apoptosis, which is considered to be among most important mechanism of actions of FK228 on cell growth inhibition [19,20]. Further, FK228 has been reported to increase p21 protein in a p53independent manner in cancer cells [21,22]. Several studies have revealed that p53-21 signaling pathways plays an important role in the induction of apoptosis in different types of tumor cells [23,24]. Moreover, in the present study, FK228 inhibited the cell growth through increase of G2/M-phase arrest, as shown by the increase of cells with G1 DNA content and inducing apoptosis by increase of cleaved caspase-7 [25]. The upregulation of pro-apoptotic and/or downregulation of anti-apoptotic proteins are the major biochemical events involved for the induction of apoptosis. Furthermore, activation of caspases such as 3, 7 and 8 are frequently reflected as point-of-no-return in apoptotic signaling cascade [26]. Our study is consistent with previous studies that FK228 increased the apoptotic cells and number of cells at G0/G0 phase. Our findings suggest that FK228 treatment may potentially induce cell death through activation of p53-p21 signaling cascades. DNA methylation and histone modifications are plays an important role in cancer development. Epigenetic silencing triggered by HDAC and DNMTs are the major targets of cancer prevention and therapeutic approach. The new classes of HDAC inhibitor drugs are currently in early phase clinical trials. Despite these HDAC inhibitor agents demonstrate many features of anti-cancer activity including fewer toxicity for normal cells and an ability to suppress tumor cell growth and their mechanisms of action are largely unknown [27]. It has been shown that FK228 increased the levels of the active chromatin markers such as acetyl-H3, acetyl-H3K9 and acetylH4 and subsequently decreased the levels of the inactive chromatin markers such as trimethyl-H3K9 and trimethyl-H3K27 in cancer cells (Reviewed in [28]). Our study also showed that FK228 significantly increased the protein levels of acetylated histones H3 and H4taht confirms the HDAC inhibition of FK228 in EC cells It is concluded from the present study that FK228 has the potential to regulate p53/caspase signaling cascades which regulates cell-cycle, leads to cell death. Thus, our findings suggest that FK228 might use as a chemopreventive agent for EC cancer treatment. However, clinical trials are warranted to determine the chemopreventive effect for the treatment of EC. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics 2015, CA. Cancer J. Clin. 65 (2015) 5–29. [2] L.R. Duska, A. Garrett, B.R. Rueda, J. Haas, Y. Chang, A.F. Fuller, Endometrial cancer in women 40 years old or younger, Gynecol. Oncol. 83 (2001) 388–393. [3] N.K. Lee, M.K. Cheung, J.Y. Shin, A. Husain, N.N. Teng, J.S. Berek, D.S. Kapp, K. Osann, J.K. Chan, Prognostic factors for uterine cancer in reproductive-aged women, Obstet. Gynecol. 109 (2007) 655–662. [4] V.E. von Gruenigen, K.M. Gil, H.E. Frasure, E.L. Jenison, M.P. Hopkins, The impact of obesity and age on quality of life in gynecologic surgery, Am. J. Obstet. Gynecol. 193 (2005) 1369–1375.
[5] F.P. Vendetti, C.M. Rudin, Epigenetic therapy in non-small-cell lung cancer: targeting DNA methyltransferases and histone deacetylases, Expert Opin. Biol. Ther. 13 (2013) 1273–1285. [6] U. De, S. Kundu, N. Patra, M.Y. Ahn, J.H. Ahn, J.Y. Son, J.H. Yoon, H.R. Moon, B.M. Lee, H.S. Kim, A new histone deacetylase inhibitor, MHY219 inhibits the migration of human prostate cancer cells via HDAC1, Biomol. Ther. (Seoul) 23 (2015) 434–441. [7] T. Liu, S. Kuljaca, A. Tee, G.M. Marshall, Histone deacetylase inhibitors: multifunctional anticancer agents, Cancer Treat. Rev. 32 (2006) 157–165. [8] Y. Shao, Z. Gao, P.A. Marks, X. Jiang, Apoptotic and autophagic cell death induced by histone deacetylase inhibitors, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 18030–18035. [9] E. Verdin, F. Dequiedt, H.G. Kasler, Class II histone deacetylases: versatile regulators, Trends Genet. 19 (2003) 286–293. [10] J.E. Bolden, M.J. Peart, R.W. Johnstone, Anticancer activities of histone deacetylase inhibitors, Nat. Rev. Drug Discov. 5 (2006) 769–784. [11] A.J. Frew, R.W. Johnstone, J.E. Bolden, Enhancing the apoptotic and therapeutic effects of HDAC inhibitors, Cancer Lett. 280 (2009) 125–133. [12] A. Schech, A. Kazi, S. Yu, P. Shah, G. Sabnis, Histone deacetylase inhibitor entinostat inhibits tumor-Initiating cells in triple-negative breast cancer cells, Mol. Cancer Ther. 14 (2015) 1848–1857. [13] M. Haberland, R.L. Montgomery, E.N. Olson, The many roles of histone deacetylases in development and physiology: implications for disease and therapy, Nat. Rev. Genet. 10 (2009) 32–42. [14] E. Lech-Maranda, E. Robak, A. Korycka, T. Robak, Depsipeptide (FK228) as a novel histone deacetylase inhibitor: mechanism of action and anticancer activity, Mini Rev. Med. Chem. 7 (2007) 1062–1069. [15] H.C. Kitchener, E.L. Trimble, Endometrial Cancer Working Group of the Gynecologic Cancer, Endometrial cancer state of the science meeting, Int. J. Gynecol. Cancer 19 (2009) 134–140. [16] J.I. Sorosky, Endometrial cancer, Obstet. Gynecol. 111 (2008) 436–447. [17] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007) 495–516. [18] L. Ouyang, Z. Shi, S. Zhao, F.T. Wang, T.T. Zhou, B. Liu, J.K. Bao, Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis, Cell Prolif. 45 (2012) 487–498. [19] Y. Kobayashi, M. Ohtsuki, T. Murakami, T. Kobayashi, K. Sutheesophon, H. Kitayama, Y. Kano, E. Kusano, H. Nakagawa, Y. Furukawa, Histone deacetylase inhibitor FK228 suppresses the Ras-MAP kinase signaling pathway by upregulating Rap1 and induces apoptosis in malignant melanoma, Oncogene 25 (2006) 512–524. [20] K. Sutheesophon, Y. Kobayashi, M.A. Takatoku, K. Ozawa, Y. Kano, H. Ishii, Y. Furukawa, Histone deacetylase inhibitor depsipeptide (FK228) induces apoptosis in leukemic cells by facilitating mitochondrial translocation of Bax, which is enhanced by the proteasome inhibitor bortezomib, Acta Haematol. 115 (2006) 78–90. [21] R. Hu, V. Hebbar, B.R. Kim, C. Chen, B. Winnik, B. Buckley, P. Soteropoulos, P. Tolias, R.P. Hart, A.N. Kong, In vivo pharmacokinetics and regulation of gene expression profiles by isothiocyanate sulforaphane in the rat, J. Pharmacol. Exp. Ther. 310 (2004) 263–271. [22] D.S. Son, A.J. Wilson, A.K. Parl, D. Khabele, The effects of the histone deacetylase inhibitor romidepsin (FK228) are enhanced by aspirin (ASA) in COX-1 positive ovarian cancer cells through augmentation of p21, Cancer Biol. Ther. 9 (2010) 928–935. [23] L. Farrand, S. Byun, J.Y. Kim, A. Im-Aram, J. Lee, S. Lim, K.W. Lee, J.Y. Suh, H.J. Lee, B.K. Tsang, Piceatannol enhances cisplatin sensitivity in ovarian cancer via modulation of p53, X-linked inhibitor of apoptosis protein (XIAP), and mitochondrial fission, J. Biol. Chem. 288 (2013) 23740–23750. [24] E. Oda, R. Ohki, H. Murasawa, J. Nemoto, T. Shibue, T. Yamashita, T. Tokino, T. Taniguchi, N. Tanaka, Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis, Science 288 (2000) 1053–1058. [25] J.A. Karam, J. Fan, J. Stanfield, E. Richer, E.A. Benaim, E. Frenkel, P. Antich, A.I. Sagalowsky, R.P. Mason, J.T. Hsieh, The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapy, Int. J. Cancer 120 (2007) 1795–1802. [26] D.R. Green, G.P. Amarante-Mendes, The point of no return: mitochondria, caspases, and the commitment to cell death, Results Probl. Cell Differ. 24 (1998) 45–61. [27] R.K. Lindemann, B. Gabrielli, R.W. Johnstone, Histone-deacetylase inhibitors for the treatment of cancer, Cell cycle 3 (2004) 779–788. [28] W.S. Xu, R.B. Parmigiani, P.A. Marks, Histone deacetylase inhibitors: molecular mechanisms of action, Oncogene 26 (2007) 5541–5552.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Histone deacetylase inhibitor, Romidepsin (FK228) inhibits endometrial cancer cell growth through augmentation of p53-p21 pathway Lu-Hong Lia,* , Pei-Ru Zhanga , Pei-Ya Caia , Zhi-Chao Lib a b
Department of Obstetrics and Gynecology, Second Affiliated Hospital of Fujian Medical College, China Department of Radiology, Second Affiliated Hospital of Fujian Medical College, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 March 2016 Received in revised form 25 April 2016 Accepted 25 April 2016
Objective: Romidepsin (FK228), a Histone Deacetylase (HDAC) inhibitor, has been used for anti-cancer therapies. However, the anti-cancer effect of FK228 and its underlying mechanism in endometrial carcinoma (EC) have not been studied. The aime of this study was to investigate the anti-cancer effects of FK228 and the associated mechanism(s) in EC. Methods: Ishikawa and HEC-1-A endometrial cancer cells were treated with 8 nM concentration of FK228 and cell growth was measured by XTT assay. The cell cycle distribution and cell death were measured by flow cytometry, immunofluorescence, respectively. The mNRA and protein expressions were analyzed by quantitative RT-PCR and western blot, respectively. Results: Based on assays carried out in EC cell lines, it was observed that FK228 inhibited EC cell proliferation in a dose and time-dependent manner. Furthermore, following treatment with FK228 for 48 h, there were significant induction of apoptosis and cell cycle arrest at G0/G1 phase in EC cells. Moreover, FK228 treatment significantly increased the mRNA and protein expressions of p53, p21, cleaved caspases such as 3, 7 and 8 and PARP. Further, FK228 treatment increased the levels of acetylated histone H3 and H4 that confirms the HDAC inhibition. Conclusion: In conclusion, FK228 inhibits EC tumor cell proliferation and induces apoptosis by activation caspase/PARP via the induction of p53/p21 signaling cascades, suggesting that FK228 is a potential therapeutic agent for EC. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Endometrial cancer FK228 Apoptosis Cell-cycle and p53
1. Introduction Endometrial cancer (EC) is the most common malignancy diagnosed in women and the incidence of EC is increasing in the world wide. In the United States, EC is now the fourth common cancer among women, with an estimated 54, 870 new cases and 10, 170 deaths in 2015 [1]. The most EC cases are diagnosed after menopause, however; 14% of EC are diagnosed before menopause and the age of 40 [2,3]. The standard treatment for EC are chemotherapy with cytotoxic agents and surgical staging such as hysterectomy, bilateral salpingo-oophorectomy and sampling of lymph nodes[4]. However, EC patients with surgery and long-term exposure of chemotherapeutic drugs that leads to have significant medical comorbidities. Therefore, finding new targets for therapy
* Corresponding author at: Department of Obstetrics and Gynecology, Second Affiliated Hospital of Fujian Medical College, No. 34, Zhongshan North Road, Licheng District, Quangzhou, Fujian 362000, China. E-mail address: [email protected] (L.-H. Li). http://dx.doi.org/10.1016/j.biopha.2016.04.053 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
and therapeutic options are necessary to decrease the morbidity and mortality observed with advanced stage EC. Histone deacetylase (HDAC) and DNA methyltransferases (DNMTs) are important factors for the dysregulation of epigenetic process which leads to altered gene expression and play important role in a multiple cancers [5,6]. The differential activities of HDAC are associated with certain types of human cancer, including EC human cancer thereby affecting signal transduction pathways and the expression of tumor suppressor genes such as p53 and p21/ WAF, [7–9]. The drugs including HDAC inhibitors are a promising class of anti-cancer drugs because of their ability to inhibit/activate cancer cell proliferation, cell cycle arrest, apoptosis, metastasis and angiogenesis [10–12]. The HDAC inhibitors such as such as valproic acid, suberoylanilide hydroxamic acid (SAHA), LBH-589 and romidepsin (FK228) have been shown to have anti-neoplastic effects in a variety of tumor [10]. The advantage of HDAC inhibitors are less toxic alternative to standard therapy due its selective targeting effect of cancer cells versus normal cells [13]. Recently, FK228 has been approved by FDA for the treatment of T cell
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cutaneous lymphoma and now it is in phase II clinical trial in solid tumors and hematopoietic malignancies [14]. To best of our knowledge, the role(s) and mechanism(s) of action of FK228 in EC cells has not been examined. Hence, in the present study, we intended to determine the effect of FK228 on the suppression of EC cell growth and its mechanism(s) of action. We show here that FK228 inhibits EC cell growth, induces cell death and arrest cells at G0/G1 phase by augmenting p53-p21 signaling pathway. 2. Materials and methods 2.1. Reagents Romidepsin (FK228), penicillin/streptomycin and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide were as purchased from Sigma (St. Louis, MO). All cell culture media were obtained from Invitrogen Life Technologies (Grand Island, NY). Antibodies including p53, p21, cleaved caspase-7, cleaved PARP, acetylated H3 and H4 and b-actin were procured from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). 2.2. Cell culture
Fig. 1. FK228 reduces endometrial cell proliferation in a dose-dependent manner. (A) Ishikawa and (B) HEC-1-A cells were treated with different concentrations of FK228 (0, 4, 8, 16, 32 and 64 nM) for 48 h and their survival rate was then measured using a XTT assay as described in materials and methods. Data are reported as the means standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
Ishikawa and HEC-1-A, the human endometrial cell lines were procured from Chinese Academy of Sciences Committee Type Culture Collection cell bank (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 10% FBS, 1X penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 C. 2.3. Cell proliferation assay Ishikawa and HEC-1-A cells were plated in 96-well plate at 50,000 cells/ml and treated with different concentrations (0, 4, 8 and 16 nM) of FK228 for 48 h. After 72 h of treatment, 50 ml of XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide) was added and incubated at 37 C for 2 h and then read absorbance at 450. 2.4. Annexin V staining Cell death were determined using apoptosis cell quantitation kit (Biotium, Hayward, USA). Ishikawa and HEC-1-A cells were seeded in 2-well chamber coverglass slides. After 24 h of cells attached to the slides, cells were treated either vehicle (DMSO) or 8 nM of FK228 for 48 h. After 48 h of treatment, cells were stained with 5 ml of Annexin V and Hoechst 33342 dye to 100 ml of 1 binding buffer and incubate for 15 min at room temperature in the dark. The cells were washed with 1 binding buffer and observe under fluorescence microscope at 20 objective. 2.5. Cell death analysis Cell death were analyzed by using FITC Annexin V kit according to the manufacturer’s instruction. FK228 treated cells were pelleted by centrifugation at 1000 rpm for 5 min. The pellets were suspended in binding buffer and add Annexin V-FITC (5 ml) and propidium iodide (50 mg/ml) and incubated at room temperature for 5 min in the dark. FITC Annexin V stained cells were analyzed in a FACScan.
Fig. 2. FK228 reduces endometrial cell proliferation in a time-dependent manner. (A) Ishikawa and (B) HEC-1-A cells were treated with 8 nM of FK228 for 24, 48 and 72 h and their survival rate was then measured using a XTT assay as described in materials and methods. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
2.6. Cell cycle analysis Ishikawa and HEC-1-A cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 48 h and then fixed with 75% ethanol at 20 C. The ethanol fixed cells were resuspended in PBS with
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Fig. 3. FK228 induces apoptosis in EC cells. (A, B) Ishikawa and (C, D) HEC-1-A cells were treated with 8 nM of FK228 for 48 h and their apoptotic index was measured using Annexin V staining by immunofluorescence and FACS as described in materials and methods. Green fluorescent staining shows apoptotic cells. All nuclei are stained with blue fluorescence. Bar graph show the quantitation of apoptotic cells as expressed relative green fluorescence. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control group (0).
addition of RNase A (1 mg/ml) and incubated for 1 h at 37 C. Propidium iodide (50 mg/ml) was used for staining the fixed cells for 30 min at room temperature. The DNA contents of the stained cells were analyzed by using the CELL Quest Software with a FACScan flowcytometer.
2.7. Quantitative RT-PCR (qRT-PCR) Ishikawa cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 24 h and total RNA was isolated by using AxyPre TM Multi-source RNA miniprep kit (Axygen, USA). Two mg of total RNA was converted into complementary DNA (cDNA) with PrimeScript
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RT reagent kit (Takara, Japan). P53, p21, caspase-3, 7 and 8 and PARP mRNA expressions were measured by using specific TaqMan Probes and TaqMan assay kit (Life Technologies) according to manufacturer’s protocol with real-time PCR machine. GAPDH was used as a control to determine relative mRNA expression. 2.8. Immunoblot analysis Ishikawa cells were treated with either vehicle (DMSO) or 8 nM of FK228 for 24 h and cells were lysed with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 1 mM PMSF. The protein concentrations were measured using the Bio-Rad assays. An equal amount proteins were separated in 10% SDS-PAGE, transferred to a nitrocellulose membrane. The membranes were incubated with indicated primary antibodies (p53, p21, cleaved caspase 3, 7, and 8 cleaved PARP, acetylated histone H3 and H4 and b-actin) for overnight at 4 C. After 5, 10 and 15 min washes with TBST, the membranes were incubated with corresponding secondary IgG antibodies for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescence reagent (ECL). 2.9. Statistical analysis Statistical analysis was performed by using SPSs 16.0 software. The data are expressed as mean standard deviation (SD). Oneway ANOVA followed by post hoc test was used to analysis significant differences between each sample. p < 0.05 values were considered statistically significant. 3. Results 3.1. FK228 inhibits EC proliferation Fist, to examine the different concentrations of FK228 on cell survival of two different endometrial cells, we treated Ishikawa and HEC-1-A cells with 0, 4, 8, 16, 32 and 64 nM concentrations of FK228 for 48 h and measured cell proliferation by XTT assay. As shown in Fig. 1A and B, the percentage of cell survival rate was decreased in a dose-dependent manner and 8 nM of FK228 was inhibited 50% of cell growth in both Ishikawa and HEC-1-A. Next we determine the time dependent cell growth inhibition of FK228. The cells were treated with 8 nM of FK228 for 24, 48 and 72 h and measured cell proliferation. The cell survival rate were significantly decreased in a time-dependent manner and 50% of inhibition was observed at 48 h (Fig. 2A and B). 3.2. FK228 induces EC cell death To investigate the effect of FK228 on apoptosis, we measured cell death by measuring apoptotic cells by immunofluorescence and FACS. Ishikawa and HEC-1-A cells were treated with 8 nM of FK228 for 48 h and measured apoptotic cells. There were significant increases of apoptotic cells as seen green fluorescence (Fig. 3A and C) and percentage of apoptotic cells measured by FACS (Fig. 3B and D) in FK228 treated both cells when compared to vehicle treated (Control) cells, indicating that FK228 induces cell death in both endometrial cells. 3.3. FK228 arrests cells at G0/G1 phase Fig. 4. FK228 regulates cell-cycle in EC cells. (A) Ishikawa and (B) HEC-1-A cells were treated with 8 nM of FK228 for 48 h. After 48 h, cells were harvested and fixed with 75% ethanol as described in materials and methods. Cells were stained with propidium iodide. Cell cycle distribution was assessed by flow cytometry. Data are reported as the mean standard deviation of three independent experiments. *P < 0.05 compared with control.
To confirm the cell death effect of FK228, we performed cellcycle to determine the positions of cells in the cell cycle were determined by flow cytometry. Consistent with apoptosis data (Fig. 3A and B), treatment of cells with FK228 for 48 h significantly increased the percentage of cells in G0/G1 from 40% to 80% (Fig. 4A
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increased in FK228 treated cells (Fig. 6A), indicating that activation of p53-p21 pathway due to FK228 treatment may be responsible for the induction of apoptosis by FK228 treatment. Further, treatment of FK228 significantly increased the acetylated histone H3 and H4 (Fig. 6B), suggesting that FK228 inhibits deacetylase activity that increases acetylation of histone H3 and H4. 4. Discussion
Fig. 5. FK228 increases the mRNA expression of p53, p21, caspases 3, 7 and 8 and PARP in EC cells. Ishikawa cells were treated with 8 nM of FK228 for 24 h and then subjected to qPCR analysis as described in materials and methods. GAPDH was used as endogenous control to analysis mRNA expression and expressed as relative mRNA expression. Values represent the mean standard deviation of three independent experiments. *P < 0.05 compared with control.
and B), showing that FK228 arrests Ishikawa and HEC-1-A cells at G0/G1 and consistent with cell death as shown in Fig. 3A and B. 3.4. FK228 increases the expression of p53, p21, caspase-7, PARP, Achistone H3 and H4 in EC cells To further study the effect of FK228 on the expression of genes involved in apoptosis, we measured the mRNA and protein levels of p53, p21, caspase 3, 7 and 8 and PARP. Ishikawa cells were treated with 8 nM of FK228 for 24 h and measured mRNA and protein expressions as described in materials and methods. The mRNA expressions of p53, p21, caspase 3, 7 and 8 and PARP were significantly increased in FK228 treated cells as compared to control cells (Fig. 5). Further, the protein levels of p53, p21, cleaved caspase 3, 7, 8 and cleaved PARP as the indicator for apoptosis were also
EC is the most common gynecological malignancy with increasing incidence in developed countries[15]. Over the past 20 years, the EC incidence has also increased in China with an increase of over 100% in the overall mortality rate [16]. The current chemotherapy and radiotherapy has been the strength for cancer treatment but has severe side effects and drug resistance that limit its clinical effectiveness. Therefore, identification of novel drugs and the mechanisms underlying the invasion of EC is likely to assist in the development of novel therapeutic approaches. HDAC inhibitors, including FK228 enhance histone acetylation and this response contributes to the anticancer activities against numerous tumors in vitro and in vivo [10,14]. However, to date there are no studies into the anti-cancer effect of FK228 on EC. In the present study, FK228 inhibited the proliferation of the Ishikawa and HEC-1-A EC cell lines in a dose and time-dependent manner and induced cell death. Further, we investigated the molecular mechanisms underlying the anti-cancer of effect of FK228 on the inhibition of endometrial cell growth. FK228 treatment markedly increased the number of cells at G0/G0 phase and consequently induces apoptosis. Further, FK228-increased G0/G0 arrest and apoptotic responses are appeared to be the regulation of p53-p21 pathway which leads to activation of PARP and caspase-7. Apoptosis, called programmed cell death, including the intrinsic mitochondrial pathway and the extrinsic cell death pathway and the activation of the p53-p21 is also related to
Fig. 6. FK228 increases the protein levels of p53, p21, cleaved caspases 3, 7 and 8 and PARP, acetylated H3 and H4 in EC cells. Ishikawa cells were treated with 8 nM of FK228 for 24 h and then subjected to western blotting to analysis the protein levels (A) p53, p21, cleaved caspase-7 and PARP (B) acetylated histone H3 and H4 as described in materials and methods. B-actin was used as loading control.
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apoptosis [17,18]. Here, FK228 treatment resulted in the cleavages of caspase 3, 7 and 8 and PARP and upregulation of p53 and p21 as apoptotic protein in Ishikawa cells, demonstrating the apoptotic effect of FK228 via p53-p21 pathway. Studies reported that FK228 increases cell cycle arrest and apoptosis, which is considered to be among most important mechanism of actions of FK228 on cell growth inhibition [19,20]. Further, FK228 has been reported to increase p21 protein in a p53independent manner in cancer cells [21,22]. Several studies have revealed that p53-21 signaling pathways plays an important role in the induction of apoptosis in different types of tumor cells [23,24]. Moreover, in the present study, FK228 inhibited the cell growth through increase of G2/M-phase arrest, as shown by the increase of cells with G1 DNA content and inducing apoptosis by increase of cleaved caspase-7 [25]. The upregulation of pro-apoptotic and/or downregulation of anti-apoptotic proteins are the major biochemical events involved for the induction of apoptosis. Furthermore, activation of caspases such as 3, 7 and 8 are frequently reflected as point-of-no-return in apoptotic signaling cascade [26]. Our study is consistent with previous studies that FK228 increased the apoptotic cells and number of cells at G0/G0 phase. Our findings suggest that FK228 treatment may potentially induce cell death through activation of p53-p21 signaling cascades. DNA methylation and histone modifications are plays an important role in cancer development. Epigenetic silencing triggered by HDAC and DNMTs are the major targets of cancer prevention and therapeutic approach. The new classes of HDAC inhibitor drugs are currently in early phase clinical trials. Despite these HDAC inhibitor agents demonstrate many features of anti-cancer activity including fewer toxicity for normal cells and an ability to suppress tumor cell growth and their mechanisms of action are largely unknown [27]. It has been shown that FK228 increased the levels of the active chromatin markers such as acetyl-H3, acetyl-H3K9 and acetylH4 and subsequently decreased the levels of the inactive chromatin markers such as trimethyl-H3K9 and trimethyl-H3K27 in cancer cells (Reviewed in [28]). Our study also showed that FK228 significantly increased the protein levels of acetylated histones H3 and H4taht confirms the HDAC inhibition of FK228 in EC cells It is concluded from the present study that FK228 has the potential to regulate p53/caspase signaling cascades which regulates cell-cycle, leads to cell death. Thus, our findings suggest that FK228 might use as a chemopreventive agent for EC cancer treatment. However, clinical trials are warranted to determine the chemopreventive effect for the treatment of EC. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics 2015, CA. Cancer J. Clin. 65 (2015) 5–29. [2] L.R. Duska, A. Garrett, B.R. Rueda, J. Haas, Y. Chang, A.F. Fuller, Endometrial cancer in women 40 years old or younger, Gynecol. Oncol. 83 (2001) 388–393. [3] N.K. Lee, M.K. Cheung, J.Y. Shin, A. Husain, N.N. Teng, J.S. Berek, D.S. Kapp, K. Osann, J.K. Chan, Prognostic factors for uterine cancer in reproductive-aged women, Obstet. Gynecol. 109 (2007) 655–662. [4] V.E. von Gruenigen, K.M. Gil, H.E. Frasure, E.L. Jenison, M.P. Hopkins, The impact of obesity and age on quality of life in gynecologic surgery, Am. J. Obstet. Gynecol. 193 (2005) 1369–1375.
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