The putative G-protein coupled estrogen receptor agonist G-1

Am J Transl Res 2012;4(4):390-402 www.ajtr.org /ISSN:1943-8141/AJTR1207005

Original Article The putative G-protein coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner Cheng Wang1,2, Xiangmin Lv1,2,4, Chao Jiang1,2, John S Davis1,2,3 1Olson

Center for Women’s Health, 2Department of OB/GYN, University of Nebraska Medical Center, Omaha, NE 68198, USA; 3VA Medical Center, Omaha, NE 68105, USA; 4Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China. Received July 17, 2012; accepted September 19, 2012; Epub October 10, 2012; Published October 30, 2012 Abstract: G-protein coupled estrogen receptor 1 (GPER) plays an important role in mediating estrogen action in many different tissues under both physiological and pathological conditions. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl]-ethanone) has been developed as a selective GPER agonist to distinguish estrogen actions mediated by GPER from those mediated by classic estrogen receptors. In the present study, we surprisingly found that G-1 suppressed proliferation and induced apoptosis of KGN cells (a human ovarian granulosa cell tumor cell line), actions that were not blocked by a selective GPER antagonist G15 or siRNA knockdown of GPER. G-1 also suppressed proliferation and induced cell apoptosis in GPER-negative HEK-293 cells and MDA-MB 231 breast cancer cells. Our results demonstrate that G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. G-1 may be a candidate for the development of drugs against ovarian and breast cancer. Keywords: GPR30/GPER, G-1, G15, ovarian cancer, breast cancer, estrogen receptors

Introduction Estrogens play vital roles in the body under both physiological and pathological conditions [1, 2]. Recently, several groups have shown that the Gprotein coupled estrogen receptor GPR30, now called GPER, is able to mediate the rapid actions of estrogen [3-5], although this concept has been questioned by other researchers [6-8]. More recent studies have shown that GPER plays a role in the function of the nearly every system of the body, including the immune system, nervous system, skeletal system, renal system, cardiovascular system, endocrine system and reproductive system [9]. The mechanism by which GPER regulates physiological actions in the body is still unclear because estrogen is a ligand for both classic estrogen receptors (ERα and ERβ) and GPER. It is difficult to separate the estrogen actions mediated by GPER from those mediated by the classic estrogen receptors. A non-steroidal, high

-affinity GPER agonist G-1 (1-[4-(6-bromobenzo [1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro-3Hcyclopenta-[c]quinolin-8-yl]-ethanone) has been developed to dissect GPER-mediated estrogen responses from those mediated by classic estrogen receptors [10]. It has been shown that G-1 binds to GPER, but it does not bind to classical estrogen receptors [10]. The selectivity of G-1 has also been demonstrated by a very recent report, which showed that G-1 did not activate estrogen response elements (ERE) [11] and that it did not bind to 25 other G-protein coupled receptors [12]. Activation of GPER by G-1 has been shown to increase mobilization of intracellular calcium in GPER overexpressing COS-7 cells, activate PI3 kinase in SKBR-3 cells (GPER positive, ER negative) and MCF7 cells (GPER and ER positive), and inhibit migration in both SKBR-3 and MCF-7 cell lines [10]. The effects of G-1 on cell survival and proliferation appear to be cell type specific. For example, Balhuizen et al. showed that in mouse pancre-

GPER agonist G-1 suppresses growth of ovarian and breast cancer cells

atic islets, G-1 was able to abolish cytokineinduced cell apoptosis [13]. Albanito et al. showed that activation of GPR30 by G-1 stimulated proliferation of BG1 and 2008 ovarian cancer cells and SKBR-3 breast cancer cells [14]. On the contrary, Chan et al. showed that G1 suppressed growth of PC-3 cells in a GPERdependent manner [15]. Therefore, the function of G-1 and G-1-activated GPER under the normal and pathological conditions need further investigation. The ovarian granulosa cell is the main source and also a major target of estrogen. Under physiological conditions, ovarian granulosa cells convert the androgen produced in the theca cells into estrogen [1, 2]. Therefore, granulosa cells are exposed to an environment with very high level of estrogen [2]. Under pathological conditions, such as in patients with granulosa cell tumors (GCT), GCT cells produce large amount of estrogen leading to symptoms of estrogen excess [16]. The role of estrogen in the progression of GCT is unclear. One recent study with genetically modified mice showed that the loss of the classic estrogen receptor ERβ was associated with the development of pituitary tumors and GCT in aged mice [17]. However, the role of GPER in the initiation and development of GCT is unknown. The KGN cell is a well-characterized cell line used for the study of GCT [16, 18]. These cells were derived from a GCT tumor and maintain many features of normal granulosa cells and GCT cells [18]. They have ability to respond to gonadotropin stimulation and can produce steroid hormones [16, 18]. Therefore, using KGN cell as a GCT cellular model and G-1 as a GPER agonist, we initiated a study to investigate GPER function on GCT cell proliferation. Our results showed that knockdown of GPER suppressed KGN cell proliferation. Surprisingly, we found that G-1, the selective agonist of GPER, suppressed KGN cell proliferation by arresting KGN cells in the G2/M phase and inducing KGN cell apoptosis regardless of GPER expression. In cell lines without GPER expression, G-1 also suppressed cell proliferation, arrested cell cycle progression and induced cell apoptosis. These novel findings clearly suggest that G-1, the putative GPER selective agonist, suppressed ovarian and breast cancer cell proliferation in a GPERindependent manner. The ability to inhibit proliferation of tumor cells makes G-1 a promising candidate drug for ovarian and breast cancer

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therapy. Materials and methods Chemicals G-1 was purchased from Tocris Bioscience (Ellisville, MO). DMEM and other cell culture medium were from Invitrogen (Carlsbad, CA). FBS was from HyClone laboratories Inc. (Logan, UT). Antibodies against GPER and β-tubulin were from Sigma (St. Louis, MO). Second antibodies for Western blotting chemiluminescence were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA); ECL AdvanceTM Western blotting Detection kit was from GE Healthcare Bio-Science Corp (Piscataway, NJ); Vybrant® MTT Assay Kit was from Invitrogen (Carlsbad, CA). All other molecular-grade chemicals were purchased from Sigma (St. Louis, MO), Fisher (Pittsburgh, PA), or United States Biochemical (Cleveland, OH). Cell lines and culture The KGN cell line was from the Riken Biosource Center (Tsukuba, Japan). The IGROV-1 cell line (an ovarian epithelial cancer cell line) was obtained from Dr. Bo Rudea (Massachusetts General Hospital, Boston, MA). The HEK 293 cell line (Human Embryonic Kidney cell) and MDAMB 231 cell line (human breast cancer cell) were purchased from ATCC. Cells were maintained in DMEM/F12 medium with 10% FBS and incubated at 37°C in a humidified, 5% CO2 incubator. The media were replaced with phenol red-free DMEM supplemented with 10% steroidfree FBS (from Hyclone, Logan, UT) 24 h before treatments. Cells were treated in fresh phenol red-free DMEM supplemented with steroid-free FBS as indicated in the figure legends. Western blot detecting GPER protein expression in KGN cells GPER protein was detected by Western blot according to a methods used in our laboratory [19, 20]. Briefly, cultured cell lines were directly lysed in the dishes, homogenized by sonicating in 100 μl of lysis buffer (10mM Tris PH7.4, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1mM NaF, 20mM Na4P2O7, 1% triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate) with protease inhibitor cocktails and PMSF and kept on ice for 20 min. After centrifugation, the supernatant was collected and the protein was

Am J Transl Res 2012;4(4):390-402

GPER agonist G-1 suppresses growth of ovarian and breast cancer cells

measured with Micro BCATM Protein Assay Kit (PIERCE, Rockford, IL). Protein (20μg) was fractioned with 10% polyacrylamide gels, electrotransferred to Optitran membranes and probed with the primary antibodies at 4°C for overnight. Peroxidase conjugated donkey anti-rabbit secondary antibodies were applied on the membrane and the bound secondary antibody was detected with the Enhanced Chemiluminescence (ECL) Advance Western blotting detection kit (GE Healthcare). The signal was recorded by a UVP gel documentation system (UVP, Upland, CA). β-tubulin was used as a loading control. Cell proliferation and viability assays To detect the effect of G-1 on ovarian and breast cancer cell proliferation, 60% confluent cells were incubated in phenol-red free DMEM supplement with 10% steroid-free FBS with or without G-1 at the indicated concentrations for 48 or 72 h. Cell number was counted with a Countess® Automated Cell Counter (Carlsbad, CA). To detect the effect of GPER on KGN cell proliferation, KGN cells were plated in 6-well cell culture plates and incubated until 40% confluent. Cells were then transfected with siGLO (a labeled non-target siRNA as control) or GPER siRNA for 6 h using METAFECTENE (BiontexUSA, San Diego, CA) as a transfection reagent according to the manufacturer’s instruction. Cells were then incubated in DMEM-10%FBS for 48 h before G-1 treatment and subsequent determination of cell morphometry and proliferation. The MTT assay was used to detect cell viability. Cells were plated in 24-well cell culture plates and incubated until approximately 70% confluent. Cells were then treated with or without G-1 for 24 or 48 h. The MTT assay was performed with a Vybrant® MTT Assay Kit according to the manufacture’s instruction. Flow cytometry detecting the progression of cell cycle Flow cytometry was used to detect the effects of G-1 on cell cycle progression. Ovarian and breast cancer cells were cultured as described above with or without G-1 at the indicated concentrations for 24 h. Cells were then trypsinized, fixed and permeabilized with 70% ethanol overnight at -20°C. Cells were then la-

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beled with propidium iodide for 30 min at 37°C and analysis was performed by the University of Nebraska Medical Center flow cytometry core facility. Caspase 3/7 activity assay Cell apoptosis was also monitored by detecting caspase3/7 activities with a Caspase-Glo® 3/7 assay kit (Promega, Madison, WI). Ovarian and breast cancer cells were treated with DMSO or G-1 at the indicated concentrations for 24 h. The media were then removed, 100µl of phenolred free medium (no serum) and equal volume of caspase assay reagent were directly added to the culture plate. After shaking for 30 min, the luminescence was measured with a FLUOstar OPTIMA luminometer. Statistics Morphological analysis and Western blot experiments were repeated at least three times and representative images were presented. All assays were repeated at least three times and the quantitative data were analyzed using one-way ANOVA with Tukey’s post hoc test. P