Abstract B82: G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly

Abstract

Abstract Accumulating evidence indicates that the G-protein coupled estrogen receptor (GPER) mediates the non-genomic actions of estrogen in many different cells and tissues. The selective GPER agonist G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro- 3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed to distinguish estrogen actions mediated by GPER from those mediated by the traditional nuclear estrogen receptors. Our recent data showed that G-1 suppresses the growth of breast and ovarian cancer cells in a GPER-independent manner. However, the mechanisms underlying the suppressive actions of G-1 on cancer cell growth are still unclear. The aim of the present study was to explore potential mechanisms of G-1 action in human ovarian granulosa tumor cells. The KGN cell line, a cell line derived from a metastatic adult ovarian granulosa cell tumor (aGCT), and the COV434 cell line, a cell line derived from a juvenile granulosa cell tumor (jGCT), were used as cellular models. Cell proliferation and viability were determined by cell number counting and MTT assay, respectively. Cell cycle analysis was monitored by flow cytometry. Key proteins associated with cell cycle progression were analyzed with Western blot. Cell apoptosis was detected by analysis of caspase activity and DNA integrity. Cellular cytoskeleton changes were monitored by fluorescent immunocytochemistry, confocal microscopy and in vitro microtubule assembly assays. Consistent with our previous results, the putative GPER agonist G-1 suppressed proliferation in a concentration- and time-dependent manner in both KGN and COV434 cells. G-1 inhibited GCT cell growth via suppressing cell viability, which was indicated by a significant decrease in the MTT metabolism, an increase in DNA fragmentation, and an increase in PARP cleavage. G-1 treatment did not affect the expression of cyclin D1, cyclin D2 and cyclin E, suggesting that G-1 treatment had no effect on the early stage cell cycle progression, a result further confirmed by flow cytometry. However, flow cytometry showed that G-1 treatment arrested the cell cycle in the G2/M phase. G2/M phase arrest of KGN cells was confirmed by a significant increase in the phosphorylation of histone H3 after G-1 treatment. Surprisingly, the Western blot analysis showed that G-1 treatment did not affect the activation of mitotic promoting factors (MPF) and had no effect on G2/M entry checkpoint proteins and kinases. Fluorescent immunocytochemistry and confocal microscopy showed that G-1 treatment disrupted the segregation of chromosomes during mitosis, an effect that may be attributed to the abnormal assembly of microtubules. In vitro microtubule assembly assays demonstrated that G-1 treatment indeed suppressed tubulin polymerization and thereby inhibited the assembly of the microtubules. In conclusion, our results from the present study demonstrate that G-1, the putative GPER agonist, suppresses GCT cell proliferation and induces tumor cell death via blocking microtubule assembly. The ability of G-1 to suppress tumor cell proliferation and induce tumor cell apoptosis by targeting microtubules makes it a promising candidate drug for the treatment of GCT. Citation Format: Xiangmin Lv, Guohua Hua, Chunbo He, John S Davis, Cheng Wang. G-protein coupled estrogen receptor (GPER) agonist G-1 inhibits growth of human granulosa cell tumor cells via blocking microtubule assembly. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B82.