Investigation On The Function Of ER-α36 In Endometrial Carcinoma Cells And Icaritin Induces Apoptosis

Estrogen receptor (ER) belongs to the steroid hormone family of the nuclear receptor superfamily and expressed in various tissues.ER plays an important role in tissue development and tumor occurrence.ER consists of ER-a and ER-(3. Previously, we identified and cloned a novel variant of ER-a. ER-a36.ER-a36 gene is localized on chromososme 6q24.2-25.3 and contains a 5.4 kb cDNA, which encode a 310 amino acid open-deading frame and a a protein with a molecular weight of 36 kDa that is transcribed from previously unidentified promoter located in the first intron of the original 66 kDa ER-a (ER-a66) gene.ER-a36 lacks both transcriptional activation domains of ER-a66 (AF-1 and AF-2).but it retains the DNA-binding domain and partial ligand-binding domain. It possesses a unique 27 amino acid domain that replaces the last 138 amino acids encoded by exons 7 and 8 of the ER-a66 gene.The purpose of this study was to investigate the function and the underlying mechanisms of ER-a36 in growth regulation of endometrial Ishikawa cancer cells. The cellular localization of ER-a36 and ER-a66 were determined by immunofluorescence in the Ishikawa cells.Immunofluorescence staining of Ishikawa cells demonstrated that ER-a36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-a66 was predominantly localized in the cell nucleus. PKC isoforms are involved in a variety of cellular functions, including growth, differentiation, tumor promotion, aging, and apoptosis.Although early studies of the effect of PKC8 on cell proliferation suggested that PKC8 suppresses proliferation. However, several reports have demonstrated that PKC5 could act as a positive regulator of cell proliferation. Here, we found that both E2 and E2-BSA stimulated the activation of PKC5 signaling pathway in Ishikawa cells, and knockdown of ER-a36 expression with the shRNA abrogated E2-induced PKC5 phosphorylation. These indicated that ER-a36 mediates membrane-initiated PKC8 pathway induced by E2. In general, increased PKCαactivity is associated with increased motility and proliferation of cancer cells. In addition, PKCαhas been shown to inhibit or facilitate apoptosis of cancer cells. Here, we found that E2 was unable to induce PKCa phosphorylation. cAMP is a second messenger that plays a role in intracellular signal transduction of various stimuli. A major function of cAMP in eukaryotes is activation of cAMP-dependent protein kinase (PKA). In the present study, only E2 was able to induce cAMP-dependent protein kinase A (PKA) phosphorylation. E2-and E2-BSA-induced ERK phosphorylation required ER-α36 and PKCδ. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-α36.Our results demonstrated that E2 activates the PKCδ/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-α36, suggesting a possible involvement of ER-α36 in E2-dependent growth-promoting effects in endometrial cancer cells.Icaritin, a compound from Epimedium Genus, Icaritin exhibits many pharmacological and biological activities, such as stimulation of neuronal and cardiac differentiation, enhancement of osteoblastic and suppressed osteoclastic differentiation and activity, prevention of steroid-associated osteonecrosis, induction of human prostatic smooth muscle cells apoptosis via ERK1/2 pathway. Previously, it was reported that icaritin exhibits estrogen-like activity in estrogen receptor-positive breast cancer MCF-7 cells at sub-micromolar concentrations. At micromolar range, however, icaritin inhibited growth of prostate cancer PC-3 cells. These results indicated that icaritin has both agonist and antagonist activities depending on concentrations and may function as an estrogen receptor modulator to regulate cell growth. However, there are no reports on activity of Icaritin against endometrial cancer.Mitogen-activated protein (MAP) kinases participate in diverse cellular functions such as cell proliferation, cell differentiation, cell motility, and cell death. There are three major MAPK family subgroups:extracellular signal-regulated kinase1/2 (ERK1/2),c-Jun N-terminal of stress-activated protein kinasesl/2 (JNK1/2) and the p38 protein kinases. The signaling cascades involving JNK and p38, activated by extracellular stress signals, are involved in cell differentiation and apoptosis. Previous studies have demonstrated that transient activation of ERK1/2 plays a pivotal role in cell proliferation and that sustained ERK1/2 activation induces cell cycle arrest and differentiation.Here,we examined Icaritin effect on cell growth of human endometrial cancer Hecl A cells and found that Icaritin potently inhibited proliferation of Hecl A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinDl and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, Icaritin induced sustained phosphorylation of extracellular signal-regulated kinase 1/2 (the MAPK/ERK1/2) in Hecl A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK activation by Icaritin and abolished the Icaritin-induced growth inhibition and apoptosis. Our results demonstrated that Icaritin induced sustained ERK activation and inhibited growth of endometrial cancer Hecl A cells, and provided a rational for preclinical and clinical evaluation of Icaritin for endometrial cancer therapy.