| BackgroundEndometrial carcinoma is one of the most common female genital tract malignancies with increasing morbidity reported worldwide in recent years. It is well-known that risk for endometrial adenocarcinoma increases in patients with high estrogen levels that are unopposed by progestin, since estrogen exhibits growth-promoting properties in endometrial cancer cells. But, the mechanism of estrogen inducing endometrial carcinoma is unclear. In the classical model, the biological functions of estrogens are mediated by the estrogen receptor (ER), which function as hormone inducible transcription factors binding to the estrogen-responsive element (ERE) located within the promoter region of target genes, induce gene transcription and proteins synthesis. This process is named "transcriptional effect" or "genomic effect" or "nuclear effect". However, this model does not benefit to estrogen-dependent cancers, such as endometrial cancer and breast cancer. It cann’t explain why anti-estrogen therapy is inefficient to some patients with ER-positive endometrial cancer, efficient to some patients with ER-negative endometrial cancer.It has been commonly believed that estrogen affects cell proliferation mainly through nuclear events. In addition to its ability to mediate gene transcription, estrogen also elicits rapid, non-transcriptional effects involving activation of signal transducing pathways. Non-transcriptional effect, also named "non-genomic effect" or "non-nuclear effect" does not include gene transcription and protein synthesis. Recent report indicated that estrogen immediately activated PI3K/AKT (phosphatidylinositol3-kinase-AKT) pathway in endometrial cancer cell lines, Ishikawa and HEC-1Acells. This effect was ER-dependent in Ishikawa cells, but ER-independent in HEC-1A cells. Non-transcriptional effect of estrogren could activate several pathways including Ras/Erk (extracellular-regulated kinase) pathway. This effect would help to treat endometrial cancer, especially for hormone therapy. However, what mediates activation of PI3K/AKT pathway induced by estrogen is unclear.G protein coupled estrogen receptor, GPER is a estrogen-binding protein, also named protein coupled receptor30, GPER. GPER was found in breast, ovary, placenta, prostate, heart and uterus. GPER is not ralated with nuclear ER, has high affinity with estrogen.GPER is one of the breast cancer progression markers. GPER involved in MAPK (mitogen activated protein kinase) pathway activated by TGF-β in breast cancer cells. Estrogen activated PI3K/AKT pathway via GPER in ER-negative and GPER-positive breast cancer SKBR3cells, promoted tumor growth and progression. However, GPER inhibited cell proliferation in ER a-positive breast cancer MCF-7cell, enhanced cell proliferation in ER a-negative breast cancer SKBR3cell. GPER expression was high in thyroid cancer and ovarian cancer cells. There was report GPER was involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6secretion via the MEK/ERK MAPK pathway. High GPER expression was found in atypical hyperplasia endometrium and endometrial cancer tissue, especially in the endometrial cancer with poor differentiation, deep myometrial invasion, and late clinical stages. And the expression of GPER was positively correlated with AKT activation. However, the relationship of GPER with activation of PI3K/AKT pathway induced by estrogen is unclear.In this thesis, we investigated GPER mediated the non-transcriptional effect of estrogen in ER-positive and ER-low expression endometrial cancer cells.Part I The expressions of GPER and ERs in endometrial carcinoma cells1ObjectiveTo detect expressions of GPER and ERs in endometrial cancer cells.2Methods2.1The expressions and localizations of GPER, AKT, p-AKT, ER α and ER β.Immunocytochemistry assays were performed to investigate the expressions and localizations of GPER, AKT, p-AKT, ER α and ER β in Ishikawa and HEC-1A cells.Immunoblot was performed to determine the expressions of GPER, ER α and ER β in Ishikawa and HEC-1A cells.3. Results3.1The expressions and localizations of GPER, AKT, p-AKT, ER α and ER β.ER α expressed and localized in the nucleus of both cell lines, positive staining for ER β was clearly observed in the nucleus of Ishikawa cells, in contrast, expression of ER β was barely detectable in HEC-1A cells. GPER expressed and localized to the endoplasmic reticulum of both cell lines. AKT and p-AKT expressed in both cells, the former localized in cytoplasm and cell membrane, the latter only localized in cytoplasm.The expressions of GPER, ER α and ER β were positive in Ishikawa cells. The expressions of GPER and ER α were positive in HEC-1A, but ER β was negtive.4ConclusionThe data conformed the expressions of target proteins. Part Ⅱ GPER mediated the non-transcriptional effect of estrogen on activation of PI3K/AKT pathway1ObjectiveTo investigate whether the non-transcriptional effect of estrogen on activation of PI3K/AKT pathway was mediated by GPER in endometrial cancer cells.2Methods2.1The Effect of E2on the expression of GPER, ER a and ER β.Immunoblot was performed to determine the expressions of GPER, ER a and ER β induced by1μM17β-estradiol (E2) at0,15,30,60and120min in Ishikawa and HEC-1A cells.2.2Whether GPER directly binds to PI3K in activated PI3K/AKT pathway.Immunoprecipitation was performed to investigate whether GPER directly bind to PI3K in activated PI3K/AKT pathway.2.3Down-regulation of GPER affected PI3K/AKT pathway.Down-regulation of GPER expression was performed by transfecting GPER shRNA. Immunoblot was performed to investigate the activation of PI3K/AKT pathway in Ishikawa and HEC-1A cells.3Results3.1The Effect of E2on the expression of GPER, ER a and ER β.In ERs-positive Ishikawa cells, GPER reached the peak at30min after the stimulation by E2; In ER a-positive HEC-1A cells, GPER reached the peak at15min after the stimulation of E2(p<0.05). Short treatments did not modify the levels of ER a and ER P in both cells.3.2Whether GPER directly binds to PI3K in activated PI3K/AKT pathway. GPER did not bind to PI3K in activated PI3K/AKT pathway.3.3Down-regulation of GPER affected PI3K/AKT pathway. Low-expression of GPER in stable transfected cells verified by immunoblt, led to significantly reduce phosphorylation of AKT. Taken together, down-regulation of GPER inactivated PI3K/AKT pathway.4Conclusions4.1GPER mediated the non-transcriptional effect of estrogen on the activation of PI3K/AKT pathway in endometrial cancer cells.4.2GPER did not bingd to PI3K, how to activate PI3K/AKT was unclear.Part Ⅲ the effect of GPER on the proliferation and apoptosis of endometrial cancer cells and tumor formation in nude mice1ObjectiveTo investigate the effects of GPER on the proliferation and apoptosis of endometrial cancer cells and tumor formation in nude mice.2Methods2.1MTS assay detected the effected of down-regulation of GPER on the proliferation of Ishikawa and HEC-1A cells.To determine whether GPER affect growth of endometrial cancer cells, we carried out a proliferation assay to test whether down-regulation of GPER could have effects on endometrial cancer cell proliferation. Cells were incubated in DMEM containing5%(v/v) DCC-FBS and1μM E2for1d,2d,3d,4d or5d or100n g/ml of the GPER inhibitor PTX (pertussis toxin) and10μM of the PI3K inhibitor WM (wortmannin) for5d. Cell proliferation was evaluated using MTS.2.2The effect of down-regulation of GPER on apoptosis and cell cycle.Flow cytometry analysis was performed to investigate the effect of GPE30on cell apoptosis and cell cycle of Ishikawa and HEC-1A cells. 2.3The effect of down-regulation of GPER expression on tumor formation in nude mice.To further verify the down-regulation of GPER expression mediated the proliferation inhibition in vivo, Ishikawa, Ishikawa-shGPER, Ishikawa-Vector, HEC-1A, HEC-1A-shGPER and HEC-1A-Vector cells were implanted into the right flank of nude mice. The tumor volumes were monitored every three days for3weeks.2.4The effect of down-regulation of GPER expression on apoptosis of endometrial carcinoma cells via PI3K/AKT pathway in vivo.Immunohistochemistry, TUNEL and immunoblot were performed to determine the effect of GPER on PI3K/AKT pathway in Ishikawa and HEC-1A xenografts of nude mice.3Results3.1MTS assay detected the effected of down-regulation of GPER on the proliferation of Ishikawa and HEC-1A cells.E2increased the proliferation of endometrial cancer cell lines, Ishikawa and HEC-1A. Ishikawa-shGPER, HEC-1A-shGPER, Ishikawa and HEC-1A cells treated with GPER inhibitor PTX and PI3K inhibitor WM, their proliferation declined even in the presence of E2, versus the negative control group.3.2The effect of down-regulation of GPER on apoptosis and cell cycle.Down-regulation of GPER expression clearly induced early apoptosis, and mediated cell cycle arrest of Ishikawa and HEC-1A cells.3.3The effect of down-regulation of GPER expression on tumor formation in nude mice.Down-regulation of GPER expression obviously decreased the volumes and weights of tumors in Ishikawa-shGPER and HEC-1A-shGPER groups, compared to that in the untransfected or Ishikawa/HEC-1A-Vector groups (P＜0.05). However, there were no differences between untransfected and Ishikawa-Vector group in the volumes of tumors (P＞0.05), suggesting down-regulation of GPER could significantly inhibit the tumor formation. 3.4The effect of down-regulation of GPER expression on apoptosis of endometrial carcinoma cells via PI3K/AKT pathway in vivo.Tumors of Ishikawa/HEC-1A-shGPER group compared with tumors of untransfected group and Ishikawa/HEC-1A-Vector group showed increased numbers of apoptotic cells with condensed and irregularly shaped nuclei, staining positively for TUNEL. Immunohistochemistry assay found the expression of p-AKT and caspase-3increased, and p-Bad expression decreased in the xenografts of nude mice.4Conclusions4.1Down-regulation of GPER expression mediated cell cycle arrest and apoptosis.4.2Down-regulation of GPER expression inhibited the growth of endometrial cancer xenografts via PI3K/AKT pathway. |
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