Mechanism Of Inhibitory Effects Of Proliferation On Endometrial Carcinoma(HEC-1A) Cells

Endometrial carcinoma is the most lethal gynecologic malignancy,the major strategy for endometrial cancer treatment is including surgery,radiotherapy,chemotherapy and hormone.Although traditional combination chemotherapy,such as platinum medicine,has improved the prognosis of the initial treatment of endometrial cancer,the survival and replase rate of advanced-stage endometrial cancer is still unsolved,due to extensive primary and secondary drug resistance and adverse reaction of chemotherapy.Therefore,the identification of new sensitive drugs and chemotherapy optimization programs for endometrial cancer is imperative.Cyclopamine?CYP?is the specific inhibitor of Sonic hedgehog signaling pathway?Shh?by changing the spatial structure of the smoothened?Smo?to block Shh signaling pathway activation and to prevent its downstream target gene expression.At present,it has been confirmed that CYP can inhibit tumor cell proliferation in various human cancer.Also,the CYP can inhibit the cell growth in Ishikawa and HHUA cells,but the effects of CYP on endometrial cancer cells and its therapeutic function are poorly understood.In our study,human endometrial cancer cell line HEC-1A,was treated to various CYP dose including 0,5,10,20 and 40?mol·L^-1 for 24 h,48 h or 72 h.Cell morphologies were observed by microscopy and Wright Giemsa staining.The rates of dead cell was observed by AO/EB double staining assay.Additionally,cell growth was determined using the CCK-8 assay,and the apoptosis rate was monitored by Annexin V-FITC/PI method using flow cytometry.The expression levels of Bax and Bcl-2 genes was analyzed by quantitative real-time polymerase chain reaction?Q-PCR?,and tumor cell migration was measured by wound healing assay.The cell cycle and the expression of autophagy related protein LC3-B gene and protein were analyzed by Q-PCR and Western blot analyse.Results: After treatment with various dose of CYP,the morphology of HEC-1A displayed significant morphology changes in a concentration-dependent manner.The dead cell rate of CYP treated-cell was increased by AO/EB staining,and the proliferation ability was dramatic decreased by CCK-8 assay.Flow cytometry assay determined that treatment with CYP induced the formation of apotosis in a dose-dependent manner?P<0.05?.As indicated by Q-PCR,a significant increase in Bax expression and a parallel decrease in Bcl-2 expression was observed following CYP treatment.The migration rate of HEC-1A cell treated with CYP was descending,while the cell cycle was blocked in the G₀/G₁ phases.Western blot analysis revealed that treatment with CYP increased the expression of LC3-B protein in a dose-dependence.Conclusion: CYP inhibits growth of endometrial cancer cell line HEC-1A,causes cell-cycle arrest,diminishes migration capacity,induces apoptosis and autophagy.