Study On The Correlation Between Insulin/Shc/Ras/ERK Signaling Pathway And Endometrial Carcinogenesis

It is accepted that the risk factors for endometrial carcinoma include obesity, hypertension and diabetes mellitus.The same pathophysiological basis of these risk factors is insulin-resistance.We hypothesized a correlation between insulin resistance, hyperinsulinemia and endometrial carcinoma,we want to investigate the role of Insulin/Ras/ERK signaling pathway in the endometrial carcinogenesis.SectionⅠ.Expression and activation of Shc,Grb2 in endometrial carcinomaObjective:This study was designed to explore the role of src homology domain 2 containing protein(Shc)and growth factor receptor-bound protein 2(Grb2)expression and activation as well as Shc·Grb2 level in endometrial carcinogenesis.Methods:62 endometrial carcinoma cases(EC),17 atypical hyperplasia patients (AHE)and 25 normal controls(NE)were from General Hospital of Tianjin Medical University.Shc,Grb2 expression and Shc activation were determined by western blotting.Immunoprecipitation/WB was used to measure the level of Shc·Grb2.Result:Fast serum levels of C-peptide and insulin of EC were higher than those of the controls.Tyrosine phosphorylation of Shc and level of Shc·Grb2 were correlated with fasting serum C peptide and insulin level positively.There was a positive relationship between Shc activation and Shc·Grb2.Tyrosine phosphorylation of Shc and level of Shc·Grb2 in EC were higher than AHE and NE groups.Shc tyrosine phosphorylation and Shc·Grb2 level were significantly higher in patients with poor clinical-pathologic features.Conclusions:Insuin maybe stimulate Shc activation and Shc·Grb2 association in endometrial carcinoma.Shc activation and Shc·Grb2 association maybe are involved in carcinogenesis and development of endometrial carcinoma. SectionⅡ.Expression and activation of Ras and extracellular signal regulated kinase in endometrial carcinomaObjective:The objective of this study was to evaluate the expressions of Ras and extracellular signal regulated kinase(ERK)as well as ERK activation in endometrial carcinoma.Methods:Western blot was used to measure Ras,ERK expression and ERK activation.Result:Expressions of Ras and ERK as well as ERK phosphorylation were often present in endometrium.Serum C-peptide and insulin concentrations were all positively correlated with ERK activation.The activation of ERK in endometrium was positively correlated with the activation of Shc and level of Shc·Grb2.ERK activation in EC group was more significant than that in AHE and NE groups.Abnormal activation of ERK in endometrial carcinoma was associated with poor clinical-pathologic features.Conclusions:The results imply insulin promote ERK phosphorylation via Shc activation and Shc·Grb2 formation.ERK overactivation maybe is involved in carcinogenesis and development of endometrial carcinoma.SectionⅢ.Expression of Cyclin D1 in endometrial carcinomaObjective:The aim of this study is to explore the role of Cyclin D1 expression in endometrial carcinogenesis.Methods:Western blot was used to measure Cyclin D1 expression.Results:Cyclin D1 often expressed in endometrium.Serum C-peptide and insulin levels were all positively correlated with Cyclin D1 expression.The protein level of Cyclin D1 in endometrium is positively correlated with the activation of p-Shc, Shc·Grb2 and p-ERK.Cyclin D1 expression of in EC was higher than AHE and NE groups.Abnormal expression of Cyclin D1 in endometrial carcinoma was associated with poor clinical- pathologic features. Conclusions:The results suggest insulin increase Cyclin D1 level by Insulin/Shc/Ras/ERK signaling pathway.Cyclin D1 maybe is involved in carcinogenesis and development of endometrial carcinoma.SectionⅣ.Effect of insulin on Shc/Ras/ERK signaling pathway in endometrial carcinoma cellsObjective:The aim of this study was to investigate the effect of insulin on Shc/Ras/ERK signaling pathway.Methods:Levels of p-Shc,Shc,Shc·Grb2,p-ERK,ERK and Cyclin D1 were examined in cells after stimulation with insulin/PD98059.Protein levels were examined by western blotting and mRNA levels were observed by RT-PCR.Result:Insulin induced Shc phosphorylation,Shc·Grb2 formation and ERK phosphorylation in a time-dependent manner.The maximal took place at 15,15 and 30 min respectively.Insulin increased Cyclin D1 mRNA and protein level,the maximal levels took place at 12 h and 24 h respectively.PD98059 blocked ERK phosphorylation,Cyclin D1 mRNA transcription and protein expression induced by insulin in a dose-depedent manner.Conclusions:insulin can activate Shc/Ras/ERK signaling pathway in endometrial carcinoma cell line Ishikawa3-H-12 and promote Cyclin D1 mRNA transcription and protein expression.Activation of ERK is MEK-dependent.Increase of Cyclin D1 mRNA and protein are ERK-dependent.SectionⅤ.Mitogenic,anti-apoptotic and invasion effects induced by insulin in endometriai cancer cells are via Ras/ERK signaling pathwayObjective:We explore the role of Ras/ERK signaling pathway in the insulin-induced regulation of proliferation,apoptosis and invasion in endometrial cancer cells in vitro.Methods:Cell proliferation,apoptosis rate and invasion were determined by MTT assay,flow cytometric analysis and transwell chamber respectively.Result:Insulin promoted the proliferation of Ishikawa cells significantly,this effect was inhibited by PD98059 in a concentration-dependent manner.Flow cytometric analysis showed a reduction in apoptotic cell population in cells treated with insulin. PD98059 blocked this anti-apoptotic effect of insulin.Invasion of Ishikawa cells was increased with insulin treatment and was partly prevented by PD98059.Conclusions:Insulin-induced mitogenic and anti-apoptotic effects in endometrial cancer cells are ERK-dependent.Insulin-induced invasion is ERK-dependent partly.