Study Of Insulin Effects On The Carcinogenesis Of Endometrial Carcinoma

Endometrial carcinoma is a common gynecological malignancy of genital tract while the etiology is not clear yet. Epidemiologic studies show that obesity, diabetes, hypertension and polycystic ovary syndrome(PCOS) are risk factors of endometrial carcinoma. The pathophysiological basis of these risk factors is insulin resistance. Insulin resistance is defined as a clinical state in which a normal or elevated insulin level produces an attenuated biologic response. Insulin resistance results in hyperinsulinemia. As the risk factors mentioned above are corralated with endometrial carcinoma, we hypothesized a correlationship between insulin resistance,hyperinsulinemia and endometrial carcinoma. Insulin is a kind of multi-functional peptide and elicits multiple responses. As a growth factor, it acts not only on metabolic response but also on mitogenic response and apoptosis. In this study, we tried to investigate the possible roles of insulin in the carcinogenesis of endometrial carcinoma in four parts.Part1. Insulin level and risk of endometrial carcinomaTo study the possible relationship between fast serum of C-peptide(a marker of pancreatic insulin secretion),insulin and endometrial carcinoma risk by a case-control strdy.Cases were 85 women with newly-diagnosed endometrial carcinoma from Tianjin General Hospital and Tianjin Obstet & Gynecol Hospital. 81 controls were female patients admitted for non-hormone related, non-neoplastic conditions from the same hospitals and some healthy women. All cases and controls met a certain criteria. Information of cases and controls was collected by a standard questionnaire. All participants were also measured for current heights,weights,circumferences of thewaist, abdomen and hips. Fast serumC-peptide,insulin(INS),estrogen(El),estradiol(E2),androstenedione(A),testerone(T) and sex hormone binding globin(SHBG) were measured. All statistical analysis were based on Spss 11.5 for Windows.Results showed: ?Fast serum levels of C-peptide and INS of the cases were higher than those of the controls,with odds ratio(OR) of 2.911(95%CI=1.960-4.324) and 1.178(95%CI=1.089-1.275) respectively. The association of C-peptide and endometrial carcinoma was attenuated but still remained after adjustment for BMI and other confounders. The risk increased with increasing levels of C-peptide,and OR from bottom quintile to top quintile were 1, 5.791, 15.329 and 19.507. The highest is 19.509 times of the lowest. The dose-response relationship between C-peptide and endometrial carcinoma risk remained after adjustment for other confounders. We concluded that C-peptide may be a risk factor independent of BMI and other confounders associated with endometrial carcinoma. There was a direct dose-response relationship between C-peptide concentration and endometrial carcinoma risk. ?C-peptide was highly and positively correlated with insulin. Adjustment for C-peptide eliminated the correlations between insulin and endometrial carcinoma while adjustment for insulin had little effect on the relationship between C-peptide and endometrial carcinoma. We confirmed that Fast serum C-peptide may reflect more accurately an individual's level of insulin secretion. (3)As for the correlations between C-peptide and steroid hormones,C-peptide was positively related to E1,E2,A and T,inversely correlated with SHBGPart2. Insulin receptor gene expression in endometrial carcinoma cell line Ishika\va3-H-12Immunocytochemistry and RT-PCR methods were used to To detect the expression of insulin receptor(INS-R) in endometrial cell line Ishikawa3-H-12 forthe following study.We demonstrated the expression of INS-R gene in Ishikawa3-H-12 cell line from protein and mRNA level.INS-R was positively stained in Ishikawa3-H-12 cell plasma;Expression of INS-R gene at mRNA level was also detected. This result was the basis for our further study.Part3. Insulin effects on proliferation and apoptosis of endometrial carcinoma cellsTo explore the possible effects of INS on proliferation, cell cycle distribution and apoptosis of endometrial carcinoma cell line Ishikawa3-H-12.We observed Ishikawa3-H-12 cell growth cultured in serum-free medium with cell starved experiments. Our results showed that Ishikawa3-H-12 cells could be cultured in serum-free medium and reached the flat period after 4 days of culture.Weobserved INS effects in various concentrations*^ lxl0't(\lxl0"8,lxl0"\ lxlO^mol/L) and at different time (24h> 48h> 721k 96h) on proliferation,apoptosis and cell cycle distribution of endometrial carcinoma cells by MTT assay and fluorescence-activated cell sorting technique. We found: (DWith increased concentration of INS and prolonged time,proliferation of endometrial carcinoma cells increased gradually. The proliferation effect of INS is most obvious at 48h. ?Cell cycle distribution analysis revealed that with increased concentration of INS and prolonged time,the percentage of Ishikawa3-H-12 cells at G0-G1 phase decreased and percentage at S phase increased significantly, whereas that of G2-M phase didn't show significant alteration. ?With increased concentration of INS, apoptosis rate of Ishikawa3-H-12 cells decreased gradually,while there is no significant alteration at different time.Our results demonstrated:INS can promote the proliferation and G0-G1/S phase transition in a dose- and time-dependent manner;INS can inhibit the apoptosis of Ishikawa3-H-12 cells in a dose- dependent manner.Part4. Insulin signaling pathway in endometrial carcinoma cellsIn this prat we tried to explore the possible preliminary signaling pathway of INS in endometrial carcinoma cells. Levels of phosphorylated Akt(Ser473) (p-Akt)N MEK(Ser217/221) (p-MEK) and total AkU MEK were examined by western blotting in cells after stimulation with 10^mol/L INS for different time (0min,5min,15min,30min,60min,120min) and with various doses of INS (Omol/L,lx 10"10mol/L, 1 x 10'8mol/L, 1 x 10'6mol/L, 1 x lO^mol/L) for optimal time.Our results showed: ?Activation of Akt: the maximal p-Akt/Akt took place at 15min and the activation persisted at least 120min after stimulation with lO^mol/L INS. The activation of Akt increased gradually with increased doses of INS. (2) Activation of MEK: the maximal p-MEK/MEK took place at 30min and the activation persisted at least 120min after stimulation with lO^mol/L INS.Our results confirmed:INS can activated PI-3K/Akt and Ras-MEK-MAPK signaling pathway in endometrial carcinoma cell line Ishikawa3-H-12. INS may activated Akt and MEK promptly in a dose-dependent manner.Conclusions1. Fast serum C-peptide may reflect more accurately the level of insulin secretion;C-peptide may be a risk factor independent of BMI and other confounders associated with endometrial carcinoma;there was a direct dose-response relationship between C-peptide concentration and endometrial carcinoma risk. C-peptide was positively related to E1,E2,A and T, inversely correlated with SHBG2. INS-R gene was expressed in Ishikawa3-H-12 cell line from protein level and mRNA level. This conclusion was the basis for our further study.3. INS can promoted the proliferation and G0-G1/S phase transition in a dose-and time-dependent manner. INS can inhibit the apoptosis of Ishikawa3-H-12 cells in a dose- dependent manner.4. INS can activated PI-3K/Akt and Ras-MEK-MAPK signaling pathway in endometrial carcinoma cell line Ishikawa3-H-12. INS may activated Akt and MEK promptly in a dose-dependent manner.