| Incidence of colorectal cancer is related with lifestyle,eating habits. Epidemiological survey suggested that the incidence of colorectal cancer is related with excessive energy intake, obesity, less exercise and so on. In developing countries, eating"Westernization" is an important reason for the rapid increase in incidence of colorectal cancer. It is predicted that China’s morbidity and mortality of colorectal cancer in the future will gradually rise to become one of the most common malignant tumor. Leptin is the product of obesity gene encoded protein, which is mainly secreted by fat tissue, regulate fat metabolism and energy balance. In recent years, studies have found that it is closely related to the development of many malignant tumors. In this study, we first examined the expression of leptin and its receptor in colon carcinoma, and further study of leptin on the proliferation and apoptosis of colon cancer cell line HCT-116, so as to explore its possible mechanism, which provide new experimental evidence for the effect and mechanism of leptin on colorectal cancer.Chapter1Expression of leptin and its receptor on colorectal carcinoma tissuesObjective To investigate the expression of leptin and its receptor in human colorectal carcinoma tissues at different clinical stages so as to show the relationship between leptin and clinical pathological stages of colorectal carcinoma tissues.Methods108specimens of colorectal carcinoma had been collected. To study the expression location of leptin and its receptor in colorectal carcinoma tissues using immunohistological methods, and the expression intensity was grading. The expression of p-Akt, p-GSK3, p-mTOR, p-P70S6K was detected in each specimen, the relationship between expression of leptin,its receptor and p-Akt, p-GSK3, p-mTOR, p-P70S6K was analysed.Results Positive expression of leptin and its receptor in colorectal cancer tissues are brown-yellow particle in cytoplasm. The positive expression rate of leptin in normal intestinal mucosa, adenomatous polyps and colorectal cancer tissues was10%,27.5%,71.3%, and the differences between groups was statistically significant (p<0.05); the positive expression rate of leptin receptor in normal intestinal mucosa, adenomatous polyps, colorectal carcinoma was0%,20%,52.8%, respectively, which were statistically significant between groups (p<0.05). Statistical analysis of expression of leptin, leptin receptor and its clinicopathological parameters found that the expression of leptin was related with the depth of invasion in colorectal cancer, clinical stage, lymph node metastasis, distant metastasis, degree of differentiation. Also, expression of leptin, leptin receptor had a significant correlation with PI3K/Akt/mTOR signaling pathway related molecules p-Akt, p-GSK3, p-mTOR, p-P70S6K (p<0.01), not related with the patient’s age and gender (p>0.05). All datas suggest that leptin may be a indicator of development, metastasis of colorectal cancer, inhibition of leptin or its receptor may be used as an aid for clinical treatment of colorectal cancer.Conclusion A higher expression of leptin and its receptor was existed in colorectal carcinoma tissues. There is a significant correlation between expression of leptin, leptin receptor and invasion depth of colorectal cancer, clinical stage, lymph node metastasis, distant metastasis, differentiation degree.Chapter2Effect of leptin on the proliferation and apoptosis of colorectal carcinoma cell HCT-116Objective From both positive and negative detection of leptin on colorectal cancer cells HCT-116proliferation and apoptosis, and to explore its possible mechanism to provide a new method and theoretical basis for the treatment of colorectal cancer.Methods HCT-116colorectal cancer cells were cultured and detected the expression of ObRa, ObRb by RT-PCR and Western blot. The proliferation of colorectal cancer cells HCT-116stimulated with different concentration of leptin (0ng/mL,10ng/mL,20ng/mL,50ng/mL,100ng/mL,200ng/mL) was detected by the MTT, colony formation assay, soft agar colony formation assay; flow cytometry was used to detect the apoptosis of colorectal cancer cells HCT-116under the stimulation of different concentrations of leptin. Meanwhile, in order to further determine the molecular mechanism of the proliferation, and inhibition of apoptosis of colorectal cancer cell line HCT-116stimulated by leptin, we examined the change of key molecules p-Akt, p-GSK3, p-mTOR, p-P70S6K in PI3K/Akt/mTOR signaling pathway of HCT-116cells under stimulation of leptin; PI3K and mTOR molecule inhibitor LY294002and rapamycin were used to block PI3K/Akt/mTOR signaling pathway to detect the impact of HCT-116cells proliferation and apoptosis by stimulation of leptin with MTT assay, and flow cytometry method. Furthermore, the effect of different doses of leptin on HCT-116cell proliferation, apoptosis-related genes at different time by RT-PCR method.Results Expression of ObRa and ObRb in HCT-116cell line was detected, and we found that ObRa and ObRb were highly expressed in HCT-116cells. Under stimulation of different concentrations of leptin, the cell survival ratio of HCT-116cells was significantly higher than HCT-116cells without leptin stimulation, and when the leptin concentration was50ng/mL,100ng/mL,200ng/mL, the cell survival ratio of HCT-116cells were statistically different with that without leptin stimulation(p<0.05). Colony formation assay showed that compared with HCT-116cells without stimulation of leptin, the clone formation rate of HCT-116cells under the stimulation of100ng/mL,200ng/mL increased significantly which was statistically significant (p<0.05). Under stimulation of200ng/mL leptin, colony formation number was39.5±3, the colony formation rate was3.95%, however, the colony formation number of HCT-116cells without leptin was22±2, colony formation rate was2.2%. Flow cytometry showed that leptin could significantly inhibition of the apoptosis of HCT-116cells serum-free cultured. Under the stimulation of100ng/mL leptin for30min, the related molecules p-Akt, p-GSK3, p-mTOR, p-P70S6K in PI3K/Akt/mTOR signaling pathway in HCT-116cells was activated strongest, so we inhibited the molecules in the PI3K/Akt/mTOR signaling pathway by molecule inhibitor LY294002and rapamycin of PI3K and mTOR, we found that LY294002and rapamycin could inhibit leptin-induced proliferation of HCT-116cells, and inhibition of apoptosis of HCT-116cells. Leptin could promote proliferation, apoptosis-related genes Survivin, Mcl-1, Al, NF-κB expression was time-dependent, with the extension of the stimulation time of leptin, above gene expression gradually increased. With stimulation of different concentrations of leptin on colon cancer cells for48h, leptin could promote the expression of proliferation, apoptosis-related genes Survivin, Mcl-1, A1, NF-κB in a dose-dependent manner, with the increasing doses of leptin, the gene expression gradually increased.Conclusion Leptin can promote the proliferation, inhibition of apoptosis of human colorectal cancer cell line HCT-116, and this effect is carried out through the PI3K/Akt/mTOR signaling pathway. |
PDF Full Text Request