| Endometrial cancer(EC)is one of the most common gynecological malignancies which ranks first in western developed countries,with an increasing morbidity and further research needed to elucidate pathogenesis.Epidemiological investigation revealed that well accepted EC risk factors,including obesity or overweight,hypertension,type 2 diabetes mellitus and PCOS,are also metabolic syndrome(MS)related diseases.The pathophysiology basis of MS lies in the hyperinsulinemia resulting from insulin resistance,which is not only recognized as risk factors for cardiovascular diseases but also closely involved in the pathogenesis of many cancers and cancer related death.Varieties of adipokines secreted by adipose tissue have roles in cancer genesis.Visfatin,a new member of adipokines,is over expressed in many MS related cancers,drawing widely attention as key connection between MS and cancer.Insulin resistance and hyperinsulinemia are involved in the pathogenesis and development of cancer.Our previous work showed that visfatin and insulin are related with the pathogenesis and development of EC.Whether there is a synergistic effect between them remained unclear.This study aimed to investigate the synergistic effect between visfatin and insulin on EC cell lines and xenografts in nude mice through in vitro and in vivo experiments and find out effected cell signaling pathway by detecting related key proteins.Section One The effect of visfatin and insulin on biological behaviors of EC cellsObjective: To investigate the effect of visfatin and insulin on biological abilities of proliferation,apoptosis,migration and invasion in EC cell lines in vitro experiments.Methods: The effect of visfatin and insulin on the proliferation of Ishikawa and KLE cells was detected by CCK-8 kit,and effects on cell cycle and apoptosis were measured by flow cytometry.Transwell chamber was used to detect the effects of visfatin and insulin on the ability of migration and invasion.Results: Visfatin and insulin can promote the proliferation of Ishikawa and KLE cells,promoting G1/S progression in cell cycle,protecting against apoptosis and promoting migration and invasion of Ishikawa and KLE cells.Compared with the control group,the difference was statistically significant(P<0.05).Synergistic effect was observed between visfatin and insulin in these process when compared with solo group(P<0.05).Conclusions: Visfatin and insulin have synergistic effect in promoting cells proliferation,protecting against apoptosis and promoting migration and invasion of Ishikawa and KLE cells,working together to promote the malignant process.Section Two Visfatin promotes the progression of EC via PI3K/Akt and MAPK/ERK1/2 signaling pathways and has synergistic effect with insulinObjective: To investigate the mechanism of visfatin involved in EC progression and the synergistic effects with insulin.Methods: The expression of relevant proteins of PI3K/Akt and MAPK/ERK1/2 signaling pathway including Ins R、p-Ins R、IRS1、p-IRS1、IRS2、p-IRS2 and Akt、p-Akt、ERK1/2、p-ERK1/2p-IRS2 were detected by Western Blot.The proliferation of Ishikawa and KLE cells after insulin receptor silenced by Sh-RNA was detected by CCK-8 kit.Flow cytometry was used to detect the effect of visfatin on the cell cycle of Ishikawa and KLE cells after Ins R down-regulated.Ishikawa cells were transfected with a flag-labeled liposomal overexpression of lentivirus.Immunoprecipitation was used to detect whether visfatin bind with Ins R directly.Results: Visfatin and insulin can activate the downstream PI3K/Akt and MAPK/ERK1/2 signaling pathways by promoting the phosphorylation of Ins R,IRS1 and IRS2,promoting the expression of p-Akt,p-ERK1/2.Synergistic effect was observed between visfatin and insulin in the activation of signaling pathways.After the down-regulation of the Ins R expression,there was an inhibited tendency of pro-proliferative effect of visfatin in EC cells.The difference was not statistically significant between Sh-Ins R and negative control.Immunoprecipitation showed that visfatin did not bind with Ins R directly.Conclusions: Visfatin can activate PI3K/Akt and MAPK /ERK1/2 signaling pathway in EC cells and has synergistic effect with insulin.Section Three The effect of visfatin and insulin on xenografts of EC cells in nude miceObjective: To investigate the effect of FK866,visfatin and insulin on xenografts of EC in nude mice.Methods: Subcutaneous injection of Ishikawa cells in nude mice was done to construct xenografts model.Observe the effect of FK866,visfatin and insulin on the xenografts of EC in nude mice.ELISA was used to detect the levels of serum visfatin in nude mice.The expression of VISFATIN and Ki-67 in subcutaneous xenografts of nude mice was measured by immunohistochemistry.Results: Visfatin can promote the growth of xenografts in nude mice,while FK866 can inhibit that.The volume and weight of xenografts in nude mice treated by combination of visfatin and insulin were significantly higher than those treated by visfatin or insulin alone(P<0.05).Serum levels of visfatin treated by visfatin were significantly higher than those in control group and FK866 group(P<0.05).Immunohistochemical results showed that VISFATIN was positive in tumor tissue.The expression of VISFATIN in visfatin group and visfatin and insulin co-action group were highly expressed when compared with control group and insulin group.The positive rate of Ki-67 expression in FK866 group was lower than that in control group.The positive rate of Ki-67 expression in visfatin or insulin alone group was higher than control group but lower than visfatin and insulin co-action group,which indicates that visfatin and insulin has synergistic effect.Conclusions: In vivo experiments revealed that visfatin and insulin can promote the growth of subcutaneous xenografts in nude mice,and synergistic effect was observed.Nampt inhibitor FK866 can inhibit the growth of xenografts in nude mice,which may provide a new therapeutic way targeting visfatin for EC treatment. |
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