Overexpression Of HepaCAM Inhibits Bladder Cancer Proliferation And Viability Via Downregulation Of AKT/FOXO Signaling

PART ONE: THE EXPRESSION AND CORRELATION OF HEPACAM AND FOXO3 IN BLADDER CANCER TISSUESObjective Collect the clinical samples to study the expression and correlation between hepa CAM and FOXO3 in bladder cancer.Method Collected 22 tumor tissues and 14 adjacent normal bladder tissues of bladder cancer patients, immunohistochemistry(IHC) assay was used to detect the expression of hepa CAM and FOXO3。Imaging with the use of upright microscope, analyzed the correlation of hepa CAM and FOXO3 with each clinical parameter and the relation between the two of them.Result The expression of hepa CAM and FOXO3 were deficiency and with high expressed in adjacent normal tissues. The statistic analysis showed that hepa CAM and FOXO3 positive correlation in bladder cancer(r=0.898, p<0.01) and there has no significant with clinical parameter(p>.05).Conclusion The low expression of FOXO3 in bladder cancer was positive correlation with hepa CAM. This result established clinical foundation for exploration the mechanism of hepa CAM regulates FOXO3 in bladder cancer.PART TWO: THE REGULATION OF HEPACAM FORAKT/FOXO AND PROLIFERATION RELEVANCEDPROTEINObjective Detect the regulation of overexpressed hepa CAM on AKT/FOXO and proliferation protein in bladder cancer cell. Then, combined treatment with small molecular inhibitor of AKT and overexpression of hepa CAM adenovirus to identified the mechanism of hepa CAM suppressed bladder cancer cell proliferation and viability via AKT/FOXO.Method Tested the adenovirus-hepa CAM induced the expression changes of p-AKT, p-FOXO1, p-FOXO3 and proliferation relevanced protein CDK2 and PCNA by Western blot; Immunofluorescence(IF) was used to determine the location of FOXO3 in bladder cancer T24 and BIU-87 cell, meanwhile verification the distribution by western blot. MTT assay was used to identified the effect of LY294002 on viability in BIU-87, T24 cell and detected the inhibition of bladder cancer cell when combined treatment with hepa CAM and LY294002. In addition, proved the hepa CAM and LY294002 combined effect on proliferation in BIU-87 and T24. Then, detect the protein expression of p-AKT, p-FOXO1, p-FOXO3, CDK2 and PCNA with combined treatment of hepa CAM and LY294002, statistic analyzed the results.Result Overexpression of hepa CAM downregulated p-AKT, p-FOXO1, p-FOXO3 and small molecular protein CDK2 and PCNA significantly(P<0.05) in bladder cancer cell T24 and BIU-87; Immunofluorescence test proved that hepa CAM improved the nuclear translation of FOXO3 increased in nuclear and reduced in cytoplasm, the same as western blot. The inhibition of bladder cancer cell BIU-87 and T24 had concentration and time dependent by LY294002. Combined treatment with hepa CAM and LY294002 decreased cell viability remarkable when compared with hepa CAM or LY294002 used alone. The colony formation assay found that hepa CAM and LY294002 used together blocked cell proliferation more markedly compared with LY294002 or hepa CAM, respectively. Western blot showed that combination of hepa CAM and LY294002 increased the inhibition effect of LY294002 on p-AKT, p-FOXO1, p-FOXO3, CDK2 and PCNA.Conclusion Overexpressed hepa CAM induced nuclear translocation of FOXO3 and inhibited proliferation and viability of bladder cancer cell T24 and BIU-87 via downregulated AKT/FOXO.