The Role Of FOXO3a In The Proliferation,Transition And Migration Of Hepatocellular Carcinoma Cells

The First PartThe role of FOXO3a in pro-proliferation of hepatocellular carcinoma cells induced by serotoninAim:Peripheral serotonin (5-hydroxytryptamine,5-HT) plays an important role in the carcinogenesis and proliferation of hepatocellular carcinoma (HCC). While the mechanism of serotonin to promote HCC cells’proliferation remains to be obscure. And FOXO3a is reported as a vital transcription factor involving in regulating some key signaling pathways in various cells. In this study, we explored the role of FOXO3a in serotonin-mediated HCC cells’proliferation and its mechanism.Methods and materials:Three HCC cell lines, HuH-7, HepG2and Hep3B, were selected and cultured. The influence of serotonin in the viability and proliferation of HCC cells culturing with/without serum was measured by the cell counting kit-8(CCK-8) assay, the cell proliferation assay (Edu) and cell cycle analysis by flow cytometry. And the FOXO3a-relevant mRNA expression, phosphorylation level, subcellular localization and the phosphorylation of upstream regulatory factor AKT were all detected. The siRNA interfering and plasmid transfection were applied to inhibit and up-regulate the expression of FOXO3a in all three HCC cells respectively and then the cell viability, proliferation and intracellular FOXO3a expression (mRNA and protein) in different culture conditions were all detected. The expression of serotonin2B receptor (5-HT2BR) was tested and the specific inhibitor of5-HT2BR was used to assess the role of serotonin in promoting cells’proliferation and regulating expression of intracellular FOXO3a in HCC cells.Results:The proliferation of all HCC cells culturing without serum was significantly inhibited and serotonin could reverse the inhibited proliferation of HuH-7cells culturing without serum, while adding serum to the medium failed to promote HuH-7cells proliferation. And the inhibited proliferation of HepG2and Hep3B cells induced by deprivation of serum could not be reversed by serotonin. Serum deprivation significantly down-regulated FOXO3a expression in HCC cells and serotonin enhanced the expression of FOXO3a in HuH-7cells but not in HepG2and Hep3B cells. In deprived-serum HuH-7cells, serotonin up-regulated the expression of FOXO3a and the AKT phosphorylation but had no effect on FOXO3a’s nuclear localization; And in deprived-serum HepG2and Hep3B cells, serotonin failed to alter the FOXO3a phosphorylation. Plasmid transfection of non-phosphorylated FOXO3a successfully increased FOXO3a expression and promoted the proliferation of all deprived-serum HCC cells. Compared with HepG2and Hep3B cells,5-HT2BR was highly expressed in HuH-7cells and its specific inhibitor could block serotonin-induced FOXO3a up-regulation and reversed the proliferation promotion.Conclusion:The results demonstrated that in the serum-deprived circumstance FOXO3a acts as the factor promoting the proliferation of HCC cells. And serotonin functions to promote cells’proliferation via up-regulating FOXO3a in HCC cells, which is mediated by5-HT2bR. Drugs targeting the pathway of serotonin-5-HT2BR-FOXO3a in HCC cells may be a new promising strategy for the treatment of HCC.The Second PartThe role of FOXO3a in epithelial mesenchymal transition in hepatocellular carcinoma cells induced by doxorubicinAim:The induced adaptive drug resistance in the chemotherapeutic treatment is mainly responsible for the high mortality of hepatocellular carcinoma (HCC). Doxorubicin is one of the most commonly used drugs in HCC’s chemotherapy, while it easily induces HCC’s epithelial-mesenchymal transition (EMT), which is a main cause of doxorubicin-induced drug resistance in HCC. Salinomycin, an ionophore antibiotic, is able to selectively kill cancer stem cells.In this study, we aimed to explore whether ionophore could exert synergistic cytotoxicity with doxorubicin in HCC cells and the underlying mechanisms.Methods and materials:Four HCC cell lines, HuH-7, HepG2, SNU-449and SNU-387, were selected and studied. After treatment of doxorubicin or doxorubicin plus salinomycin, HCC cells’ viability and EMT-associated proteins were assessed by cell count kit-8(CCK-8) and Western blot, respectively. The expression, activation and subcellular localization of FOXO3a were detected by western blotting and immunofluorescence in HCC cells treated by doxorubicin or doxorubicin plus salinomycin. FOXO3a expression was up-regulated and down-regulated by transfecting FOXO3a siRNA and FOXO3a-TM plasmid, respectively, and then the cell viability and EMT-associated proteins were both assessed after doxorubicin treatment alone or combined treatment. In addition, we detected the interaction of FOXO3a with β-catenin using Co-Immunoprecipitation (Co-IP) and measured the expression of β-catenin/TCF target genes (ZEB1, c-Myc and CyclinD1) in HCC cells. At last, we using the xenograft model of HCC in nude mice to further confirm the synthetic curative efficacy of salinomycin and doxorubicin to treat HCC in vitro.Results:Doxorubicin treatment significantly reduced expression of E-cadherin and upregulated vimentin in HCC cells compared to untreated control, whereas salinomycin reversed doxorubicin-induced expression changes of EMT-markers and significantly enhanced the cytotoxicity of doxorubicin. Moreover, doxorubicin upregulated phosphorated-FOXO3a expression and promoted the cytoplasm localization of FOXO3a, whereas salinomycin reversed phosphorylation of FOXO3a and maintained FOX03a location in cell nucleus. These observations were further confirmed by alteration of the expression of FOX03a. After inhibiting FOXO3a expression, salinomycin failed to reverse doxorubicin-induced EMT or the enhanced cytotoxicity combined with doxorubicin, while FOX03a activation resulted in the opposite effect. The interaction of β-catenin/TCF and its targeting downstream proteins were key factors to regulate doxorubicin-induced EMT and PKF118-310, the specific inhibitor of β-catenin/TCF complex, significantly reversed doxorubicin-induced EMT and enhanced HCC cells’sensibility to doxorubicin. Activation of FOXO3a or salinomycin treatment promoted the interaction of FOXO3a/β-catenin to block the β-catenin/TCF complex, and attenuated the transcription of TCF4dependent genes to finally trigger the progress of EMT.Conclusion:This study demonstrated that inactivation of FOXO3a deviating from cell nucleus is essential for doxorubicin-induced EMT in HCC cells. And salinomycin enhances cytotoxicity of doxorubicin in HCC cells by preventing EMT process via binding intranuclear aggregation of activated FOXO3a to β-catenin, inhibiting the transcription of TCF dependent target genes to regulate EMT.