TFEB Reduces Sensitivity Of Cancer Cells To Doxorubicin By Regulating Autophagy

Aim: To observe the effect of doxorubicin on TFEB expression.To explore whether TFEB plays a role in chemosensitivity of cancer cells to doxorubicin by regulating autophagy.Methods: Immunofluorescence was used to observe the expression of TFEB protein after doxorubicin treatment in cancer cells.Nuclear and cytosolic extracts were prepared according to the manufacturer's instruction after doxorubicin treatment,then cytoplasmic and cell nuclear TFEB protein level were determined with western blot analysis.Transient transfection of plasmid GFP-TFEB and TFEB siRNA was used to overexpression and knockdown TFEB expression,respectively,then CCK8 analysis was used to detect the proliferation inhibition effect of doxorubicin on cancer cells;colony formation analysis was used to detect the effect of doxorubicin on cancer cell survival after TFEB was knocked down.Hoechst dye was used to stain the nucleus of cancer cells to observe the effect of doxorubicin on cancer cell apoptosis after TFEB was knocked down.Western blot analysis was used to detect apoptosis-related protein Caspase 3 after TFEB was knocked down with or without treatment of doxorubicin.The effect of TFEB on autophagy was evaluated with western blot analysis by detecting autophagy-related LC3 protein level after doxorubicin treatment.Results: Immunofluorescence results showd that the endogenous TFEB and the exogenous GFP-TFEB both translocated from the cytoplasm to the nucleus in Lovo cells after treatment with doxorubicin;Western blot analysis results showed that TFEB protein level decreased in the cytoplasm while increased in the nucleus after Lovo cells were treated with doxorubicin;CCK8 assay showed that,after overexpression of TFEB,the inhibitory effects of doxorubicin on proliferation of colon cancer Lovo cells and cervical cancer HeLa cells were reduced,while after knockdown of TFEB,the inhibitory effects of doxorubicin on proliferation of Lovo cells and HeLa cells were increased;cell colony formation assay showed that the effects of doxorubicin on Lovo cells survival increased after TFEB was knocked down;Hoechst dye staining results showed that knockdown of TFEB increased cell apoptosis after Lovo cells treated with doxorubicin;Western blot analysis results showed increased activated Caspase 3 after TFEB was knocked down with or without treatment of doxorubicin in Lovo cells;Western blot analysis showed that autophagy-related protein LC3 upregulated after doxorubicin treatment,overexpression of TFEB increased doxorubicin-induced upregulation of LC3 and knockdown of TFEB decreased doxorubicin-induced upregulation of LC3,and knockdown of TFEB caused TFEB downregulated;after using autophagy inhibitor 3-MA and knockdown of ATG5 to inhibiting autophagy,CCK8 results showed that overexpression TFEB lost its impact on the proliferation inhibitory effects of doxorubicin on Lovo cells.Conclusions: doxorubicin induced tumor cell TFEB nuclear translocation;TFEB overexpresson reduced the chemosensitivity of cancer cells to chemotherapeutic drug doxorubicin,decreased doxorubicin-induced apoptosis,promote tumor cell survival;TFEB regulated doxorubicin-induced autophagy activation;Inhibition of autophagy activation abolished the survival effects of TFEB on cancer cells in the presence of doxorubicin.TFEB reduced the chemosensitivity of cancer cells to doxorubicin by regulating autophagy.