Quercetin Induces Cancer Cell Apoptosis By Regulating MTORC1-TFEB-Lysosome Signaling Pathway

OBJECTIVE To investigate the molecular mechanisms involved in Quercetin-induced apoptosis in tumor cells,and to explore the role of mTORC1-TFEB-lysosome pathway in the anticancer effect of Quercetin.METHODS Human hepatocellular carcinoma(HCC)cell lines HepG2 and Hep3B cells,human breast cancer MDA-MA-231 cells,human colon cancer HCT116(both HCT116 p53 wild-type cells and HCT116 p53 knockout cells)were treated with different doses of Quercetin for various time courses.Then the effects of Quercetin on cell death,autophagy and TFEB-lysosome functions were determined by the following procedures.Firstly in the study of Quercetin induced cell death,MTT assay and flow cytometry were applied to investigate the cytotoxicity of Quercetin in several cancer cells;colony formation assay to evaluate the long-term effect of Quercetin on cell proliferation;then Hoechst staining and Western blotting to further dissect the types of Quercetin-induced cell death.Secondly in the study of Quercetin induced autophagy,co-treatment with autophagy inhibitors and then fluorescence microscope observation were conducted to observe the changes of autophagic flux;Western blotting to determine the effect of Quercetin on autophagy and the regulation of related pathways;knocking-down of essential genes involved in autophagy by siRNA to determine the role of autophagy in Quercetin-induced apoptosis.Lastly in the study of TFEB-lysosome signaling pathway,Western blotting was applied to determine TFEB nuclear translocation and lysosomal protein expression;Flow cytometry and confocal microscopy to detect lysosomal activity with lysotracker Red and MagicRed dyes;TFEB siRNA transfection to confirm the role of lysosome in Quercetin-induced cell deaths.RESULTS Firstly,Quercetin inhibits the proliferation of tumor cells in a dose-and time-dependent manner.Quercetin induced G2/M cycle arrest in tumor cells at lower concentrations(12.5-25 μM),but mainly apoptosis at higher concentrations(50-100 μM).At the same time,typical apoptosis characteristics such as nuclear chromatin condensation and fragmentation were observed.The Western blotting results further demonstrated that Quercetin induces caspase activation and PARP cleavage.Consistently,co-treatment with caspase inhibitor Z-VAD blocks Quercetin-induced cell death and the cleavages of caspase-3 and PARP.More importantly,the above changes were not affected by p53 protein.Secondly,the autophagy study found that Quercetin induces GFP-LC3 puncta formation;co-treatment with lysosomal inhibitor chloroquine further enhances this effect.Western blotting results showed that Quercetin inhibits mTOR and the phosphorylation levels of its downstream proteins S6,S6K,and 4EBP1 to induce autophagy.However,knocking-down Atg7,a key autophagic gene,has no significant effects in Quercetin-induced cell death,suggesting that autophagy may not be essential for Quercetin-induced cells death.Lastly,the study of TFEB-lysosome signaling pathway found that Quercetin promotes nuclear translocation of TFEB from cytoplasm to nuclei,subsequently enhances TFEB activity and up-regulates its target genes(mRNA transcription and protein expression)involved in lysosomal activity.Flow cytometry results and confocal fluorescence imaging together suggested lysosomal activation(as indicated by lysosome acidification,enhanced activity of cathepsin B).By transfection with TFEB siRNA in HepG2 cells,TFEB was found to be essential for Quercetin-induced apoptosis.CONCLUSIONS 1.Quercetin induces p53-independent apoptosis in cancer cells;2.Quercetin induces autophagy by inhibition of mTOR,but Quercetin-induced autophagy does not affect its apoptotic cell death;3.Quercetin promotes TFEB nuclear translocation and lysosomal activation.Furthermore,TFEB activation plays a key role in Quercetin-induced cell death.