Effect Of Epigallocatechin-3-gallate On Proliferation,Apoptosis,Migration And Invasion Of Tongue Squamous Cell Carcinoma Cells And Its Molecular Mechanism

Backgrounds and ObjectivesThe aim of this research is to investigate the anti-tumor effects of epigallocatechin gallate(EGCG)on the biological characteristics of tongue squamous cell carcinoma(TSCC)cells,including cell proliferation,apoptosis,migration and invasion,and to explore its molecular mechanism��whether the effect of EGCG is going on through TAZ and its possible upstream regulation mechanism.By investigating the connections of EGCG and TAZ in TSCC cells,we can enrich the molecular mechanism of anti-oral cancer drugs,provide the molecular basis for EGCG as a potential new chemotherapy drug of TSCC,and offer more choices and possibilities for drug therapy of TSCC.Methods1.The effect of EGCG on the proliferation of CAL27 and SCC15 cells.After incubating with different concentrations of EGCG for 24 h,the proliferative changes of CAL27 and SCC 15 cells were evaluated by CCK-8 assay.After incubating with different doses of EGCG for 4-5 days,the changes of growth curve in CAL27 and SCC15 cells were also examined by CCK-8 assay.The changes of proliferation-related proteins Erk,p-Erk,Akt,p-Akt were detected.2.The effect of EGCG on the apoptosis of CAL27 and SCC 15 cells.After culturing with different concentrations of EGCG for 24 h,the apoptotic changes of CAL27 and SCC15 cells were measured by flow cytometry experiment,and the results were performed with statistical analysis.The changes of apoptosis-related proteins Bcl-2?Bax?PARP?Cleaved PARP were also detected.3.The effect of EGCG on the migration and invasion of CAL27 and SCC15 cells.After incubating with different times and different concentrations of EGCG,the migratory and invasive changes of CAL27 and SCC15 cells were determined by using scratch and transwell assays,respectively.The results were also performed with statistical analysis.The changes of related proteins Vimnetin?E-Cadherin were measured.4.The connections between EGCG and TAZ in TSCC cells,and its upsteam effect mechanism.After culturing with different concentrations of EGCG for 24 h,the Hippo-related changes of CAL27 and SCC15 cells were determined by Western blotting assay,including TAZ,p-TAZ,MOB1,LATS1,MST1 and SAV1,as well as JNK and p-JNK,and the results were performed with statistical analysis.Extracting cytoplasmic and nuclear proteins,respectively,and the TAZ protein levels were also evaluated using Western blotting assay,which were performed with statistical analysis.5.Whether enforced TAZ can,to a certain degree,abolish the inhibitory effects exerted by EGCG or not.CAL27 cells were treated with TAZ overexpression(OE TAZ)or negative control(NC)lentiviral particles,the transfection efficiency can be examined using Western blotting and qRT-PCR assays.Cells were divided into four groups:OE TAZ group;OE TAZ and EGCG group;NC group;NC and EGCG group.Cell proliferation was examined by CCK-8 and EdU assays;cell apoptosis was evaluated using flow cytometry assay;cell migration and invasion were measured by scratch and transwell assay,respectively.We also did statistical analysis of relative results.6.The effects of EGCG on biological characteristics of CAL27 cells when combining EGCG and simvastatin treatment.Previous research demonstrated that simvastatin can exert inhibitory effect though Hippo-TAZ in vitro,and now the combined effects on biological behaviors on CAL27 cells were examined,including cell proliferation,apoptosis,migration and invasion.The results were performed with statistical analysis.Results1.EGCG treatment inhibited proliferation of CAL27 and SCC15 cells.It demonstrated that cell proliferation was significantly decreased in response to EGCG treatment in time-and dose-dependent manners.The experimental results were statistically significant.The changes of proliferation-related proteins were basically consistent with phenotypes.2.EGCG treatment promoted apoptosis of CAL27 and SCC15 cells.It revealed that cell apoptosis was decreased in a dose-dependent manner with EGCG treatment.The results were statistically significant.The apoptotic protein changes concurred with phenotypic variations.3.EGCG treatment inhibited migration and invasion of CAL27 and SCC15 cells.It revealed that the migratory distances were reduced in time and dose dependent manners;the cell numbers of invading into lower chamber were decreased.The experimental results were in statistical significance.The related protein changes were mainly consistent with phenotypic changes.4.EGCG downregulated the protein expressions of TAZ and upstream signaling effectors.The protein levels of total TAZ and p-TAZ were decreased in response to different doses of EGCG,as well as upstream effectors MOB1 and LATS1.However,EGCG had minimal or no effect on MST1 and SAV1 expressions.The protein level of p-JNK was also reduced,while no or minal difference was observed in JNK expression.The TAZ protein levels in cytoplasm and nucleus were dramatically reduced.5.Western blotting and qRT-PCR experiments indicated that CAL27 cell line,which TAZ was overexpressed,was successfully constructed.TAZ overexpression can attenuate the effects of EGCG on CAL27 cells,including proliferation,apoptosis,migration and invasion.6.EGCG and simvastatin additively inhibited proliferation,migration and invasion,and promoted apoptosis compared to single EGCG treatment in CAL27 cells.ConclusionEpigallocatechin-3-gallate can affect the proliferation,apoptosis,migration and invasion of TSCC through TAZ effector,and its upstream might be mediated by p-JNK.