Disulfiram Inhibits TGF-? Induced Epithelial-mesenchymal Transition Of Oral Cancer KB

Objectives:Oral cancer is the sixth most common cancer worldwide.Metastasis,relapse as well as chemotherapy tolerance are the main cause of the failure and deaths of oral cancer.However,there has been lack of useful methords to solve theses problems.Transforming growth factor-?(TGF-?)is one of the major inducer of epithelial-mesenchymal transition(EMT)in many researches.Disulfiram(DSF)is an effective treatment for alcohol abuse.Recently,it has been found to promote apoptosis,inhibit invasion and matastasis as well as reverse drug resistance in tumors.In this study,we used TGF-? to induce EMT in oral cancer cell line KB to explore whether DSF could reverse this process,and then to inhibit the invasion and migration of oral cancer and reverse the effect of drug resistance.Methods:In this study,oral cancer cell line(KB)and oral cancer cell resistant cell lines(KBV)were used in this research.First,the cells were pretreated with different concentrations of DSF(1 ?M,5 ?M,10 ?M,20 ?M,40 ?M)and 10 ng / ml TGF-?for 24 h.Methods were used as following:MTT assay was used to test cell proliferation;Cell colony formation was used to test cell colony ability and cell proliferation;Annexin V / PI staining was used to test apoptosis and necrosis;Transwell assay and wound healing assay were used to test invasion and migration;Quantitative Real-time PCR was used to test expression of epithelial,mesenchymal markers(E-cadherin,vimentin)and stem cell associated gene SOX2 and OCT4 on m RNA level.All experimental results are repeated at least 3 times.Statistical analysis was performed using SPSS 17.0 statistical software for t test.Results:1.TGF-?(10 ng / ml)can effectively induce the KB cells from polygonal to long spindle,and promote invasion and migration.With the increase of DSF concentration,the morphology of KB cells gradually restored to morphology before TGF-?treatment.When DSF concentration ? 5 ?M,the expression of TGF-?-induced invasion of kb was significantly inhibited(P <0.05)in a concentration-dependent manner.2.TGF-? can increase the expression of mesenchymal marker(vimentin)and decrease the expression of epithelial cell marker(E-cadherin)in KB cells.This suggests that TGF-? can induce the occurrence of EMT in KB cells.DSF can significantly reduce the expression of vimentin and increase the expression of E-cadherin at high doses.This suggests that DSF can inhibit TGF-?-induced EMT in KB cells.3.When DSF concentration ? 5 ?M,it can significantly inhibit the proliferation of KB cells in vitro(P <0.05).When the concentration of DSF ? 10 ?M,it can significantly inhibit the number and diameter of clones in KB cells(P <0.05).However,DSF(concentration ? 20 ?M)can significantly promote the apoptosis of KB cells(P <0.05).4.KBV cells are resistant to tumor chemotherapeutic drugs vincristine.When vincristine combined with 5 ?M DSF,KBV cells will be killed by vincristine.5.The m RNA expression of SOX2 and OCT4 was down-regulated by DSF in KB cells(P <0.05).Conclusion:DSF can not only inhibit EMT by TGF-?,inhibiting invasion and migration,but also inhibit cell proliferation,colony formation and promote apoptosis in oral cancer in vitro.Apart from that,DSF can reverse drug resistance in oral multi-drug resistant cancer cell line,and decrease the stemness in oral cancer cell line.