Nutlin-3 Reverses The Epithelial-mesenchymal Transition In Gemcitabine-resistant Hepatocellular Carcinoma Cells

Objective: The incidence of hepatocellular carcinoma(HCC)is very high in China.Besides,the treatment of advanced HCC is a difficult problem to be solved.Sorafenib is the standard treatment of advanced HCC.However,sorafenib can only achieve a moderate degree of clinical benefit.In recent years,the new cytotoxic drugs represented as oxaliplatin(OXA)and gemcitabine(GEM)has excellent application prospects.Nevertheless,acquired resistance is the main factor that restricts the maximum efficiency of the drugs in the treatment of advanced HCC.In order to explore the mechanism of drug resistance and seek the treatment strategy to overcome resistance in HCC,we developed gemcitabine-resistant(GR)HCC cells(Hep G2 GR and SMMC-7721 GR cells).In the same time,we observed that gemcitabine-resistant cells acquired epithelial-mesenchymal transition(EMT)phenotype.Further research shows that EMT may be the main mechanism of resistance to GEM in GR HCC cells.In this study,we elucidated a role of Nutlin-3 in reversing the epithelial-mesenchymal transition(EMT)and the correlation mechanism in gemcitabine-resistant(GR)HCC cells.Nutlin-3 could be an attractive agent for the treatment of HCC and offers new prospects to overcome chemoresistance.Methods: 1.We have cultured GR HCC cells(Hep G2 GR and SMMC-7721 GR cells)in our laboratory as described previously.To determine whether Nutlin-3 inhibited cell proliferation in GR HCC cells,we performed MTT assays following Nutlin-3 treatment.2.The morphological change of GR HCC cells were studied after being treated with different concentrations of Nutlin-3.Besides,we observed the changes of thebiological behavior before and after intervention through cell wound healing,migration,invasion,attachment and detachment assays.3.RT-PCR and western blotting analysis were used to measure the expression of EMT markers including e-cadherin,vimentin,snail and slug in GR HCC cells after being treated with different concentrations of Nutlin-3.4.RT-PCR and western blotting analysis were used to measure the expression of Smad2 and m RNA in HCC parental cells(Hep G2 and SMMC-7721)and GR HCC cells(Hep G2 GR and SMMC-7721 GR).RT-PCR and western blotting analysis also were used to measure the expression of Smad2 and m RNA in GR HCC cells after being treated with different concentrations of Nutlin-3.5.We depleted Smad2 using specific si RNAs in GR Hep G2 cells.After verify the jamming efficiency,we used RT-PCR and western blotting analysis to measure the expression of EMT markers including e-cadherin,vimentin,snail and slug in GR HCC cells transfected with Smad2 si RNA1.We observed the effect of Smad2 instantaneous silence on EMT molecular phenotype.6.We observed the effect of Smad2 instantaneous silence on the biological characteristics of EMT in Hep G2 GR after Smad2 si RNA1 transfection by the morphology observation,migration or invasion assay,scratch assay,detachment and attachment assays.Results: 1.Nutlin-3 significantly inhibited cell proliferation in both Hep G2 and SMMC-7721 cells.2.Nutlin-3 altered morphology of GR HCC cells.Specifically,pseudopodia were reduced and cells became ovoid and clustered during growth in Hep G2 GR and SMMC-7721 GR cells following Nutlin-3 treatment.3.The motility and invasion ability were decreased in Hep G2 GR and SMMC-7721 GR cells following Nutlin-3 treatment.4.Nutlin-3 reversed EMT phenotype in Hep G2 GR and SMMC-7721 GR cells Furthermore,Nutlin-3 treatment increased E-cadherin protein levels,but decreased the protein levels of Vimentin,Snail and Slug in Hep G2 GR and SMMC-7721 GR cells.5.We found higher expression of Smad2 in Hep G2 GR and SMMC-7721 GR cells compared with their parental control cells.Nutlin-3 inhibited the expression of Smad2 in both Hep G2 GR and SMMC-7721 GR cells.6.Depletion of Smad2 reversed EMT molecular phenotype and changed biological characteristics of Hep G2 GR cells.Conclusion: 1.Nutlin-3 reversed EMT in Hep G2 GR and SMMC-7721 GR cells.Besides,the motility and invasion ability were decreased in Hep G2 GR and SMMC-7721 GR cells following Nutlin-3 treatment.2.Nutlin-3 reversed EMT in GR cells through regulation of Smad2 expression.Nutlin-3 reversed EMT through influence the expression of Snail and Slug.