Role Of TP63α In Sulforaphane Prevention Of TGF-β-induced Lung Cancer EMT

Objective:Lung cancer is one of the highest incidence and death rates in the world.TGF-β is an classic inducant that occurs in cancer cell EMT.TP63a is closely related to the transfer of multiple cancers.Sulforaphane is a phytochemical for multiple cancers in multiple stages of cancer development.We established the model of TGF-β-induced lung cancer cells.Exposure of TGF-β-induced lung cancer cells to sulforaphane,we explore the preventive effect of sulforaphane to EMT.Treated the TGF-β-induced lung cancer cells with TP63α and sulforaphane,we found if TP63αplays an important role in sulforaphane suppress TGF-β-induced lung cancer EMT.Methods:1.The A549 and H1299 lung cancer cells were treated with different concentrations of sulforaphane for 3days in 96-well plates.We treated A549 and H1299 cells with sulforaphane that the concentration of which was nontoxic.We observed the morphologic changes of A549 and H1299 cells.We used Wound-Healing and Transwell experiment to explore the invasion and metastasis abilities of A549 and H1299 cells which were treated with sulforaphane.And then we observed the changes of the expression of epithelial markers,E-cadherin and ZO-l;the mesenchymal markers,N-cadherin and Vimentin;TAp63a,ANp63a in A549 and H1299 cells by Western blotting experiment.2.The TGF-β-induced A549 and H1299 cells were treated with△Np63a plasmid combined with sulforaphane for 3days,we observed the morphologic changes of A549 and H1299 cells.We used Wound-Healing and Transwell experiment to explore the invasion and metastasis abilities of TGF-β-induced A549 and H1299 cells which were treated with ANp63a plasmid and sulforaphane.And then we observed the changes of the expression of epithelial markers,E-cadherin and ZO-l;the mesenchymal markers,N-cadherin and Vimentin inTGF-β-induced A549 and H1299 cells by Western blotting experiment.3.The TGF-β-induced A549 and H1299 cells were treated with TAp63a-siRNA combined with sulforaphane for 3days,we observed the morphologic changes of A549 and H1299 cells.We used Wound-Healing and Transwell experiment to explore the invasion and metastasis abilities of TGF-β-induced A549 and H1299 cells which were treated with TAp63a-siRNA and sulforaphane.And then we observed the changes of the expression of epithelial markers,E-cadherin and ZO-1;the mesenchymal markers,N-cadherin and Vimentin inTGF-β-induced A549 and H1299 cells by Western blotting experiment.Result:1.Impact of TGF-β on EMT in A549 and H1299 cellsA549 and H1299 cells were treated with lOng/ml TGF-p.A549 and H1299 cells which treated with TGF-p go through the change from elliptical-like epithelial morphology to long spindle-like mesenchymal morphology.The ability of invasion increased after treated with TGF-β.2.Impact of TGF-β on the expression of TP63a in A549 and H1299 cellsA549 and H1299 cells were treated with 10ng/ml TGF-β.The protein level of△Np63a wereincreased and TAp63a were decreased after the treatment of TGF-β.3.Cell viability of sulforaphane treatment in A549 and H1299 cellsMTT assay showed 3.5μmol/Lsulforaphane did not show cell toxicities in A549 cells;4μmol/Lsulforaphane did not show cell toxicities in H1299 cells.According to this,we chose 1μmol/L,2μmol/L sulforaphane for the following experiments.4.Sulforaphane prevented the TGF-β-induced EMT in A549 and H1299 cellsA549 and H1299 cells were treated with sulforaphanefor 3 days.The cells went through the change from long spindle-like mesenchymal morphology to elliptical-like epithelial morphology.The ability of invasion decreased after treated with sulforaphane.Western blotting experiment showed that the epithelial markers,E-cadherin and ZO-1 were increased while the mesenchymal markers,N-cadherin and Vimentin were decreased after the treatment of sulforaphane.5.Sulforaphane changed the expression level of TAp63α and △Np63a in TGF-β-induced A549 and H1299 cellsA549 and H1299 cells were treated with sulforaphanefor 3 days.The protein level of△Np63a were decreased and TAp63a were increased after the treatment of sulforaphane.6.Impact of transfection of TP63α on EMT in A549 and H1299 cellsA549 and H1299 cells were treated with △Np63a plasmid or TAp63a-siRNA Transfection of ANp63a plasmid or TAp63a-siRNA made A549 and H1299 cells go through the change from elliptical-like epithelial morphology to long spindle-like mesenchymal morphology.Wound-Healing and Transwell experiment presented that the ability of invasion increased after treated with △Np63a plasmid or TAp63a-siRNA.Western blotting experiment showed that the epithelial markers,E-cadherin and ZO-1 were decreased while the mesenchymal markers,N-cadherin and Vimentin were increased after the treatment of △Np63a plasmid or TAp63a-siRNA.7.Role of △Np63a in sulforaphane prevention of TGF-β-induced EMT in A549 and H1299 cellsThe TGF-β-induced A549 and H1299 cells were treated with △Np63a plasmid combined with sulforaphane for 3days.Transfection of △Np63a plasmid reversed the effection of sulforaphane in TGF-β-induced A549 and H1299 cells.The cells went through the change from elliptical-like epithelial morphology to long spindle-like mesenchymal morphology.Wound-Healing and Transwell experiment presented that the ability of invasion increased after treated with △Np63a plasmid.And sulforaphane did not show preventive effect on them.Western blotting experiment showed that the epithelial markers,E-cadherin and ZO-1 were decreased while the mesenchymal markers,N-cadherin and Vimentin were increased after the treatment of △Np63 a plasmid.8.Role of TAp63a in sulforaphane prevention of TGF-β-induced EMT in A549 and H1299 cellsThe TGF-β-induced A549 and H1299 cells were treated with TAp63a-siRNA combined with sulforaphane for 3days.Transfection of TAp63a-siRNA reversed the effection of sulforaphane in TGF-β-induced A549 and H1299 cells.The cells went through the change from elliptical-like epithelial morphology to long spindle-like mesenchymal morphology.Wound-Healing and Transwell experiment presented that the ability of invasion increased after treated with TAp63a-siRNA.And sulforaphane did not show preventive effect on them.Western blotting experiment showed that the epithelial markers,E-cadherin and ZO-1 were decreased while the mesenchymal markers,N-cadherin and Vimentin were increased after the treatment of TAp63a-siRNA.Conclusion:Sulforaphane prevented TGF-β-induced EMT in lung cancer through regulating the expression of TP63a.