Role Of TP63? In Lung Cancer EMT/invasion And Metastasis Induced By Cigarette Smoke Extract And The Intervention Of Curcumin

Objective:TP63 is a member of anti-oncogene p53 family.There are architectural and functional similarities betweenTP63 and p53.The study aimed to explore the role of TP63a,one of the subtypes of TP63,in lung cancer EMT/invasion and metastasis induced by cigarette smoke extract(CSE)and the intervention of curcumin.We found that exposure of lung cancer cells to CSE,a EMT phenotype would appeare and company with the decreased expression of TAp63a and increased expression of?Np63a.Exposure of lung cancer cells to TP63a and CSE simultaneously,we found that over-express of TAp63a or knockdown the ?Np63a could reverse CSE-induced EMT/invasion and metastasis.Exposure of lung cancer cells to Curcumin and CSE simultaneously,we found that Curcumin could also reverse CSE-induced EMT andthe decreased TAp63a and increased ?Np63a.We suggested that Curcumin may inhibit CSE-induced EMT/invasion and metastasis through mediating the expression of TP63a.Findings from this study could bring new insights into the regulatory mechanisms of CSE-associated lung cancer and its target prevention.Methods:1.The A549 and H1299 cells were treated with various concentrations of CSE for 4 days in 96-well plates.The effects of CSE on A549 and H1299 cells viability were examined by MTT assay.A549 and H1299 cells were treated with various concentrations of CSE for 4 days,the effects of CSE on morphology of A549 and H1299 cells were examined.The migration and invasion abilities were determined by Wound-Healing and Transwell experiment.The mRNA and protein expressions of the epithelial markers,E-cadherin and ZO-1;the mesenchymal markers,Vimentin and N-cadherin;MMP-2 and MMP-9;transcription factor,Snaill and c-myc;TAp63a and ANp63a were measured by agarose gel electrophoresis(AGE)and Western blotting respectively.2.A549 and H1299 cells were treated with TAp63a plasmid combine with CSE for 4 days,the effects of TAp63a plasoid and CSE on morphology of A549 and H1299 cells were examined.The migration and invasion abilities were determined by Wound-Healing and Trans well experiment after treated with TAp63a plasmid and CSE.The EMT related markers and TAp63a were measured by quantitative Real-time PCR and Western blotting respectively.3.A549 and H1299 cells were treated with ?Np63a-siRNA combine with CSE for 4 days,the effects of ?Np63?-siRNA and CSE on morphology of A549 and H1299 cells were examined.The migration and invasion abilities were deternined by Wound-Healing and Transwell experiment after treated with ?Np63a-siRNA and CSE.The EMT related markers and ?Np63a were measured by quantitative Real-time PCR and Western blotting respectively.4.The A549 and H1299 cells were treated with various concentrations of Curcunin for 4 days in 96-well plates.The effects of Curcumin on A549 and H1299 cells viability were examined by MTT assay.A549 and H1299 cells were treated with various concentrations of Curcumin for 4 days,the effects of CSE on morphology of A549 and H1299 cells were examined.The EMT related markers,TAp63a and ANp63a were measured by Western blotting.5.A549 and H1299 cells were treated with Curcumin combine with CSE for 4 days,the effects of Curcumin and CSE on morphology of A549 and H1299 cells were examined.The migration and invasion abilities were determined by Transwell experiment after treated with Curcumin and CSE.The EMT related markers,TAp63a and ?Np63a were measured by Western blotting·Result:1.Cell ability of cigarette smoke extract treatment in A549 and H1299 cellsA549 and H1299 cells were treated with various eoncentrations of CSE for 4 days.The effect of CSE on cell viability was examined by MTT assay.0%-l%CSE did not show cell toxicities;however,CSE at 2%led to a remarkable decrease in cell viability in A549 and H1299 cells after 4 days exposure.Since 2%CSE was toxic to A549 and H1299 cells,we chose 0%-1%CSE for the following experiments.2.Impact of cigarette smoke extract on morphology and invasion/metastasls ablility of A549 and H1299 cellsA549 and H1299 cells were exposed to various concentrations of CSE for 4 days.A549 cells went through the alteration from spindle-like epithelial morphology to more long spindle-shape;H1299 cells went through the alteration from round-shape to spindle-like mesenchymal morphology.The change presented a concentration dependent manner compared with control cells.Wound-Healing and Transwell experiment showed that the ability of migration and invasion enhanced after treated with CSE.The Wound-Healing showed that increased number of cells migrate scratches center correlate with the raised concentration of CSE.Transwell experiment showed increased number of cells pass through the membrane of transwell along with the raised concentration of CSE.3.Cigarette smoke extract activated the EMT pathway of A549 and H1299 cellsA549 and H1299 cells were exposed to different concentration of CSE for 4 days.Western blotting results showed that the epithelial markers,E-cadherin and ZO-1,were decreased,while the mesenchymal markers,Vimentin and N-cadherin;MMP-2 and MMP-9;transcription factor,Snail 1 and c-myc were increased.The AGE results showed that the epithelial markers,E-cadherin and ZO-1,were decreased,while the mesenchymal markers,Vimentin and N-cadherin were increased.4.Cigarette smoke extract changed the A549 and H1299 cells expression lerrel of TAp63a and ?Np63?A549 and H1299 cells were exposed to different concentration of CSE for 4 days.Western blotting and the AGE results showed that the protein and mRNA level of TAp63a were decreased and ?Np63? were increased.5.Transfection of TApd3? plasmid reversed cigarette smoke extract-induced A549 and H1299 cells EMT/migration and invasionA549 and H1299 cells were treated with TAp63? plasmid combine with CSE for 4 days.Transfection of TAp63? plasmid reversed CSE-induced A549 and Hl299 cells morphology EMT like changes.The Wound-Healing and Transwell experiment showed that transfection of TAp63? plasmid reversed CSE-induced increased migration and invasion ability.6.Transfection of TAp63? plasmid reversed cigarette smoke extract-induced A549 and H1299 cells changed protein and mRNA expression level of EMT related markers and TAP63?A549 and H1299 cells were treated with TAp63? plasmid combine with CSE for 4 days.The Western blotting and quantitative RT-PCR showed that transfection of TAp63? plasmid reversed CSE-induced decreased expression of epithelial markers and increased expression of mesenchymal markers as well as MMP-2 and MMP-9.7.Transfection of ?Np63?-siRNA reversed cigarette smoke extract-induced A549 and H1299 cells EMT/migration and invasionA549 and H1299 cells were treated with ?Np63?-siRNA combine with CSE for 4 days.Transfection of ?Np63?-siRNA reversed CSE-induced A549 and H1299 cells morphology EMT like changes.The Wound-Healing and Transwell experiment showed that transfection of ?Np63?-siRNA reversed CSE-induced increased invasion and metastasis ability.8.Transfection of ?Np63?-siRNA reversed cigarette smoke extract-induced A549 and H1299 cells changed protein and mRNA expression level of EMT relatedmarkers and ?NpP3?A549 and H1299 cells were treated with ?Np63?-siRNA combine with CSE for 4 days.The Western blotting and quantitative RT-PCR showed that transfection of?Np63?-siRNA reversed CSE-induced decreased expression of epithelial markers and increased expression of mesenchymal markers as well as MMP-2 and MMP-9.9.Curcumin induced morphology changes and inhibited EMT pathwayA549 and H1299 cells were treated with various concentrations of Curcunin for 4 days.The effect of Curcumin on cell viability was examined by MTT assay.A549 and H1299 cells treated with different concentration of Curcumin for 4 days.A549 cells went through the alteration from spindle-like epithelial morphology to oval-shape;H1299 cells went through the alteration from oval-shape to round-shape.The changes presented a concentration dependent manner compared with control cells.Western blotting results showed that the epithelial markers,E-cadherin and ZO-1,were increased,while the mesenchymal markers,Vimentin and N-cadherin,were decreased.Western blotting also showed that the expression level of MMP-2 and MMP-9 decreased correlate with the raised concentration of Curcumin.10.Curcumin induced changed protein expression of TAp63? and ?Np63?A549 and H1299 cells treated with different concentration of Curcumin for 4 days.Western blotting showed that TAp63a were increased and ANp63a were decreased;the changes presented a concentration dependent manner compared with control cells.11.Curcumin re,versed cigarette smoke extract-induced A549 and H1299 cells EMT/migration and invasionA549 and H1299 cells were treated with Curcumin combine with CSE for 4 days.Curcumin reversed CSE-induced A549 and H1299 cells morphology EMT like changes.Transwell experiment showed that Curcumin reversed CSE-induced increased migration and invasion ability.12.Curcumin reversed cigarette smoke extract-induced A549 and H1299 cells changed protein expression level of EMT related markers,TAp63? and ?Np63?A549 and H1299 cells were treated with Curcumin combine with CSE for 4 days.Western blotting showed that Curcumin reversed CSE-induced decreased expression of epithelial markers and increased expression of mesenchymal markers as well as increased expression of MMP-2 and MMP-9.Meanwhile Western blotting showed that Curcumin reversed CSE-induced decreased expression of TAp63? and increased expression of ?Np63?.Conclusion:TP63a played an important role in cigarette smoke extract-induced EMT/invasion and metastasis in lung cancer.As a member of anti-oncogene p53 family,TP63a played a vital role in the incidence and development of lung cancer.Transfection of TAp63? plasmid or ?Np63?-siRNA in the lung cancer,we found that TP63a could reverse the CSE-induced EMT/invasion and metastasis.Curcumin is a phytochemicals which could suppress tumor development.Lung cancer cells were treated with Curcumin,the level of TP63a were changed.We suspected that Curcumin could regulate the invasion and metastasis of tumor through mediating the activity of TP63a gene.