Experimental Study On Enhanced Anti-tumor Activity And Chemosensitivity Of Co-expression By Ad-ING4-IL-24 Double-gene For Lung Cancer In Vitro

Objective:To study the growth suppression and chemosensitivity of lung cancer cells (NCI-H460) induced by recombinant adenovirus expressing growth 4 (ING4) and interleukin-24(IL-24) (Ad-ING4-polyA+promoter-IL-24) in vitro and its mechanism.Methods:Recombinant replication-incompetent adenovirus expressing ING4 and IL-24 (Ad-ING4-polyA+promoter-IL-24) was constructed and transfected into QBI-293A cells. The adenovirus completed the amplification in QBI-293A cells. The expression of ING4/IL-24 gene in NCI-H460 lung cancer cells infected with Ad-ING4,Ad-IL-24 and Ad-ING4-IL-24 were detected by RT-PCR and Western blot; The in vitro enhanced growth-suppressing, apoptosis-inducing alteration, enhanced apoptosis effect and chemosensitivity of PBS group, Ad-GFP group, Ad-ING4 group, Ad-IL-24 group, Ad-ING4-IL-24 group, DDP group and Ad-ING4-IL-24 co-expressing DDP group in NCI-H460 lung cancer cells were assessed by MTT assay, FCM staining respectively; The Cell cycle were evaluated by flow cytometry (PI simple staining). Semi-quantitative RT-PCR was used to detected the transcription of cell apoptosis- related genes (Bcl-2,Bax,Caspase-3 and Survivin) in NCI-H460 lung cancer line. The expression of Caspase-3 was detected by Western blot.Results:After 48h of the amplification of Ad-ING4-IL-24 in QBI-293A cells, we could see the cells becoming round. The cells gathered like grapes under the light microscope. We could observe the green light under fluorescence microscope which means high efficiency of infection. ING4/IL-24 mediated by adenovirus were proved successful transcribed and translated in NCI-H460 cells by RT-PCR and Western blot; Adenovirus-mediated ING4 and IL-24 co-expression not only significantly inhibited NCI-H460 lung cancer cell growth, induced G2/M phase arrest and cell apoptosis in vitro,but also excelled obviously Ad-ING4, Ad-IL-24 single work(P<0.05); Furthermore, Ad-ING4-IL-24 had synergetic and enhanced chemosensitivity to DDP effects in suppressing NCI-H460 lung cancer cells. Ad-ING4-IL-24 had more marked effect in up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 and Survivin compared with Ad-IL-24 or Ad-ING4.Conclusion:1. Exogenous ING4 and IL-24 double gene can be effectively mediated by Adenovirus Ad-ING4-polyA+promoter-IL-24 in NCI-H460 lung cancer cells.2. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had not only suppressed statistically NCI-H460 cells induced apoptosis which excelled obviously Ad-ING4, Ad-IL-24 single work(P<0.05),but also expressed synergetic effects of anti-tumor in vitro.3. Ad-ING4-IL-24 co-expressing ING4 and IL-24 combined with DDP had significant synergetic effect in vitro. Ad-ING4-IL-24 maybe an effective chemosensitivity in lung cancer .4. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 and Survivin which may be responsible for its synergetic anti-tumor effect on NCI-H460 human lung cancer cells in vitro.