The Inhibitory Synergy And Chemosensitivity Of Adenovirus-mediated ING4 And IL-24 Co-expression For Lung Cancer And Its Mechanism

Objective:To explore the inhibitory synergy and chemosensitivity of recombinant adenovirus expressing inhibitor of growth 4(ING4) and interleukin-24 (IL-24) on lung cancer and its possible mechanisms.Methods:Recombinant replication-incompetent adenovirus Ad-ING4-IRES-IL-24 expressing ING4 and IL-24 was constructed and transfected into the human embryonic kidney 293 (QBI-293A) cells. The adenovirus completed the amplification in QBI-293A cells and the titration was also detected. The human pulmonary carcinoma cell line A549 was infected by Ad-ING4-IL-24 and combined with cisplatin (DDP) in vitro, and were randomly divided into six groups: PBS negative control group(PBS group), idling adenovirus group(Ad group), adenovirus-mediated IL-24 group (Ad-IL-24 group), adenovirus-mediated ING4 group (Ad-ING4 group), Ad-ING4-IL-24 group, DDP positive control group(DDP group) and Ad-ING4- IL-24 combined with DDP (referred to as joint group). The expression of ING4 and IL-24 in A549 cells was detected by western blotting. The effect of enhanced growth-suppressing and apoptosis-inducing by Ad-ING4-IL-24 on A549 lung cancer cells were analyzed by MTT assay and Flow cytometry. The expression of apoptosis-related genes (bcl, bax) and the Cleaved Caspase3 was detected by reverse transcription-PCR (RT-PCR) and western blotting. The human lung cancer mode was established with A549 cells in athymic nude mouse. Mice of the groups were progressed multi-points injection in tumor, the gross tumor volumes were measured dynamically. The ratios of tumor-suppression were calculated. The tumors were observed by HE staining method. The expression of apoptosis- and angiogenesis-related proteins (bcl-2, bax, CD34, HIF-1α, surviving and VEGF) in A549 transplanted tumor was determined by immunohisto- chemistry analysis.Results:After the amplification of Ad-ING4-IL-24 in QBI-293A cells, the titration was 109 pfu/mL.The expressions of ING4 and IL-24 in A549 cells were detected by western blotting. Adenovirus-mediated ING4 and IL-24 co-expression significantly inhibited A549 cell growth, induced cell apoptosis, and retarded A549 human lung cell transplanted tumor growth in athymic nude mouse, exhibiting synergetic anti-tumor effect. Furthermore, Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung cancer in vitro and vivo. Ad-ING4-IL-24 had more marked effect in up-regulating the expression of bax and Cleaved Caspase3 and down-regulating the expression of bcl-2, CD34, HIF-1α, surviving and VEGF compared with Ad-ING4 or Ad-IL-24.Conclusion:1. Ad-ING4-IL-24 had significant synergetic effect in suppressing cell growth and inducing cell apoptosis in A549 lung cell and lung cancer transplanted tumor growth in athymic nude mouse.2. Ad-ING4-IL-24 enhanced chemosensitivity to DDP effect in suppressing cell growth and inducing cell apoptosis in A549 lung cell and lung cancer transplanted tumor growth in athymic nude mouse.3. Ad-ING4-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 A549 human lung cell in vitro and in vivo in athymic nude mouse model.4. Ad-ING4-IL-24 had more potent effect in down-regulating the expression of angiogenesis-related factors CD34, HIF-1αand VEGF, which may be another important mechanism involved in its synergetic effect in suppressing lung cancer transplanted tumor growth in athymic nude mouse model.