Enhanced Anti-tumor Activity by Adenovirus-mediated ING4 and IL-24 Co-expression Combined with Anticancer Drug for Bladder Cancer Cells and Its Mechanism in Vitro
|Department of Urology
|inhibitor of growth 4 interleukin-24 adenovirus bladder cancer gene therapy
Objective:To construct a recombinant adenoviral vector co-expressing inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) and study its enhanced anti-tumor and chemosensitivity to anticancer drug mitomycin C (MMC) effects on bladder cancer cells and its mechanism in vitro.Methods:Recombinant replication-incompetent adenovirus expressing ING4 and IL-24 (pAd-ING4-polyA+promoter△-IL-24) was constructed and transfected into QBI-293A cells to abundantly amplify pAd-ING4-polyA+promoter△-IL-24, leading to formation of Ad-ING4-IL-24. The best MOI of Ad-ING4-IL-24 was chosen by Ad-ING4-IL-24 infecting the human bladder cancer cell line T24 and human embryonic lung fibroblast cell line WI-38 in vitro. Semi-quantitative RT-PCR and Western blot were used to detected the expression of ING4 and IL-24 in T24 cells. The effect of enhanced growth-suppressing, apoptosis-inducing and chemosensitivity to anticancer drug mitomycin C (MMC) effects of Ad-ING4-IL-24 co-expressing ING4 and IL-24 on T24 bladder cancer cells were assessed by MTT assay and FCM, and the apoptosis of the cell nucleus were detected by Hochest33258 staining. Semi-quantitative RT-PCR was used to detected the transcription of cell apoptosis- related genes (Bcl-2、Bax and Caspase-3) in T24 bladder cancer cell line. The expression of Caspase-3 was detected by Western blot.Results:T24 bladder cancer cells and WI-38 cells were efficiently infected by Ad-ING4-IL-24 amplified at MOI of 100 and 50. The growth of T24 bladder cancer cells was significantly inhibited by Ad-ING4-IL-24 at MOI of 100. Adenovirus-mediated ING4 and IL-24 co-expression significantly inhibited T24 bladder cancer cells growth, induced cells apoptosis compared with Ad-ING4 and Ad-IL-24 ( P<0.05 ) , exhibiting additive effect(Q=0.90). Furthermor, Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug MMC in T24 cells in vitro. 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 compared with Ad-ING4 and Ad-IL-24.Conclusions:Ad-ING4-IL-24 co-expressing ING4 and IL-24 had significant additive effect in suppressing T24 bladder cancer cells growth and inducing cells apoptosis in vitro. Ad-ING4-IL-24 had synergetic and enhanced chemosensitivity to MMC effects in suppressing T24 bladder cancer cells. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in up-regulating the expression of Bax and down-regulating the expression of Bcl-2 resulting activation of Caspase-3, which may be responsible for its synergetic anti-tumor effect on T24 bladder cancer cells in vitro.