Induction Of Apoptosis And Effects On Drug-resistance In Ovarian Drug-resistant Cancer Cell Lines By Mda-7/IL-24 Gene

Background & Objective: Ovarain cancer has the highest mortality in gynecological neoplasms. Drug-resistant to chemotherapeutics in ovarian cancer patients limits the effect of treatment in clinical. How to reverse the drug-resistant has important role for these patients. Expression of melanoma differentiation associated gene-7 (mda-7/IL-24) has a double function: highly specifically induces tumor cell apoptosis and modulates immune responses. The apparent role of anti-tumor has been found in many variety tumors. This dual functionality makes it a strong candidate for human cancer gene therapy. Here we designed to evaluate the induction of apoptosis and efficacy on drug-resistance in ovarian drug-resistant cancer cell lines by mda-7/IL-24, research the anti-tumor effect in these cells, and explore the feasibility of reversal of drug-resistance and gene therapy in clinical of mda-7/IL-24 gene..Methods: In this study we construct Ad.mda-7/IL-24 using AdEasy 1 system. Drug-resistant ovarian cancer cell lines OVCAR-3, OVCAR-8 and A2780,A2780/DDP were infected by Ad.mda-7. To evaluate its anti-tumor efficacy in ovarian drug-resistant tumor cells and the effects on the growth and drug-resistance of four cell lines. There were three parts in whole study:1. Construction of Ad.mda-7/IL-24: Amplified mda-7/IL-24 sequence from the plasmid pREP4-mda-7 with PCR and sub cloned into the pAdTrack CMV shuttle vector. The resultant plasmid was cotransfected into E.coli BJ5183 cells with pAdEasy 1 plasmid to undergo homologous recombination. The linearized recombinant plasmid was transfected into 293 cells.2. Infection of Ad.mda-7/IL-24 and effect on apoptosis inducing: The recombinant adenovirus was infected into ovarian cancer drug resistant cell line OVCAR-8. The expression of mda-7/IL-24mRNA was measured by Real time RT-PCR. Using Western-bloting analysis to detect expression of MDA-7/IL-24 protein. Then the infected cells OVCAR-3,OVCAR-8 were assayed for apoptosis using Hoechst33258 staining and flow cytometry.3. Effects on growth and drug-resistance by Ad.mda-7/IL-24: After infected by Ad.mda-7/IL-24 cells viability and resistance of four chemotherapeutic drugs, including DDP,ADM,5-FU and Taxol, were examined with metyl thiazolyl tetrazoIium(MTT) rapid photocolorimetric assay.Results : 1. The recombinant Ad.mda-7/IL-24 was constructed successfully approved by sequence analysis and electrophoresis. All OVCAR-8 and OVCAR-3 cellS had been infected under concentration of Ad.mda-7/IL-24 was 10μL.2. The expression of mda-7/IL-24mRNA in OVCAR-8 cell after infected Ad.mda-7/IL-24 was increased. There were no difference of mda-7/IL-24mRNA expression in the control groups: Ad.GFP infected and non-infected. The expression of MDA-7/IL-24 protein was obviously .After 48 and 72 hours infected, the protein expression increased. There were on expression in the control groups. The number of apoptosis cells in OVCAR-3,OVCAR-8 cell after infected were increased along with the lasting of time.Within 72 hours after Ad.mda-7 infection, the maximal apoptosis rates of OVCAR-3 and OVCAR-8 cells were 14.1% and 32.4% respectively, significantly higher than control groups.3. Cell growth was significantly inhibited after infected by Ad.mda-7/IL-24. Sensitivity to Taxol of OVCAR-3 and OVCAR-8 was increased by 2.76 and 1.52 times after infection, compared with the control. Infected OVCAR-3 cells also had higher sensitivity to ADM and 5-FU, increased 1.6 and 3.01 times respectively. Drug-resistance to DDP,5-FU and Taxol of A2780 was decreased. Sensitivity to DDP of A2780 was increased by 15.45 times, and to 5-FU and Taxol was increased by 2.15 and 3.63 times respectively. Infected A2780/DDP cells had higher sensitivity to 5-FU, increased 2.62 times.Conclusion: The recombinant adenovirus Ad.mda-7/IL-24 was successfully constructed and could infected ovarian drag-resistance cancer cell lines effectively.mda-7/IL-24 gene had apoptosis inducing capacity in drag-resistant ovarian cancer cell lines and showed cancer growth inhibition effect. Ad.mda-7/IL-24 infection is capable of reversing the resistance of OVCAR-3,OVCAR-8 cells to Taxol and increasing the sensitivity of A2780 cells to DDP,5-FU and Taxol.