Enhancing TRAIL-induced Apoptosis In Colon Cancers By Adenovirus-mediated Small Hairpin RNA Targeting Bcl-XL

Background and objective:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)is one of several members of the TNF gene superfamily that induces apoptosis through engagement of death receptors(DR).It has been shown that TRAIL can induce apoptosis selectively in a variety of cancer cell lines but not in normal cells,indicating its potential as a candidate therapeutic in cancer.Currently,both recombinant TRAIL proteins and TRAIL receptor agonistic antibodies are being tested in the clinic,showing encouraging antitumor activities and mild side effects.Unfortunately,resistance to TRAIL therapy is frequently encountered requiring combined treatments with sensitizing agents.We previously reported that BcI-XL small interfering RNA(siRNA)could increase the apoptosis-induction by TRAIL.However,delivery of chemically synthesized siRNA in vivo remains to be an obstacle for development of RNA interference therapy.In this study,our major aim was to test whether TRAIL-induced apoptosis could also be enhanced by combined use of Ad/gTRAIL and Ad/Bcl-XL shRNA,a recombinant adenovirus expressing a small hairpin RNA targeting to Bcl-XL(Bcl-XL shRNA)driven by U6 promoter.Materials and methods:Recombinant adenoviruses(Ad/CMV-GFP,Ad/gTRAIL, Ad/Bcl-XL shRNA)were amplified in HEK 293 cells and purified by centrifugation banding on cesium chloride by a standard procedure.Titers of the adenoviruses were determined by optical absorbance at A₂₆₀nm(1 A₂₆₀=10¹²particles/ml)and by plaque assay.DLD1-R,which is resistant to TRAIL,was selected by repeated treatment with Ad/gTRAIL.Cell relative viabilities were assessed by MTT assay.The expressions of apoptosis-related proteins were detected by western blot.PBS and Ad/CMV-GFP were used as blank control and vector control respectively.Differences among the treatment groups were assessed by ANOVA.Results:Treatment with Ad/gTRAIL plus Ad/Bcl-XL shRNA could dramatically suppress the growth of both TRAIL-sensitive colon cancer cell lines DLD1 and Lovo, which had a synergistic effect on apotptosis-induction.DLD1-R cells became resistant to TRAIL after repeated treatment with Ad/gTRAIL and had an increased expression of Bcl-XL.Also,combination of Ad/gTRAIL and Ad/Bcl-XL shRNA could overcome the acquired resistance toward TRAIL in the DLD1-R cells,and enhance cleavage of caspase-8,caspase-9,caspase -3,PARP,and BID.Furthermore,release of cytochrome c from mitochondria was dramatically amplified by combined treatment with Ad/gTRAIL and Ad/Bcl-XL shRNA.Conclusions:Our data demonstrated that combination treatment of Ad/gTRAIL and Ad/Bcl-XL shRNA could enhance TRAIL-induced apoptosis and should be further tested in animal models.