A novel triple-regulated oncolytic adenovirus carrying human tumor necrosis factor-related apoptusis-inducing Ugand gene exerts potent antitumor efficacy on lung cancer in vitro

Abstract

Objective To develop a triple-regnlated replicative adenovirus carrying the human TRAIL gene, a novel gene-viral therapeutic system CNHK500-hTRAIL.Methods The human TRAIL gene was cloned into the upstream.of E3 of plasmid pBHGE3 to produce pPE3-hTRAIL that was regulated by major later promoter (MLP).The plasmid pPE3-hTRAIL was eo-transfeeted with pSGS00 in 293 cells by site-specific recombination to generate recombinant conditionally replicating-competent adenovirus CNHK500-hTRAIL,in which EIA gene and E1B gene were driven by human telomerase reverse tran-seriptase promoter and hypoxia response promoter, respectively.Virus titer was measured by TCID50 meth-od.Virus replication assay was performed to evaluate the selective replication ability of CNHK500-hTRAIL.ELISA assay was used to detect the transgene expression of TRAIL.The eytotoxicity in cultured cancer and normal cells was assayed by MTT.Results A novel gene-viral therapoutie system CNHK500-hTRAIL was constructed and its titer was 2.39 x 1010 pfu/ml.Proliferative test revealed that CNHK500-hTRAIL eould be selectively proliferated in the telomerase-positive NSCLC cells A549.Furthermore, in comparison with non-replieative adenovirus Ad-hTRAIL, the transgene expression of TRAIL in the suporna-tant of cultured cells A549 mediated with CNHK500-hTRAIL was significantly higher (183.12 μg/L vs 24.53 μg/L,P <0.O1).The MTT assay showed that CNHK5OO-hTRAIL killed the A549 cells more effec-tively than Ad-hTRAIL,and the inhibitory concentration 50% (IC50) MOI was 0.197 and 17.825, re-speetively.Conclusion The novel gene-viral therapeutic system CNHK500-hTRAIL holds potential for the treatment of NSCLC. Key words: Lung carcinoma; Adenovirus; Gene therapy; TRAIL