Antitumor Effect Of RPB-DR(6) Promoter-regulated Oncolytic Adenovirus On Prostate Cancer Cells

Background and objectiveProstate cancer is one of the most common male malignant tumors. Eighty percent ofthe patients with advanced prostate cancer will develop into androgen independent prostatecancer (AIPC) after androgen-deprivation therapy6-18months. It is now still lack ofeffective methods for the treatment of AIPC. Progress has been slow for Experimentalresearch on the treatment of AIPC. So it is very important to explore new methods for AIPCtreatment.Oncolytic adenovirus-based gene-virotherapy has become a research hotspot forcancer treatment and provides a potential method for AIPC treatment. We reconstructed apromoter named rPB-DR (6) based on rPB. It kept the property of prostate specificity andcould respond to retinoic acid and retinoic acid receptor complexes. As retinoic acidreceptors are expressed in all types of prostate cancer, rPB-DR (6) promoter can be used toconstruct oncolytic adenovirus for AIPC treatment. Base on the above, we intend toconstruct conditionally replicating oncolytic adenovirus regulated by prostate specificpromotor rPB-DR(6) with an insertion of cytosine deaminase (CD) gene(a suicide gene)driven by mCMV promoter and observe its antitumor effect.MethodsrPB-DR (6) promoter and CD gene was amplified by PCR using pUC119-rPB-DR (6)and pCD2as template. With insertion of rPB-DR (6) promoter prior to early gene E1A andmCMV-driven CD gene into the adenovirus DNA sequence, recombinant plasmidp55-rPB-DR (6)-mCMV-CD (abbreviated as p55-rPC) was prepared. After identification ofthe plasmid p55-rPC, the plasmid and adenovirus backbone plasmid pPE3wereco-transfected into293cells and the recombinant oncolytic adenoviruses Ad5-rPB-DR (6)-mCMV-CD(abbreviated as Ad5-rPC) were packaged. Then the adenoviruses wereamplified, purified by CSCI gradient centrifugation and tested for PFU titer. Prostate cancercells, including androgen dependent and independent, were infected by Ad5-rPC to observe the effection of the adenovirus. Expression of E1A and CD proteins in theadenovirus-infected prostate cancer cells was detected by Western Blot. Antitumor effect ofthe recombinant adenovirus on prostate cancer cells was tested by CCK-8assay.ResultsBy restriction enzyme analysis, PCR and sequencing, rPB-DR(6) promoter and CDgene driven by mCMV promoter were proved to be inserted into recombinant adenovirussuccessfully. The titer of the purified recombinant adenovirus Ad5-rPC was1.1×1011pfu/mL. Western Blot showed positive expression of E1A and CD proteins in infectedprostate cancer cells. The titer of the progeny viruses produced by prostate cancer cellswhich were infected by Ad5-rPC was greater than3.0×106pfu/mL. CCK-8assay confirmedthat Ad5-rPC had antitumor effects on both androgen-dependent and androgen-independentprostate cancer cells. Its efficiency was greater than65%.Conclusions(1) A recombinant oncolytic adenovirus which had the ability of targeted killing effecton prostate cancer was successfully prepared. As the main regulatory elements, rPB-DR (6)promoter made the recombinant oncolytic adenovirus specific to prostate cancer. Thesuicide gene CD further enhanced the anti-tumor effect of the recombinant oncolyticadenovirus.(2) The recombinant oncolytic adenovirus Ad5-rPC was confirm to have specifickilling effect on all types prostate cancer, including AIPC, in vitro, so this study povided apotential feasible methods for the treatment of AIPC.