Therapeutic Efficacy Of An HTERT-promoter-dependent Recombinant Adenovirus That Expresses Apoptin On Prostate Cancer Cells In Vivo And In Vitro

Like many cancers, prostate cancer is a complex disease, and different types oftherapeutic strategies are required to demonstrate a benefit in a particular patient cohort. Mostof the~29,000men who succumb to prostate cancer each year in the United States die ofmetastatic disease, and this highlights the need for better systemic therapies. In recent years,with the development of molecular biology, immunology, and other concerned subjects, genetherapy has gradually emerged as a novel antitumor treatment that has a huge advantage.Adenovirus-based vectors are the most widely used cancer gene delivery platforms;however, specificity and efficacy are major challenges for this therapeutic strategy. Of theexisting adenovirus technologies, the utility of conditional replication-competentadenoviruses (CRCAs) provides an optimum approach. In our previous studies, using theRAPAd.I system, we constructed a dual-specific antitumor CRCA, designatedAd-hTERTp-E1a-Apoptin, incorporating the tumor-specific promoter hTERTp and thespecific antitumor gene Apoptin. This CRCA has the ability of both tumor-specific growthinhibition and tumor-specific replication.Apoptosis is frequently impaired in many human tumors, and is also an importantmechanism in chemotherapy-induced tumor cell death. Therefore, the modulation ofapoptosis by targeting pro-apoptotic and anti-apoptotic proteins may be a powerful andeffective method for treating cancer. Apoptin, a protein derived from chicken anemia virus(CAV), selectively induces apoptosis in a wide variety of transformed cells, but not inprimary cells.The hTERT promoter displays high activity in a majority of human cancers but not inmost host tissues and is considered an ideal tumor-specific regulator for oncolyticadenoviruses. The hTERT promoter can be used to control viral regulatory genes, such asadenoviral E1A, to restrict the replication of oncolytic adenoviruses to malignant cells andtissues. Dual-specificity adenoviral promoters that regulate E1A expression in response tomultiple stimuli. The cancer-specific promoter hTERT can both confer tumor-specific replication and regulate E1A expression, and several tumor cell-replicating. However, none ofthese viruses combines both promoter elements into a single virus to regulate E1A expressionand viral replication. As hTERT is expressed in>90%of cancers, an oncolytic virus thatcombines both of these features has the potential to induce oncolytic activity across a broadrange of human tumors and tumor cell populations. Cancer gene therapy based on oncolyticadenoviruses has been widely studied in pre-clinical and clinical trials in recent years.To investigate the antitumor effects of the recombinant adenovirusAd-hTERTp-E1a-Apoptin on human prostate cell line, PC-3, and murine cell line, RM-1.Using MTT assay to measure viability of PC-3and RM-1cells which are infected byrecombinant adenovirus. To evaluate the antitumor effects of Ad-hTERTp-E1a-Apoptin onPC-3and RM-1cells. Using AO/EB staining, DAPI staining, Annexin V assay to investigatethe lethal effect and style of Ad-hTERTp-E1a-Apoptin on PC-3and RM-1cells in vitro;Caspase detective method is that, we can extract total protein from PC-3and RM-1cellswhich are infected by recombinant adenovirus for48h, to examine the activity of Caspase. Inthis way, we can investigate the lethal effect of Ad-hTERTp-E1a-Apoptin on PC-3and RM-1cells. In addition to Ad-CMV-EGFP, Ad-hTERTp-E1a-Apoptin, Ad-CMV-Apoptin andAd-hTERTp-E1a-EGFP which is able to inhibit the proliferation of PC-3and RM-1cells invitro. In contrast, Ad-hTERTp-E1a-Apoptin and Ad-hTERTp-E1a-EGFP were more effectivethan Ad-CMV-Apoptin in inhibiting cell growth, about3times. P＜0.05. The inhibition effectof Ad-hTERTp-E1a-Apoptin on PC-3and RM-1cells exhibited dose and time relationship.The infection of Ad-hTERTp-E1a-Apoptin resulted in the exposure of phosphatidylserine, theshrink of nucleus, the permeation of membrane and the activation of caspase. ThehTERT-promoter-dependent oncolytic adenovirus Ad-hTERTp-E1a-Apoptin could effectivelysuppress prostate cancer cells growth by inducing apoptosis. In the animal models,Ad-hTERTp-E1a-Apoptin significantly inhibited tumor growth and extended the lifespan ofanimals. Experimental results indicate that Ad-hTERTp-E1a-Apoptin has a potentialapplication in tumor gene therapy.