Anti-tumor Effects Of A Dual Specific Oncolytic Adenovirus On Human Esophageal Cancer EC-109in Vivo And In Vitro

Esophageal cancer (EC), the sixth leading cause of cancer deaths worldwide, ranksamong the most common malignancies, and is mostly incurable by conventional treatmentmodalities. Indeed, overall survival rates for esophageal cancer have not changed substantiallyduring the past several decades, despite the introduction of aggressive multimodalityinterventions. Incidence and mortality rates of EC are equivalent, suggesting a failure ofcurrent therapies, despite the variety of combined modality approaches which have beenintroduced to complement surgical treatment. There is an urgent need for novel cancertherapies and clinical trials to improve the treatment of this condition.Adenovirus-based vectors are the most widely used cancer gene delivery platforms;however, specificity and efficacy are major challenges for this therapeutic strategy. Ofexisting adenovirus technologies, the utility of conditional replication-competentadenoviruses (CRCA) provides an optimum approach. In our previous studies, we constructeda dual specific anti-tumor CRCA, designated Ad-VP,(Ad-PEG-3p-E1a-Apoptin) using theRAPAd.I system, incorporating the tumor specific promoter PEG-3p and specific anti-tumorgene Apoptin. This CRCA has the ability of both tumor specific growth inhibition and tumorspecific replication. Further investigation showed that Ad-VP anti-tumor activity had moresignificant than replication defective adenoviruses (Ad-Apoptin and Ad-EGFP).Apoptosis is the proactive phenotype of programmed or physiologic cell death, theprocess used to remove excess or defunct cells during normal tissue maintenance. Apoptosisoccurs frequently in many human tumors; therefore, induction of apoptosis may be a powerfuland effective method of cancer treatment. Apoptin is a chicken anemia virus-derived,p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induceapoptosis in human cells derived from a variety of tumors including hepatomas, lymphomas,cholangiocarcinomas, melanomas, breast, lung and colon carcinomas. In contrast, apoptindoes not induce apoptosis in normal, non-transformed cells such as fibroblasts, keratinocytes,or smooth muscle cells. The mechanism by which apoptin selectivity targets malignant cells is not yet fully elucidated, but is known to involve the caspase-3pathway. Apoptosis-inducingactivity has correlated to the subcellular localization of apoptin protein expression. In normalcells apoptin is predominantly cytoplasmic, but is detected in the nucleus of transformed cells.Interestingly, several inhibitors of upstream caspases or the p53pathway failed to inhibitapoptin-induced cell death. Moreover, overexpression of anti-apoptotic genes such as Bcl-2,BAG-1, or Bcr-Abl does not protect cells from apoptin-induced apoptosis. The apoptin genecan be inserted into various vectors such as parvoviruses, papovaviruses, and adenovirusesdue to its small size, indicating apoptin presents a promising candidate for anti-tumor therapy.PEG-3p is a minimally active promoter region of the progression elevated gene-3(PEG-3). PEG3p acts as a tumor specific promoter and can drive gene expression in humanhepatoma carcinoma (HepG-2) cells, human lung carcinoma (A549) cells, human cervicalcarcinoma (HeLa) cells, but not in normal cell types including human embryonic lung (WI-38)cells or African green monkey kidney (Vero) cells. Cancer gene therapy based on oncolyticadenoviruses has been widely studied in pre-clinical and clinical trials in recent years. In ourprevious studies, we constructed a CRCA Ad-VP using the RAPAd.I system incorporatingPEG-3p and the specific anti-tumor gene apoptin, which demonstrated tumor-specific growthinhibition ability.In this work, we investigated the anti-tumor effects of Ad-VP on EC-109cells in vitro.MTT assays indicated infection with Ad-VP at100MOI significantly inhibited the growth ofEC-109cells after72h infection, whereas infections of1or10MOI had less effective growthinhibition effects. This data indicated that growth inhibition of EC-109cells is related to theMOI of Ad-VP and time period post-transfection. In contrast, Ad-Apoptin and Ad-EGFPinfected tumor cells resumed proliferation after72h treatment at all MOI doses tested.AO/EB, DAPI and Annexin V assays indicated the Ad-VP could suppress growth of EC-109cells through induction of apoptosis. Consistent with the MTT assay, AO/EB, DAPI andAnnexin V staining assays demonstrated Ad-VP had the most significant growth inhibitioneffect on EC-109cells. We further tested whether Ad-VP infection affects the normal humanliver cell line L02. The results confirmed our previous observations, indicating that normalcells hardly show sensitivity to Ad-VP.The mitochondrial pathway is mainly triggered by cell apoptotic signals and these signals frequently cause the loss of ΔΨm and the release of cytochrome c. Furthermore, theactivation of caspases plays a central role in most apoptotic pathways. Upon formation of thedeath-inducing signaling complex in the extrinsic death receptor pathway, or the apoptosomein the intrinsic mitochondrial pathway, initiator caspases are autoproteolytically processedresulting in the activation of downstream caspases and cleavage of numerous death substrates.In this study, we investigated the Ad-VP triggered mitochondrial/caspase-dependent apoptoticpathway. The results showed that EC-109cells treated with Ad-VP showed significant loss ofΔΨm and release of cytochrome c. At the mean time, the activation of caspases and itscorresponding substrates were also detected in Ad-VP infected EC-109cells. However, inL02cells, Ad-VP had no effect on the loss of ΔΨm and the release of cytochrome c, as wellas the activation of caspases. This experiment indicates that Ad-VP mediated tumor specificcell death is associated with mitochondrial, and the study further suggests that Ad-VP kills thecancer cells dependently of caspase-3,6and7.The animal model presented in this study showed that intratumoral injections of Ad-VPlead to effective tumor growth inhibition and increased survival, but did not result in completeelimination of the human xenograft tumors, confirming and extending the results of the invitro studies.Overall, the dual specific recombinant adenovirus Ad-VP induced significant apoptosisof EC-109cells and the toxicity of Ad-VP involved the activation of caspases by amitochondirial dependent pathway. The unique action of the Ad-VP may provide a novel,promising candidate for cancer gene therapy in clinical trials for esophageal cancer.