| Gastric cancer is a worldwide health burden. Treatment has limited progress and75%ofpatients are diagnosed only at the unresectable stage. Following curative resection alone oreven after adjuvant therapy, nearly60%of affected patients succumb to gastric cancer. Allthese factors pose the challenge of whether to choose a strictly supportive approach or toexpose patients to the side effects of a potentially ineffective treatment.Cancer gene therapy based on adenoviruses has been studied extensively in pre-clinicaland clinical trials. Oncolytic virotherapy is a promising approach for patients who areresistant to traditional cancer therapies. Many strategies have been evaluated in pre-clinicaland clinical trials, for example treatment with conditional replication-competent adenovirus(CRCA). The application of CRCAs has gained increased attention for a number of reasonsdetailed here. Importantly, because the promoters in these vectors are selective for cancercells, these oncolytic viruses have the ability to replicate and to spread to adjacent tumorcells. Furthermore, infection with CRCA generates anti-tumoral immune responses that cancomplement chemotherapy and radiotherapy. Importantly, CRCAs are capable of achievingthe destruction of primary and distant tumors given the proper therapeutic transgenes.However, insufficient levels of efficacy and poor specificity of treatment remain the majorchallenges for this targeted cancer gene therapy strategy and have highlighted the need forthe development of novel cancer therapies.The human telomerase reverse transcriptase (hTERT) promoter is highly activated inimmortalized cell lines and in over85%of human cancers and has therefore been usedwidely for the construction of CRCAs. In these studies, early region1A (E1A) gene, whichplays a central role in virus infection by regulation of virus replication and cell cycle, wasthe gene used most frequently in the construct and was essential for adenovirus replication.In the present study, we describe the anti-tumor effects of a dual cancer-specificoncolytic adenovirus, Ad-hTERT-E1a-apoptin, in which replication was driven by thehTERT promoter that replicates selectively and induces apoptosis specifically in tumor cells.The anti-tumor effects were evident within24h when administered to gastric cancer cells invitro. Treatment with100MOI or10MOI doses completely inhibited the growth of SGC7901cells4days later, although a single Ad-hTERT-E1a-apoptin treatment at1MOIwas less effective. In contrast, growth inhibition was not observed in GES-1cells aftertreatment with Ad-hTERT-E1a-apoptin at any MOI dose. These findings indicate that theAd-hTERT-E1a-apoptin replicates specifically and induces growth suppression selectively inSGC7901cells compared with the normal counterparts.The applicability of cancer therapies is not only determined by their efficiency asspecificity is an equally important prerequisite. Apoptin, a protein of13.6kDa in mass thatwas derived from chicken anaemia virus (CAV) VP3gene and has been thought to induceapoptosis selectively in a variety of tumor cells, such as hepatomas, lymphomas,cholangiocarcinomas, melanomas, and breast, lung, oral and colon carcinomas. Preliminarystudies have demonstrated that more than70different transformed and malignant cells ofhuman origin were sensitive to apoptin-induced apoptosis. However, apoptin did not act onnormal non-transformed human cells, such as primary fibroblasts, smooth muscle cells, Tcells, hepatocytes, hematopoietic stem cells, keratinocytes, or endothelial cells. Furthermore,long-term expression of apoptin in normal human fibroblasts has shown that it has no toxicor transforming activity in these cells. In contrast to our findings, Guelen et al. have showntoxicity of apoptin towards non-cancerous cells, however this study demonstrated cell deathonly in a fetal cell type. Furthermore, the ability of apoptin to induce tumor-specificapoptosis is independent of p53. Thus, apoptin is similarly effective at killing tumor cellsthat are p53deficient or that express either wild-type or mutant p53. The role ofanti-apoptotic molecules in apoptin-induced apoptosis is still a matter of debate. In certaintumor cell lines, apoptin-mediated cell death is independent of the bcl-2status and is evenstimulated by bcl-2or is insensitive to bcr-Abl and bcl-xL. Then, apoptin can be used tocomplement radiotherapeutic and chemotherapeutic approaches. All these factors makeapoptin an attractive candidate for cancer gene therapy and many studies have demonstratedthe effects of apoptin inserted into various vectors on the restriction of manifold tumors.Chromatin condensation and nuclear fragmentation remain the hallmarks of apoptoticcells. Fluorescence light microscopy with differential uptake of fluorescent DNA-bindingdyes (such as EB/AO staining) is the method of choice due to its simplicity. Here, using theAO/EB method, we quantified the percentage of live, necrotic, and apoptotic cells afterrecombinant adenoviruses treatment. As shown in result, infection of SGC7901cells with therecombinant adenoviruses may, in part, inhibit the cells by causing necrosis. We expected, therefore, that the effects of the apoptin gene might be masked. However, the effects of therecombinant virus did not impede apoptin-induced apoptosis. After Ad-hTERT-E1a-apoptininfection,38.6%of apoptin-expressing transformed cells became apoptotic after only48h,and27.1%of infected cells exhibited necrosis. In contrast, although24.3%of cells wereapoptotic48h after Ad-hTERT-E1a infection,25.5%cells died as a result of necrosis. Theresults indicated that, although replication-component adenovirus infection stimulatesnecrosis, this event does not appear to render tumor cells sensitive to apoptin. Furthermore,classification of cell death should always include morphological examination coupled with atleast one other assay. Apoptin induces apoptosis in a wide variety of human cancer cell linesvia the classical apoptotic pathways. Caspase-3, caspase-6and caspase-7are the importantdownstream effecter caspases that cleave major cellular substrates in apoptotic cells andamplify these substrates via intrinsic or extrinsic pathways. In the present study, a westernblot analysis of total protein extracts showed activation of caspase-3, caspase-6andcaspase-7in recombinant virus-infected SGC7901cells. However, no caspase activation wasobserved in GES-1cells, except for non-specific replication-competent adenovirus infection.GAPDH expression was used to confirm equal loading of the gel. All results validated thespecific apoptosis-inducing effects of the dual cancer-specific oncolytic adenovirusAd-hTERT-E1a-apoptin construct.We also evaluated anti-tumor activity in vivo in a human gastric cancer xenograftmouse model, which confirmed and extended the results of the in vitro studies. In this test,tumors derived from mice gastric cancer cells transplanted into BALB/c nude mice wereinfected with various recombinant adenoviruses. We found that all recombinant adenoviruseshad a significant anti-tumor effect, despite the fact that the replication-defectiveadenoviruses infected only part of the tumors. Injection of Ad-hTERT-E1a-apoptin intotumors resulted in a complete response to treatment. However, infection withAd-hTERT-apoptin, Ad-CMV-apoptin, Ad-hTERT-E1a or Ad-CMV-E1a was less effective. Itis plausible that application of replication-component adenoviruses allows virus dispersioninto any tumor tissues in the animal and that apoptin expression enhances the capability ofthe oncolytic adenovirus. When infection was carried intravenously, however, treatment ofAd-hTERT-E1a-apoptin did not lead to complete tumor regression, but growth of the tumorswas delayed significantly. Suppression of the indirectly injected tumor may also reflect asecondary virus infection by the CRCA. The oncolytic activity of Ad-hTERT-apoptin, Ad-CMV-apoptin, Ad-hTERT-E1a or Ad-CMV-E1a was limited, which was similar to that ofintratumoral injection groups. No tumor regression was observed in mice that had SGC7901tumors when injected intravenously with saline and Ad-mock. Furthermore, we did notobserve any toxic effects after injection of Ad-hTERT-E1a-apoptin in the in vivoexperiments described here. Thus, our data indicate that there is great potential for animprovement in the safety and efficacy of adenovirus vectors for wide application in thetreatment of neoplastic diseases.In conclusion, gene therapy with apoptin offers unique advantages over currentapproaches for cancer therapy. Factors such as:(1) apoptin does not need a functional p53pathway;(2) it is not hindered by the commonly found blockage of apoptosis by bcl-2orbcr-abl,(3) it apparently acts downstream of most other factors, and (4) it has unparalleledpotency, suggest that apoptin will be applicable for the treatment of a wide range of tumors.In addition, the CRCA we describe in this study induces apoptosis selectively in variouscancer cells without adverse effects on normal cells. In vivo and in vitro data described hereshow that expression of apoptin increases the effectiveness of CRCAs and that an adenovirusvector under the control of a hTERT promoter does not reduce the efficacy of the constructbut improves the global safety of CRCAs. All these factors highlight the need for furtherevaluation of this strategy for clinical trials. |
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