Antitumor Efficacy Of An Ad5/F35 Chimeric Oncolytic Adenovirus With TRIAL Gene On Gastric Cancer

Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature's own agents to find and destroy malignant cells. Infection of tumor cells with oncolytic adenovirus results in tumor-specific replication, subsequent oncolysis and release of the virus progeny. Adenovirus type 2 and 5 (Ad2 and Ad5) are most commonly used as gene transfer vectors. They bind cells with their fiber knob to the host coxsackie/adenovirus receptor (CAR). Due to low-level expression of CAR, some of the more interesting cell populations from a therapeutic standpoint, such as dendritic, cancer and hematopoietic cells, have remained difficult to infect with Ad5 alone. In contrast, B-group adenovirus type 35 (Ad35) binds to cells via the ubiquitiously expressed complement regulatory protein CD46, and has already been shown to transducer human dendritic cells, cancer cells and hematopoietic cells efficiently. To overcome the limitations of Ad5 tropism, a new chimeric replicative adenoviral vector system was developed by substation of Ad5-based vector fiber by altering the knob and shaft domains in the Ad5-based vectors, with the fiber of a human B adenovirus serotype 35. The replicative Ad5 vectors with the chimeric Ad5/35 fiber, efficiently transduce human dendritic, cancer and hematopoietic progenitor cells, etc. Therefore, Ad5/F35 vectors could be valuable tools for adenovirus-gene therapy.TNF-related apoptosis-inducing ligand (TRAIL), Apo2 ligand, is identified as a member of the TNF receptor ligand family that is capable of inducing apoptotic cell death in some tumor cell lines. Recent reports have demonstrated that many tumor cells, including the gastric adenocarcinoma cell line, acquire resistance to the apoptotic effects of TRAIL. Caspase-8 is activated in response to TRAIL and released into cytoplasm, where it initiates a protease cascade that activates effector caspases, including caspase-3 and caspase-7. Moreover, TRAIL has been shown to induce apoptosis preferentially in malignant cells, including gastric cancer cells, while sparing normal cells. Some reports have demonstrated that strong antitumoral activities could be achieved by the combination of an oncolytic adenoviral vector ZD55-TRAIL with chemotherapy or ZD55-SMAC.In this study, a new chimeric replicative adenoviral vector system SG235-TRAIL was developed with a load of TRAIL gene, and its antitumor efficacy on gastric cancer were investigated in a series of in vitro and in vivo experiments. Moreover, the synergistic antitumor efficacy of SG235-TRAIL in combination with Taxol was studied in a series of in vitro experiments.1. Construction of Ad5/F35 chimeric oncolytic adenovirus SG235-TRAIL.The fiber gene was replaced with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. To construct the new recombinant adenoviruses SG235-TRAIL, SG235-EGFP and AD5/35-TRAIL by homologous recombination, the plasmid SG235 was co-transfected with pZD-TRAIL, pZD-EGFP and pDC311-TRAIL into 293 cells respectively. The recombinant adenoviruses were confirmed by PCR analysis using specific primers, amplified at a large scale and purified by cesium chloride gradient centrifugation. The viral titer of SG235-TRAIL achieved 6.3×10~9 pfu/ml with TCID50 method.2. Antitumor efficacy of SG235-TRAIL in vitro.For the purpose of comparing the infection capability of adenoviruses, the gastric cancer cells SGC-7901, BGC-823 and normal cells BJ were cultured and transfected with SG235-EGFP and SG5-EGFP. The results were observed under the fluorescence micro-scope. Viral replication experiments were performed to evaluate the selective replication ability of SG235-TRAIL. The replication of SG235-TRAIL was remarkable in gastric cancer cells, while hardly replicated in normal cells. The expression of TRAIL gene in recombinant adenoviruses SG235-TRAIL and AD5/35-TRAIL infected host cells were demonstrated to locate to the cytoplasm by immunofluorescence. The transfection and protein expression were determined by ELISA and western blot. The results of ELISA demonstrated that SG235-TRAIL expressed large numbers of TRAIL proteins in gastric cancer cells. Western blot showed that TRAIL protein was detected only in gastric cancer cells, but not in normal cells. MTT assay and cytopathic effect (CPE) were performed to determine the killing ability of SG235-TRAIL at various viral MOIs, compared with the combination of Taxol. Gstric cancer cells apoptosis was demonstrated by Annexin- V-FITC and flow cytometry. The results showed that SG235-TRAIL had a better killing effect on gastric cancer cells, than ZD55-TRAIL, SG235-EGFP and AD5/35-TRAIL. Moreover, combination of SG235-TRAIL with Taxol exhibited an apparent synergistic cytotoxicity in gastric cancer cells, yet abolished the negative toxicity in normal cells by reducing the dosage.3. Antitumor efficacy of SG235-TRAIL in vivo.SGC-7901 cells were subcutaneously injected into the left flanks of BALB/c nude mice, with administration of 5×10 cells per mouse. When SGC-7901 tumors reached about 90mm~3 in volume, mice were allotted randomly into four groups: AD5/35-TRAIL, SG235-EGFP, SG235-TRAIL, and control groups (n=8 mice /group). Mice underwent four intratumoral injections, once every other day, with total dosage of 10~9 pfu mouse. Tumor volume was measured with calipers at days 3, 7, 10, 14, 21, 28, 35 and 42 for length and width, which should cross the central point, and then were calculated by the formula of (width×length~2/2). All animals were killed and the tumors were removed and fixed in 10% neutral formaldehyde for 6h and paraffin-embedded, and 5-μm-thick consecutive sections were cut for H&E staining, immunohistochemistry and TUNEL staining. Compared with the control group and other treatment groups, antitumor effect of SG235-TRAIL was much stronger. Obvious necrosis and apoptosis were observed by routine pathologic examination, and TRAIL protein was detected by immunohistochemistry in tumor tissues in SG235-TRAIL virus treatment group.In conclusion, Ad5/F35 chimeric oncolytic adenoviral vector SG235-TRAIL was able to infect gastric cancer cells and induce the expression of TRAIL effectively. It had an obvious killing effect on ganstric cancer cell lines. The antitumor efficacy of SG235-TRAIL was enhanced with a better biosafety when combined with a use of Taxol. Chimeric oncolytic adenovirus SG235-TRAIL proves to be promising in the treatment of gastric cancer.