Completely Eradication Of Xenograft Hepatoma With Oncolytic Adenovirus Zd55Harboring Trail-ietd-smac Gene

Background and Objective: Cancer Targeting Gene Viro Therapy (CTGVT) isconstructed by inserting the antitumor gene into oncolytic viral vector whichintegrates the advantage of both genetherapy and virotherapy. Combinationtarget therapy is necessary for cancer treatment because tumors are genetically diverse.Combination of ZD55TRAIL and ZD55Smac has potent therapeutic effect forhepatoma. This is one example of the so called Cancer Targeting DualGene Viro Therapy of Cancer (CTGVT DG). Many CTGVT DG could always getcompletive eradication of many different xenograft tumors in nude mice by the use oftwo OV genes. The key problem is to join the two genes to be one fusion gene intoone oncolytic viral vector instead of combination of two OV genes. In our currentstudy, we generated a novel E1B55K deleted oncolytic adenovirus that harbors bothTRAIL and Smac genes (ZD55TRAIL IETD Smac). In this vector, a caspase8cleavage site (IETD) was introduced between the genes. We investigated theantitumor efficiency of ZD55TRAIL IETD Smac in vitro and vivo, with the hopethat it can inhibit the tumor proliferation effectively and thoroughly and purpose toprovide more effective CTGVT DG; Meanwhile, we study IETD, a novel linker,providing an ideal connectivity tool for CTGVT DG.Method:①Activation and inhibition of caspase8affect cell viability and theexpression of TRAIL and Smac transgenes. Cancer cells were treated withZD55TRAIL IETD Smac, in combination with caspase8inhibitor (Z IETD FMK)or caspase8activator (5FU), expression of TRAIL and Smac was detected bywestern blot and cell viability was determined by a MTT assay.②ZD55TRAIL IETD was constructed and the comparison of the antitumor activitybetween ZD55TRAIL and ZD55TRAIL IETD was determined by a MTT assay. ③Antitumor activity and security analysis of ZD55TRAIL IETD Smac in vitro wasexamined by MTT assay and CPE assay.④Measuring the cancer cell apoptosis by Annexin/PI double dye.⑤Injecting ZD55TRAIL IETD Smac intratumorlly to Bel7404xenografttumor bearing nude mice to observe its anti tumor capacity in vivo.⑥Histopathologic, TUNEL and immunohistochemical analysis of tumor sections.Result and Conclusion:①The cleaving efficiency of IETD linker is induced bycaspase8inhibitor (Z IETD FMK) and increased by caspase8activator (5FU).②IETD residue in the C terminal of TRAIL could not influence the antitumor activityof transgene TRAIL, which provide a theoretical foundation for IETD mediatedCTGVT DG with TRAIL and another antitumor gene.③ZD55TRAIL IETD Smac has potent therapeutic effect for cancer cells in vitro andinduce apoptosis in cancer cells.④The results of security analysis suggest that ZD55TRAIL IETD Smac has noapparent cytotoxic effect on normal cells.⑤Intratumoral injection of ZD55TRAIL IETD Smac eliminated all xenografttumors established from a human HCC cell line Bel7404, resulting in long term,tumor free survival.⑥Oncolytic adenovirus incorporating two different therapeutic genes by introductionof the IETD sequence may be attractive candidates for clinical application intreatment of cancer..In recent years, small molecule inhibitors have bacome a hot spot in anti tumorresearch. Embelin, a small molecule XIAP (X linked inhibitor of apoptosis protein)inhibitor, can inhibit the expression and activation of XIAP, thereby removing theantiapoptotic ability of XIAP and resulting in apoptosis process smoothly. This studyis attempt to explore the mechanism of the inhibition to hepatocarcinoma cellBel7404growth by Embelin. After treated with different concentration of Embelin,the morphological change, formation of apoptotic bodies, cells viability, cell apoptosisand the apoptosis related protein expression in Bel7404cells were assessed byfluorescence microscope, Hochest33342staining, MTT assay, AnnexinV/PI flow cytometry and Western blotting et al. The results indicated that Embelin remarkablyinhibited human hepatocarcinoma cell Bel7404growth. Further experiments foundthat the mechanism of tumor cell growth inhibition by Embelin was due to the cellapoptosis with the activation of caspase apoptotic pathway and blockage of NF κBsignal pathway. The studies laid a base on the further treatment of human hepatomacarcinoma by using Embelin.