An Experimental Study Of Adenovirus-Mediated Transfer Of VEGF Promoter-Thyidine Kinase Gene For The Treatment Of Hepatocellular Carcinoma In Vitro And In Vivo

Most solid tumors have areas of low oxygen tension (hypoxia). Tumor cells under hypoxic conditions produce vascular endothelial growth factor (VEGF) to stimulate angiogenesis. The expression of the VEGF gene is regulated in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Because normal tissues are not hypoxic, the presence of hypoxic cells provides the potential for designing cancer-specific gene therapy.In the present study, we investigate whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the VEGF promoter encompassing the HRE site followed by GCV administration is effective in killing hepatocellular tumor cells in vitro and in experimental tumors.We used the AdEasyTM system , which exploits E. coil's robust, efficient recombination machinery instead of mammalian cells?to rapidly generate recombinant adenovirus without the need for time consuming plaque purification, to rapidly produce recombinant adenovirus AdVEGF-tk harboring the HSV-TK HSV-tk under the control of the human VEGF promoter 385bp sequence (from -1175bp to -790bp) encompassing the hypoxia response element (HRE). Then, recombinant adenovirus AdVEGF-tk was transduced into hepatoma cell line Hep-G2 and normal liver cell line L02 under both oxic and anoxic conditions followed by GCV administration for 5 days. Cell survival assays demonstrated GCV killing of Hep-G2 cells under both oxic and anoxic conditions, but not of L02 cells under oxic condition. Moreover, Hep-G2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under anoxicas compared with oxic conditions. In vivo, the evaluation of the efficiency of Ad.VEGGF-tk /GCV on tumor growth was assessed in a relevant model of multifocal hepatic lesions induced in rats by a potent alkylating chemical carcinogen, diethylnitrosamine using intratumoral or intrahepatic artery injection of the recombinant AdVEGF-tk. The results displayed a pronounced tumors growth delay and a prolonged survival time when Ad.VEGF-tk was injected by intratumoral (lxi O9pfu) or intrahepatic (5x1 O9pfu) artery route followed by GCV administrations. Further, no significant adverse toxicity was shown in the rats treated with AdVEGF-tk plus GCV by the intrahepatic artery route.These results indicate that the hypoxia-inducible promoter of the human vascular endothelial growth factor gene could increase the selective tumoricidal activity by GCV in gene therapy for hepatocellular carcinoma. Because hypoxia occurs in many solid tumors, this approach would be applicable to a wide range of solid tumors.