Investigation of hrDNA targeting vector-mediated tumor-specific suicide gene therapy for hepatocellular carcinoma

Abstract

Human ribosomal DNA (hrDNA) targeting vector (pHm) is one of the human derived vectors, which was devised by our lab and has got patent authority. To investigate its effect on gene therapy for hepatocellular carcinoma, a double suicide fusion gene expression cassette, CDUPRT/GFP controlled by a synthetic CMV enhancer-enhanced hTERT promoter (CeTp) which was determined by luciferase assays was constructed in pHrn backbone, creating an expression vector pHr-CeTpCDUPRT/GFP. After transfer of plasmid to hepatocellular carcinoma cell line Bel7402 in vitro, the transfection efficiency reached 30%–50% by using flow cytometer. The expression of CDUPRT/GFP was detected by RT-PCR and Western Blotting. After the administration of 5-FC, high performance liquid chromatography (HPLC) was applied to examine the level of 5-FU in supernatant, resulting in a concentration of 60.15 μg/mL. The Methylthiazolyl tetrazolium (MTT) assay was then utilized to investigate the antitumor effect of pHr-CeTpCDUPRT/GFP in Bel7402 cells, and relative cell survival of 60%–35% was observed after 5-FC treatment. In vivo experiments, the nude mouse model of hepatocellular carcinoma was constructed and in situ gene therapy was performed. The results indicated the tumor growth of treatment group was obviously suppressed, and some even shrank, when the vectors and prodrugs were injected continuously. The expression of CDUPRT in tumor tissues was also identified by RT-PCR, and the concentration of 5-FU was 7.694 μg/mL in blood serum using HPLC detection. Then the pathological section of tumor tissues revealed significant tumor cell necrosis. All of these results provide important experiment evidences of the gene therapy for hepatocellular carcinoma with the utilization of our vectors.