Oncolytic Virotherapy Of Human Malignant Glioma U251 In Vitro With The E1B-55KD Gene Deleted Adenovirus H101

Objective: Treatment of glioblastoma remains a major challenge in adults especially in male between 40-50.Tumor cells infiltrate in white matter fast. Current therapy is surgery excision and treatment such as radiation and chemotherapy therapy after operation. Because of unclear boundary, it could not be excised completely. And it would recurrence in 8 months, no wonder the median survival time rarely exceeds 1 year. Gene therapy is a newly therapeutic strategy compared to the traditional therapy. It depends on the objective gene changing in the tumor cells which are not changed in normal cells and induce apoptosis in the tumor cells. Conditionally replicative adenoviruses H101(named Onyx-015 or dl1520) which can selectively replicate and cause lysis of tumor cells but not normal cells. It's designed for the change of p53 gene in tumor cells. This type 5 adenovirus has an 800-bp deletion in the E1B region encoding the 55 kDa protein in infected cells, which binds and inactivates cellular p53.Therefore the adenovirus is thought to replicate efficiently and cause cytopathic effects in tumor cells that lacking functional p53, which could't replicate in p53 positive normal cells and finally disseminate virus particles to infect other tumor cells. The clinical trials currently undergoing for the treatment of head and neck cancers and other cancers with apparently promising results. But in the CNS glioma still remains a lot of problems which mainly focus on the neural toxicity to the neuron and whether it could cause encephalitis and encephaledema by the adenovirus H101.We mainly discuss the toxicity to the rat neuron and test the inhibition ratio to the glioblastoma cells U251 in vitro.Methods: In this experiment, we select p53 negative glioblastoma cells U251 that were established by Japanese Cell Bank. Primary cultured 12 days new born rat hippocampus neuron for the control of the experiment. Neuron is identified by the MAP-2 antibody. Cell cytotoxic experiment (MTT) and cytopathic effect (CPE) were used to detect the inhibitive ratio in different virus particle H101 in rat hippocampal neurons and glioma cells. RT-PCR was used to find H101mRNA expression in 3 time points (24h,48h,72h) in two type cells. Using t-tset to analyze significance of difference.Results: 1. H101 could cause cytopathic effects in U251 cells from 24h and shows greater oncolysis effects when much more H101mRNA expression by enhanced virus tite or prolonged time. 2. H101 could not effectively replicate in primary cultured rat hippocampal neurons. It showed no oncolytic effect on neurons. There is no difference between the H101 treated group and the control group (P>0.05),no H101mRNA was detected in 3 time points.Conclusions: 1. Oncolytic adenovirus H101 could replicate and cause oncolysis effects on glioblastoma U251 cells, it shows greater oncolysis effects and much more H101mRNA expression by enhanced virus tite or prolonged time. 2. Oncolytic adenovirus H101 could not effectively replicate in primary cultured rat hippocampal neurons. There is no cytotoxicity to the rat hippocampal neurons.