Study On The Inhibition Effect Of Adenovirus-mediated BmK CT On Glioma

BmK CT is one of the key toxins in the venom of the scorpion, Buthus martensii Karsch. It shares68%of identity with the sequence of chlorotoxin (CTX) from the Leiurus quinquestriatus and has a similar structure of protein which contains36amino acids cross-linked by four disulfide bridges. BmK CT or CTX can interact specifically with glioma cells as a chloride channel blocker and inhibit the invasion and migration of those cells via MMP-2. The current surgical treatment for malignant glioma remains difficult in that its biological characteristics of invasion into normal tissues. A significant problem is the resistance of glioblastoma cells to apoptosis induced by most of antitumor drugs. BmK CT or CTX is proposed as a novel therapeutic strategy for gliomas.Since CTX can specifically bind to glioma cells, it is believed that intensive and localized radiation will be delivered to individually infiltrated tumor cells beyond the tumor margin, leaving surrounding normal tissue cells untouched.131I-CTX targeted radiotherapy showed that it had a high concentration in tumors and low concentration in normal organs in nude mice bearing glioma cells xenografts in brain. CTX is conjugated by fluorescent material Cy5.5or super paramagnetic nanoprobes to produce the complex of CTX-Cy5.5or CTX-super-paramagnetic nanoprobes. Then CTX-Cy5.5or CTX-super-paramagnetic nanoprobes could bind to glioma cells specifically via intravenous injection, so they could be used as optical imaging by intraoperative infrared microscopy or a contrast medium for MRI to enhance the differentiation of the tumor from normal brain tissue. CTX, as a small peptide, can be rapidly to be cleared from systemic circulation or be degraded.In this study, the recombinant adenoviral system was produced by a double-recombination event, the co-transformed adenoviral backbone plasmid and the shuttle vector carrying the BmK CT gene. Ad-BmK CT was used to study its effects and mechanisms of glioma cells proliferation, migration and apoptosis. Glioma-bearing nude mice were used to identify potential therapeutic assay of Ad-BmK CT against glioma. Drug safety of Ad-BmK CT was checked using glioma-bearing rats. The main results are listed as follows:1. The recombinant adenovirus Ad-BmK CT was constructed and purified. The titer was tested by viral particle titration. The ability of BmK CT transgene expression in AD293cells mediated by adenovirus transfection was determined by RT-PCR.2. The optimum infection concentration was determined by the percentage of GFP positive cells and GFP mean fluorescence intensity. The amounts of BmK CT expression kept stable for72h. MTT assay, Boyden chamber and wound healing assay showed that it could affect cell proliferation and migration. The results showed that Ad-BmK CT decreased the proliferation of glioma cells and induced the cell death.3. The stable cell line expressing red fluorescence protein (RFP) was constructed. The glioma-bearing nude mice were intratumorally injected with Ad-BmK CT or Ad. Tumor volume analysis suggested that Ad-BmK CT inhibited glioma growth, compared with Ad and A195buffer treatment groups. There was an obvious difference in lung metastasis between the Ad treatment groups and Ad-BmK CT groups. C6/RFP cells in lungs were imaged by Xenogen IVIS Spectrum. Up to40days post-treatment, there were four mice died in the control group, two in Ad group, and no deaths in Ad-BmK CT group. The mice in Ad-BmK CT group could live longer.4. Glioma-bearing rats were used to check the drug safety of Ad-BmK CT. The models were intratumorally injected with Ad-BmK CT. After48h or72h, kidneys, spleen, liver, lung and brain were dissected and used for histological analysis. The results showed that spleen swelled and lymphocyte proliferated; infiltration of lympbocytes was observed in the liver but without obvious tissue damage; there was no influence in the kidneys, lung and brain. Long-term toxicity after injection of Ad-BmK CT would not cause abnormal rat behavior and death.5. The expression level of oncogene C-myc, MMP-2and caspase3were tested by western bloting. The results showed that C-myc was down-regulated after glioma cells infected with Ad-BmK CT. The delivered BmK CT would then interact with pro-MMP-2in the glioma cells and inhibited migration. Finally, BmK CT facilitated cytochrome c release and upregulated caspase-3, which might account for the increased apoptosis rates in BmK CT positive cells.