Roles Of TMZ/MGMT-shRNA Co-Delivery In Glioma

Glioma is the most common primary central nervous tumor. Chemotherapy is a very important part in clinic treatment. However, the expression of resistance genes in tumor cells is causing serious drug-resistance problem during chemotherapy.Temozolomide(TMZ) is a “top-pick” chemotherapy drug for gliomas. But it suffers a serious drug-resistance problem, due to the expression of O~6-m G DNA methyltransferase(MGMT) in glioma cells. To solve this problem, we established a drug/gene co-delivery system to inhibit glioma proliferation, which may overcome the drug-resistance problem. We firstly assessed the best inhibition concentration of TMZ using flowcytometry. We found the proliferation of cell lines T98 G, U251 MG and SHG44 were all inhibited by TMZ in a dosage dependent manner. However, T98 G and U251 MG were more resistant than SHG44, which could be caused by a higher endogenous expression of MGMT in T98 G and U251 MG. To verify this hypothesis, we constructed two lentivirus gene-silence systems, MGMT-sh RNA-284 and MGMT-sh RNA-396, to knockdown the expression of MGMT. Results showed virus treated U251 MG cells were more sensitive to TMZ. Therefor this gene silence strategy may be used to overcome the drug-resistance problem of TMZ.Virus systems are risk factors in clinic treatment. In order to deliver the therapeutic gene, we designed and constructed a PEI-PEG vehicle with our collaborator CAS Changchun Institute of Applied Chemistry. Transfection rate of PEI-PEG is stably higher than 75%. We further modified TMZ to a negatively charged TMZA that could also bind to PEI-PEG vehicle. Finally a drug/gene co-delivery system, "PEI-PEG-MGMT-sh RNA/TMZA ” was established. The best working concentration of TMZA is 100μM, and the best ratio between PEI-PEG and MGMT-sh RNA is 2.5:1. In vitro study showed this drug/gene co-delivery system efficiently silenced the expression of MGMT and increased the sensitivity of glioma cells to TMZA. The synergistic effect between MGMT-sh RNA and TMZA in this co-delivery system remarkably enhanced the inhibitory effect of TMZA on tumor cell proliferation. At the same time, we constructed a“T98G-Luc”cell line, which could express firefly luciferase gene in glioma cell and enable live tumor observation. Further in vivo study using “T98G-Luc”cell could validate the potential application of this drug/gene co-delivery system in clinic.