Experimental Study On The Effects Of Telomerase Inhibitor AZT In C6 Glioma Stem Cells In Vitro

ObjectivesTo investigate the effects of the inhibition of specific telomerase inhibitior 3,-azido-2',3'-dideoxythymidine (AZT)on inhibiting growth of the C6 glioma stem cells derived from the C6 glioma cell line and its possible molecular mechanism, to provide an objective test basis for the feasibility of telomerase as targets for malignant glioma to chemotherapy of targeted therapy.Methods1. Logarithmic growth phase of C6 glioma cells were seeded in serum-free medium in order to acquire BTSCs. Then, cells will be used to identify two groups of cells in the stem cell markers expression (CD133 and Nestin) by immunocytochemistry methods.2.C6 glioma stem cells and glioma cells will be treated with different concentrations of AZT(experimental group one 100umol/L,experimental group two 250umol/L,experimental group three 500umol/L,experimental group four 1000umol/L), then determination of drug sensitivity in different time by modified MTT method. The growth inhibition rate (IR)and half inhibition(IC50) are calculated respectively.3.C6 glioma stem cells and glioma cells will be treated with four different concentrations of AZT (100umol/L,250umol/L,500umol/L,1000umol/L) for 72 hours. the control group without drug group was established, denoted Oumol/L, then detected cell cycle by flow cytometry (FCM).Results1. C6 glioma stem cells grow as suspension ball-like in serum-free medium. immunocytochemistry showed that the expression of stem cell markers CD133 and Nestin; glioma stem cells in serum containing medium after induction of differentiation was adhered to the culture plate, expression of astrocyte marker GFAP with immunocytochemistry.2. MTT results have showed that AZT can inhibit various concentrations of tumor stem cells and tumor cell growth, with increasing concentrations of AZT and the role of time, the inhibition of C6 glioma stem cell proliferation gradually increased in a time-dose dependencies.3. Flow cytometry showed that:with the increase in AZT concentration, tumor stem cells and tumor cells in S phase cells increased, accompanied by an increase in apoptotic cells.Conclusions1. Preliminarily it is confirmed that the existence of brain tumor stem cells which can be obtained by serum-free culture in the C6 glioma cell line, which may be the origin of C6 glioma cells.2. AZT can inhibit growth of C6 glioma stem cells and its mechanism may act on cells through S phase, interference from the inhibition of DNA synthesis of the growth of cells.3.Compared with C6GC, C6GSC was resistant to AZT by MTT method and flow cytometry.