| Objective:Resistance phenomenon is an important reason for glioma chemotherapy, and associated with abnormal expression of resistance genes, it is important the relationship between resistance gene and chemotherapy drugs used commonly, reversal of drug resistance with regulating the gene function and increasing sensitivity to chemotherapy is a hot issue.this study is to explore the mechanism of multidrug resistance in chemotherapy and verapamil (VRP) to reversal of multidrug resistance in the human glioma cells by study of chemoresistance of temozolomide (TMZ) and carmustine (BCNU) to U251 cell.Methods:This study included four parts①Analysis of resistance of U251 glioma cells to temozolomide and carmustine:temozolomide final concentration (unit:g/L) was 0.00625,0.0125,0.025,0.05,0.1,0.2 respectively, and carmustine final concentration (unit:g/L) was 0.01,0.02, 0.05,0.1,0.2,0.5 respectively, cell active were measured by MTT, cell cycle were determined with flow cytometry;②Research of mechanism of multidrug resistance of U251 cells to temozolomide and carmustine:cell active were measured by MTT, cell cycle were determined with flow cytometry, resistance protein were detected by immunohistochemistry and west-blot;③Reseach of verapamil to reversal the chemoresistance of U251 cells in vitro:cell active were measured by MTT, cell cycle were determined with flow cytometry, resistance protein were detected by immunohistochemistry and West-blot;④Reseach of verapamil to reversal the chemoresistance of U251 cells in vivo:models of U251 cells in nude mice were established, the largest and the shortest subcutaneous tumor diameter were measured by vernier caliper. BCNU group (25mg/kg) were administered by intraperitoneal injection once weekly for two weeks,BCNU+VRP group, firstly given verapamil (4mg/kg),5min later to BCNU, were the same as BCNU dose and the Drug delivery, P-gp, MRP1, LRP expression were determined by immunofluorescence staining.Results:①Analysis of resistance of U251 glioma cells to TMZ and BCNU:concentration of Initial drug resistance to TMZ was 0.05g/L, and the time of initial drug resistance was the the third day. concentration of initial drug resistance to BCNU was 0.2g/L, and the time of initial drug resistance was the second day. compared with the control group, U251 cells can be inhibited in G0/G1 phase (P<0.05);②Research of mechanism of multidrug resistance of U251 cells to temozolomide and carmustine: U251 cells were produced drug resistance phenomenon by BCNU and TMZ; compared with the control group, U251 cells induced BCNU and TMZ were blocked in G0/G1 phase; results of West-blot show:compared with control (P-gp, MRP1, LRP), TMZ and BCNU group expression were increased, two group of P-gp, MRP1 expression level compared with the control were significantly different (P<0.05), compared with control (MGMT), BCNU expression were increased, TMZ expression reduced. Results of Immunohistochemistry showed:before using BCNU, LRP was positive, mainly was expressed in the nucleus, P-gp and MRP negative. After using BCNU, LRP was still positive, P-gp was weakly positive, MRP1 was positive; the latter two were expressed in the cytoplasm;③Reseach of verapamil to reversal the chemoresistance of U251 cells in vitro:verapamil to effects of TMZ and BCNU to U251 cell at some time points (BCNU in 3 days and TMZ in 2,5,7 days) were significantly different (P＜0.05); compared with TMZ and BCNU, respectively, U251 cells can be effectively inhibited in the GO/G1 phase by VRP+TMZ and VRP+BCNU; Results of West-blot show:compared with the control group, P-gp, MRP1, MGMT expression of VRP group were decreased, And compared with TMZ and BCNU respectively, P-gp, MRP1, MGMT expression of VRP+TMZ and VRP+BCNU were decreased, two groups of P-gp, MRP1 were significantly different (P <0.05), compared with TMZ, LRP expression of VRP+TMZ group was increased, compared with BCNU, VRP+BCNU decreased; results of Immunohistochemistry show:after plusing VRP, the resistance genes expression were not changed;④Reseach of verapamil to reversal the chemoresistance of U251 cells in vivo:compared with BCNU, BCNU+ verapamil in the 1,2,3,4 weeks were significantly different (P<0.05). compared with BCNU, LRP of VRP+BCNU group was positive, P-gp and MRP1 were weakly positive.Conclusions:①Different chemotherapeutic drugs in different concentration and time on the glioma cell lines have different sensitivity to chemotherapy;②Chemotherapy to change cell cycle produce resistance;③Resistance protein expression and type of change, especially P-gp, MRP1, are one of the causes of multidrug resistance. The phenomenon of multidrug resistance of glioma is result of primary and secondary resistance;④Experiments of vivo and vitro show that verapamil to chemotherapy drugs of glioma has some synergistic effect,verapamil can be competitive with the membrane resistance protein (P-gp, MRP1) combined to reverse the phenomenon of multidrug resistance of glioma. it can be inhibited the resistance gene (P-gp, MRP1, MGMT) expression to increase the the sensitivity of chemotherapy.
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