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The purpose of chemotherapy is one of the important means to treat tumors, it greatly increases the likelihood of cure of the tumor. However, the resistance of tumor cells (Drug resistance) often leads to the failure of chemotherapy. Tumor cells to a drug resistance, often also with different chemical structures and different mechanisms of drug resistance at the same time, this is a multi-drug resistant (Multidrug resistance, MDR). Currently being carried out extensive research on MDR mechanism encoded by the multidrug resistance gene (mdrl) molecular weight 170kD P-glycoprotein (P-glycoprotein, P-gp) overexpression caused by the resistance of tumor cells is the primary mechanism . Therefore, inhibiting or regulating the function and / or expression of P-gp is the key to reversing tumor cell MDR. Stephania base hydrochloride (Cepharanthine Hydrochloride, CH) Menispermaceae Stephania is a two-benzyl isoquinoline plant the tubers in the extraction and separation by salt derived monomer compounds, has a strong variety of biological activity. Recent overseas research has a role reversal of multidrug resistance reversal mechanism of multidrug resistance of leukemia cells with P-gp relationship has not been reported. The present study, the detection of cell and molecular biology to observe CH resistance reversal effect on tumor cells, and the molecular mechanisms of MDR-depth discussion. Methods experiment to select human chronic myelogenous leukemia cell line K562 and MDR phenotype induced by doxorubicin (Adriamycin, ADR) stable adriamycin-resistant cell lines K562/ADR. Using the MTT (3 - (4,5-dimethylthiazol) -2,5-diphenylte-2H-tetrazoloum bromide, MTT) analysis, determination of ADR alone and combined with CH, verapamil (Verapamil, VER), respectively K562 direct K562/ADR cell cytotoxicity; doxorubicin accumulation experiments, flow cytometry determination of CH role in intracellular doxorubicin volume changes; determined by flow cytometry the CH of K562 cells, K562 / P-gp expression levels of ADR cells; Rhodamine 123 (Rho123) accumulation and pump out experiments measured the intracellular accumulation Rho123 fluorescence intensity analysis of CH function of P-gp. Through the experiments to clarify the the CH reversal the K562/ADR cells of ADR resistance with the expression and function of P-gp relationship. Results 1. ADR pair the K562, K562/ADR cell cytotoxicity by MTT assay ADR cytotoxicity of K562, K562/ADR cells, its IC 50 values ??of the order of 0.45 ± 0.01μmol · L < sup> -1 and 13.97 ± 0.30μmol · L -1 resistant multiple of 31.04 times, and the difference was statistically significant (P <0.05). 2. CH, VER combined with ADR the impact of the K562, K562/ADR cytotoxic single CH weak the VER on K562 and K562/ADR, cytotoxicity, the results: CH 4μmol · L -1 inhibition rate (5.72 ± 0.31)% and (5.26 ± 0.76)%, the VER 4μmol L -1 inhibition rate of (4.10 ± 0.45)% and (3.95 ± 0.20)% . ADR and 4μmol L -1 CH combination of K562 cells IC 50 value of 0.44 ± 0.02μmol · L -1 , and single-use ADR difference was not significant (P> 0.05); The of K562/ADR cells IC 50 value of 1.88 ± 0.10 μmol · L -1 with ADR The difference was statistically significant (P <0.05), reversed multiple of 7.43 times. ADR and 4μmol L -1 VER combination of K562 cells IC 50 value of 0.46 ± 0.03μmol · L -1 , and single-use ADR difference was not significant (P> 0.05); the cells of K562/ADR IC 50 value of 6.27 ± 0.17μmol · L -1 with ADR the difference was statistically significant (P <0.05), reversing a multiple of 2.23 times. The CH combination group combined with VER the group K562/ADR cells, inhibition compare the difference was statistically significant (P <0.05), and the CH reversal effect is stronger than the VER. 3. CH on the doxorubicin accumulation of K562/ADR cells by flow cytometry 0,2,4,8 μmol · L -1 CH, respectively, with 5μg/mLADR together after 2h, K562/ADR cells within ADR fluorescence intensity were 3.1 ± 0.3,6.4 ± 0.4,16.5 ± 0.6 and 22.8 ± 0.7, there are significant differences between each dose group (P <0.05). 4. CH of P-gp function the K562, K562/ADR cells the impact using Rho123 accumulation and pump out the assay before and after treatment changes in the function of P-gp, Beckman flow cytometry on the cells were analyzed to the accumulated Rho123 intracellular fluorescence intensity as quantitative indicators. 0, 2, 4, 8, μmol · L -1 CH group and 4μmol · L -1 VER group of K562 cells Rho123 fluorescence intensity were in Rho123 accumulation experiments 81.5 ± 0.4,81.4 ± 0.3,81.7 ± 0.3,81.3 ± 0.5 and 81.2 ± 0.2, K562/ADR intracellular Rho123 fluorescence intensity were 16.9 ± 0.6,25.3 ± 0.2,44.7 ± 0.3,60.7 ± 0.7 and 23.1 ± 0.5 ; differences between each dose group of K562 cells no statistically significant (P> 0.05) difference between K562/ADR cell dose group was significant (P <0.05). Experiment Rho123 pump, 0, 4, 8 μmol · L -1 CH group and 4μmol · L , -1 VER group, the K562 cells Rho123 the fluorescence intensity were 77.0 ± 0.5,77.2 ± 0.1,76.8 ± 0.3 and 77.4 ± 0.3, K562/ADR intracellular Rho123 fluorescence intensity were 4.0 ± 0.1,7.4 ± 0.4,13.7 ± 0.2 and 4.9 ± 0.1; differences between each dose group of K562 cells no significant (P> 0.05) K562/ADR cells between each dose group difference was statistically significant (P <0.05). 5. CH K562, K562/ADR cell P-gp expression by flow cytometry analysis of the expression levels of P-gp, K562 control cells positive rate of 0.3%, plus PE-labeled monoclonal antibody P-gp, P- gp expression percentage of 0.3%, respectively, between the two groups, the difference was not significant (P> 0.05), the expression of P-gp in K562 cells; K562/ADR cells, 0,2,4,8 μmol · L -1 the CH set of and 4μmol · L -1 VER group, P-gp expression percentage were (94.5 ± 0.2)%, (94.6 ± 0.1)%, (94.4 ± 0.3) %, (94.5 ± 0.1)% and (94.1 ± 0.3)%, P-gp expression between different concentrations of CH group difference was not statistically significant (P> 0.05), lower concentration of CH has no effect on the expression of P-gp. Conclusion 1. CH can be partially reversed K562/ADR resistant cells. CH combined with ADR significantly lower the the ADR the IC of K562/ADR cells 50 values, reversing a multiple of 7.43 times stronger than the reversal of the role of the VER. 2. The CH concentration-dependent increase ADR accumulation in K562/ADR cell. 3. The CH concentration change does not affect the K562 cells Rho123 accumulation and pump out; of K562/ADR cells with increasing CH concentration, intracellular Rho123 accumulation increased, reduced pump out. The results showed that the resistance of the CH reverse K562/ADR cells may inhibit the function of P-gp. 4. K562 cells P-gp expression and higher K562/ADR cell P-gp expression; CH concentration is low, nothing to do with the expression of P-gp reversal K562/ADR cell resistance.
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