The Study On The Multidrug Resistance Reversion Of Oxymartrine Via Inhibiting P-gp In Human Colon Cancer Cell Line

Objective: To investigate the influence of Oxymatrine in chemoresistance of HCT-8 and VCR-resistant human colon cancer cell line HCT-8/VCR,and to explore the potential mechanisms involved.Methods:(1)Methyl Thiazolyl Tetrazolium(MTT)assay was performed to determine the drug resistance to Vincristine,Cisplatin and 5-Fluorouracil of HCT-8/VCR cells.(2)MTT assay was used to detect the proliferation inhibitor and the drug resistance to Vincristine,Cisplatin and 5-Fluorouracil after Oxymatrine treated with HCT-8/VCR cells.(3)The cells were divided into HCT-8 group,HCT-8/VCR group and Oxymatrine group.After the HCT-8/VCR cells were treated with Oxymatrine for 48 h,the cells' morphological were observed by inverted microscope,the expressions of P-gp mRNA and protein were detected by qPCR and western blot assays,respectively.And Flow cytometry was used to detcet intracellular concentration of Rho123.Result:(1)MTT assay showed that the IC50 of VCR,CDDP and 5-Fu were 25.63?3.13,12.33 ? 2.01 and 2.89 ? 0.62 in HCT-8 cells,and they were 320.95 ? 6.44,45.95 ? 4.76 and18.32?3.91 in HCT-8/VCR cells.There were atatistical significance between the HCT-8 and HCT-8/VCR cells(P<0.001).Compared with HCT-8 cells,HCT-8/VCR appeared multidrug resistant with resistance rates of 12.52,3.73 and 6.34 for VCR,CDDP and 5-Fu.(2)The Oxymatrine significantly inhibited the growth of HCT-8 and HCT-8/VCR cells in a concentration-dependent manner(P<0.05).We chose the 1mg/ml of Oxymatrine concentration(a inhibitor rate lower than 10%)as a experimental condition.(3)After treatment with1mg/ml of Oxymatrine for 48 hours,the sensibility of HCT-8/VCR cells to VCR,CDDP and5-Fu were increased and the IC50 of these drug were decreased(P<0.01),and the Reversal fold were 3.44,2.16 and 2.04.The cell's growth was slower,the cell death increased,and the vacuoles were seen in inverted mocroscopy.The results of qPCR showed that the relative expression of ABCB1 mRNA in HCT-8 group and Oxymatrine group were lower than HCT-8/VCR group(P<0.01).And the expression of P-gp protein also lower than HCT-8/VCR group(P<0.05).The intracellular fluorescence intensity of Rho123 in Oxymatrine group was23.45% ? 0.64%,higher than HCT-8/VCR group(8.3±2.12)%.There were atatistical significance between the HCT-8/VCR and Oxymatrine group(P<0.05).Conclusion: Oxymatrine could reverse multidrug resistance of human colon cancer cell line HCT-8/VCR,which may be related to down regulate the expression of P-gp gene and protein,enhance the intracellular concentration of drug.