环孢素通过抑制H2AX表达减少DNA损伤后修复逆转K562／A02细胞对多柔比星的耐药性

Reversing Mechanism of Adriamycin Resisitance of K562/A02 Cell Line by Ciclosporin through H2AX Suppression to Attenuate DNA Damage Repair

目的探讨环孢素(CsA)通过减少DNA损伤后修复而逆转K562/A02对多柔比星(ADM)耐药性的可能性及其机制。方法K562或K562/A02与不同水平ADM和CsA共培养后,采用四甲基偶氮唑盐微量酶反应比色(MTT)法计算半数抑制水平(IC50)、耐药倍数及增敏倍数。K562/A02细胞经CsA处理后,应用反转录(RT)-PCR检测细胞mdr1mRNA及H2AXmRNA表达水平;应用流式细胞仪测定细胞P-糖蛋白(P-gp)及H2AX蛋白表达水平;中性彗星实验法检测经兔抗γH2AX抗体处理后K562/A02细胞双链DNA损伤情况。结果2×10-3g/LCsA即可增加K562/A02细胞对ADM的敏感性,增敏倍数达5.28倍。RT-PCR结果显示CsA下调K562/A02细胞mdr1mRNA和H2AX mRNA的表达(P<0.05);流式细胞仪检测结果显示CsA作用K562/A02细胞72h后使P-gp表达下降(17.8±1.5)%(P<0.05),H2AX蛋白表达下降(13.3±1.7)%(P<0.05);0.3×10-6g/L的抗体即可加剧ADM造成的双链DNA损伤,代表性的指标尾矩和尾DNA%明显增高。结论抗γH2AX抗体阻止双链DNA损伤后修复;CsA可抑制H2AX的表达而减少DNA损伤后修复,从而增加K562/A02细胞对ADM的敏感性起逆转耐药的作用;此外,CsA尚可下调mdr1mRNA、P-gp表达减少、细胞内药物溢出而逆转耐药。

Objective To explore the reversing mechanism of muhidrug resistance of ciclosporin （CsA） on K562/A02 cell line, attenuating DNA damage repair through H2AX suppression. Methods K562 and K562/A02 respectively co - cultured with adriamycin （ADM） of different concentrations and CsA. MTT assay was employed to determine the inhibitory concentration of 50 percent （ IC50 ）, the resistance times and the reversal times. The K562/A02 cells treated with CsA, and reverse transcfiptase （RT）-PCR technique was used to examine the mdrl and H2AX mRNA level. Flow cytometry was used to measure P - glycoprotein（ P - gp） and H2AX expression. Neutral comet assay was used to detect the level of DNA double strands break （DSBs） of the K562/A02 treated with γH2AX antibody. Results 2 × 10^-3 g/L CsA could increase the sensitivity of K562/A02 to ADM, and the reversal times was 5.28. Mdrl mRNA level and H2AX mRNA level were decreased when treated with CsA （ P 〈 0.05 ）. P - gp and H2AX expression was significantly reduced by （ 17.8 ± 1.5 ） % （ P 〈 0.05 ） and （ 13.3± 1.7 ） % （ P 〈 0.05 ） in K562/A02 cells treated with CsA. The level of DSBs increased significantly when treated with ADM and 0.3 × 10^ -6g/L antibody than that with ADM alone, and the tail moment and tail DNA% increased. Conclusions Phospho - Histone H2AX （ Ser139 ） antibody can reduce the repair of DSBs, and CsA can suppress the expression of H2AX to decrease the repair of DSBs to reverse the drug resistance. The reversing mechanism of CsA also includes supressing the expression and function of P - gp to reduce the drug overflow from cells.