环孢菌素D衍生物PSC833逆转K562/A02细胞多药耐药的研究

Reversal of multidrug-resistance in human leukemia cell line K562/A02 by a cyclosporin D analogue PSC 833

目的 证实PSC 833逆转剂的高效性 ,探讨PSC 833逆转肿瘤细胞多药耐药的机制。方法 以人红白血病细胞系K5 6 2及其耐药细胞系K5 6 2 /A0 2 (耐阿霉素 )为实验研究对象 ,采用MTT法检测细胞毒性 ;直接免疫荧光测定法检测P糖蛋白 (P gp)表达水平 ;RT PCR法检测mdr1mRNA水平 ;以流式细胞术测定两种细胞系内柔红霉素 (DNR)的潴留来反映P gp的外排功能。结果 与K5 6 2细胞系相比 ,K5 6 2 /A0 2耐药细胞系mdr1mRNA及P gp高表达 ,DNR潴留减少。 1μmol/L的PSC 833对K5 6 2 /A0 2细胞的mdr1mRNA及P gp表达水平无明显影响 (P >0 .0 5 ) ,PSC 833对K5 6 2 /A0 2细胞的DNR细胞毒性有剂量依赖性增敏作用 ,其增敏作用至少是环孢菌素A(CsA)、维拉帕米 (Ver)的 3倍。PSC 833能增加K5 6 2 /A0 2耐药细胞系的DNR潴留。 1μmol/L的PSC 833能使K5 6 2 /A0 2细胞内DNR潴留量恢复至K5 6 2细胞的 10 0 .9% ,而 10 μmol/LCsA只恢复至K5 6 2细胞的 86 .9% ,PSC 833对K5 6 2细胞系的DNR细胞毒性及DNR潴留均无明显影响 (P >0 .0 5 )。结论 PSC 833较CsA、Ver逆转活性至少高 3～10倍 ,其逆转K5 6 2 /A0 2多药耐药的机制可能是通过抑制P gp功能 ,而非直接下调mdr1mRNA及P

Objective To explore the efficacy of PSC 833 on multidrug resistance (MDR) reversal and its mechanism. Methods Human erythroleukemic cell line K562 and its doxorubicin resistant counterpart K562/A02 were used in the study. Cytotoxicity was assessed by MTT assay, P gp expression by direct immunofluorescence and mdr1 mRNA expression by reverse transcriptase polymerase chain reaction (RT PCR) with β actin as internal control. Intracellular DNR retention was measured with flow cytometry. Results K562/A02 cells displayed high levels of mdr1 mRNA and P glycoprotein and reduced DNR retention compared to their parental K562 cells. 1 μmol/L of PSC 833 had no effect on the levels of mdr1 mRNA and P gp expression in K562/A02 cells (P>0.05). PSC 833 conferred a dose dependent increase on chemosensitivity of K562/A02 to DNR, and its effect was at least 3 fold more potent than that of CsA or Ver. PSC 833 could increase DNR retention in K562/A02 cells. A 100.9% restoration of intracellular DNR retention of the level of K562 cells was gained by PSC 833 at 1.0 μmol/L in K562/A02 cells, whereas only a 86.9% restoration of DNR retention was obtained by CsA at 10 μmol/L in the K562/A02 cells. No effect on DNR sensitivity and retention was found in K562 cells (P>0.05). Conclusion PSC 833 is at least 3～10 fold more potent than CsA or Ver with respect to MDR reversing activity, and it may function by inhibiting the function of P gp and not reducing the levels of mdr1 mRNA and P gp directly.