Kars Meltem Demirel, Iseri Ozlem Darcansoy, Gündüz Ufuk, Ural Ali Ugur, Arpaci Fikret, Molnár József
Middle East Technical University, Department of Biological Sciences, 06531, Ankara, Turkey.
Anticancer Res. 2006 Nov-Dec;26(6B):4559-68.
[corrected] The effectiveness of chemotherapy is limited by the emergence of multidrug resistance (MDR). MDR is caused by the activity of various ATP binding cassette (ABC) transporters that pump anticancer drugs out of the cells in an ATP-dependent manner. Additionally some other cellular mechanisms of MDR have been reported. The purpose of this study was to investigate mechanisms of MDR in drug resistant MCF-7 cell lines and to modulate P-glycoprotein (P-gp) and MRP1-based MDR.
Paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), doxorubicin (MCF-7/Dox) and vincristine (MCF-7/Vinc) resistant sublines were developed from the parent MCF-7 cell line (MCF-7/S) by stepwise selection in dose increments over two years. Flow cytometry, MTT cytotoxicity assay, RT-PCR, caspase-3 activity assay and checkerboard combination assay were performed to investigate the degree of resistance developed in sublines and to reverse drug resistance phenotype.
The flow cytometry histograms of drug accumulation assays demonstrated that the drug-resistant cell lines are P-gp and MRPI positive. RT-PCR results showed that the resistant sublines express both MDR1 and MRP1 genes. Resistance indices of each subline to each anticancer drug were determined using the MTT cytotoxicity assay and it was found that all the sublines were resistant to their respective agents. Caspase-3 activities of the cell lines were also determined. Caspase-3 activity is an important indicator of apoptosis in the cell. The reversal of MDR was attempted by two cinnamylidene ketone and two organosilicon compounds. The results indicated that these compounds modulated P-gp effectively, but they were not very effective at reversing MRP1 activity in the MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and four synthetic MDR modulators [2-(2-methoxycinnamylidene) indan-1-one (cinnamylidene-1), 2-(2- methoxycinnamylidene)-3, 4-dihydro-2H-naphthelen-1-one) (cinnamylidene-2), ALIS 409 and ALIS 421] were applied to the sublines in combination. The fractional inhibitory indices illustrated that combined applications of cinnamylidene ketones and organosilicon compounds with paclitaxel, docetaxel or vincristine exerted significant antiproliferative effects on the resistant sublines.
This report demonstrates the development of rational models for drug resistance MCF-7 cell lines and reversal of acquired drug resistance.
[已修正]化疗的有效性受到多药耐药性（MDR）出现的限制。MDR是由多种ATP结合盒（ABC）转运蛋白的活性引起的，这些转运蛋白以ATP依赖的方式将抗癌药物泵出细胞。此外，还报道了一些其他的MDR细胞机制。本研究的目的是探讨耐药MCF-7细胞系中的MDR机制，并调节基于P-糖蛋白（P-gp）和多药耐药相关蛋白1（MRP1）的MDR。
通过在两年内逐步增加剂量的方式从亲本MCF-7细胞系（MCF-7/S）中筛选出对紫杉醇（MCF-7/Pac）、多西他赛（MCF-7/Doc）、阿霉素（MCF-7/Dox）和长春新碱（MCF-7/Vinc）耐药的亚系。采用流式细胞术、MTT细胞毒性试验、逆转录-聚合酶链反应（RT-PCR）、半胱天冬酶-3活性测定和棋盘组合试验来研究亚系中产生的耐药程度并逆转耐药表型。
药物蓄积试验的流式细胞术直方图表明，耐药细胞系P-gp和MRP1呈阳性。RT-PCR结果显示，耐药亚系同时表达MDR1和MRP1基因。使用MTT细胞毒性试验测定各亚系对每种抗癌药物的耐药指数，发现所有亚系对其各自的药物均有耐药性。还测定了细胞系的半胱天冬酶-3活性。半胱天冬酶-3活性是细胞凋亡的一个重要指标。尝试用两种亚苄基酮和两种有机硅化合物逆转MDR。结果表明，这些化合物能有效调节P-gp，但在逆转MCF-7亚系中的MRP1活性方面效果不太显著。将四种选择性抗癌药物（紫杉醇、多西他赛、阿霉素和长春新碱）和四种合成的MDR调节剂[2-（2-甲氧基亚苄基）茚满-1-酮（亚苄基-1）、2-（2-甲氧基亚苄基）-3,4-二氢-2H-萘-1-酮（亚苄基-2）、ALIS 409和ALIS 421]联合应用于亚系。分数抑制指数表明，亚苄基酮和有机硅化合物与紫杉醇、多西他赛或长春新碱联合应用对耐药亚系具有显著的抗增殖作用。
本报告展示了耐药MCF-7细胞系合理模型的建立以及获得性耐药的逆转。
