Robey R W, Medina-Pérez W Y, Nishiyama K, Lahusen T, Miyake K, Litman T, Senderowicz A M, Ross D D, Bates S E
Developmental Therapeutics Department, Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Clin Cancer Res. 2001 Jan;7(1):145-52.
We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.
我们试图描述黄酮哌啶醇与ATP结合盒（ABC）转运蛋白家族成员之间的相互作用。过表达多药耐药蛋白1（MDR-1）和多药耐药相关蛋白（MRP）的细胞未表现出明显的黄酮哌啶醇耐药性，而过量表达ABC半转运蛋白ABCG2（MXR/BCRP/ABCP1）的细胞系则对黄酮哌啶醇具有耐药性。浓度为10微摩尔的黄酮哌啶醇能够阻止MRP介导的钙黄绿素外排，而在高达100微摩尔的黄酮哌啶醇浓度下，P糖蛋白介导的罗丹明123转运不受影响。为了确定对黄酮哌啶醇耐药的假定机制，我们将人乳腺癌细胞系MCF-7暴露于逐渐增加浓度的黄酮哌啶醇中。由此产生的耐药亚系MCF-7 FLV1000在1000纳摩尔黄酮哌啶醇中维持培养，发现其对黄酮哌啶醇具有24倍的耐药性，并且对米托蒽醌（675倍）、拓扑替康（423倍）和伊立替康的活性代谢物SN-38（950倍）具有高度交叉耐药性。由于这种交叉耐药模式与报道的ABCG2过表达细胞的模式一致，因此在存在5微摩尔ABCG2抑制剂夫西地酸C（FTC）的情况下重复进行细胞毒性研究，MCF-7 FLV1000细胞对黄酮哌啶醇、米托蒽醌、SN-38和拓扑替康的敏感性得以恢复。进行了米托蒽醌外排研究，发现高水平的FTC可逆性米托蒽醌外排。Northern印迹和PCR分析显示ABCG2基因过表达。蛋白质印迹证实了ABCG2的过表达；未检测到P糖蛋白或MRP的过表达。这些结果表明ABCG2在对黄酮哌啶醇的耐药性中起作用。
