Investigation of expression regulatory mechanisms of drug transporters with its possible application for circumventing anticancer drug resistance

ABCG2/BCRP是ATP结合盒转运体G家族的成员，其功能是一种外排泵，可以跨细胞膜排除米托蒽醌、SN-38(伊立替康的活性代谢物)和拓扑替康等抗癌药物。因此，当ABCG2在癌细胞中过度表达时，会导致多药耐药。到目前为止，我们已经明确了雌激素以转录后的方式显著下调雌激素受体阳性的乳腺癌细胞中ABCG2的表达，但由于雌激素原有的生理功能，雌激素在临床上的应用似乎受到了限制。为了探索影响ABCG2表达水平的小分子，我们将ABCG2基因导入乳腺癌MCF-7细胞和胃癌MKN1、NCI-N87细胞，以探索影响ABCG2表达水平的小分子，命名为MCF-7/ABCG2、MKN1/ABCG2和NCI-N87/ABCG2细胞。外源ABCG2蛋白在MCF-7/ABCG2、MKN1/ABCG2、NCI-N87/ABCG2细胞中表达P44/p42丝裂原激活…对NCI-N87/ABCG2细胞的抑制作用蛋白激酶(MAPK)、PD98059和U0126的表达呈剂量依赖关系。这些化合物几乎完全克服了MCF-7/ABCG2和NCI-N87/ABCG2细胞对米托蒽醌或SN-38的耐药性。FACS分析表明，逆转作用是由于细胞内抗癌药物摄取增加所致。定量RT-PCR分析表明，PD98059或U0126处理不影响ABCG2mRNA水平。此外，在PD98059存在下，ABCG2蛋白的半衰期明显短于对照组。泛素/内体抑制剂MG132不影响PD98059介导的ABCG2蛋白的降解，但内涵体抑制剂巴菲罗星A1可完全阻断其降解。这些结果表明，抑制p44/p42 MAPK通路可能加速ABCG2蛋白的内体降解，从而使克服ABCG2介导的多药耐药成为可能。这些数据也提示，p44/p42 MAPK抑制剂可能有助于建立安全有效的化疗方案。较少

ABCG2/BCRP, a member of the ATP-binding cassette transporter G family, functions as an efflux pump that excludes such anticancer agents as mitoxantrone, SN-38 (an active metabolite of irinotecan), and topotecan, across cell membrane. Accordingly ABCG2 causes multidrug resistance when overexpressed in cancer cells. We have thus far clarified that estrogen markedly down-regulates ABCG2 expression in estrogen receptor-positive breast cancer cells in the post-transcriptional manner, but use of estrogen in the practical clinic seemed to be limited due to its original physiological function.We then transfected ABCG2 cDNA to breast cancer MCF-7 cells and gastric cancer MKN1 and NCI-N87 cells, which express endogenous ABCG2, in order to exploring small molecules that affects ABCG2 expression levels, and termed them MCF-7/ABCG2, MKN1/ABCG2, and NCI-N87/ABCG2 cells.Exogenous ABCG2 protein expression in MCF-7/ABCG2, MKN1/ABCG2, NCI-N87/ABCG2 cells was markedly repressed by p44/p42 mitogen-activat … More ed protein kinase (MAPK), PD98059 and U0126, in the dose-dependent manner. These compounds almost completely overcame mitoxantrone- or SN-38- resistance of MCF-7/ABCG2 and NCI-N87/ABCG2 cells. FACS analyses revealed that the reversal effects were due to increased intracellular uptake of anticancer agents. Quantitative RT-PCR analyses demonstrated that ABCG2 mRNA levels were not affected by the treatment with PD98059 or U0126. In addition, a half life of the ABCG2 protein was significantly short in the presence of PD98059 as compared with that in the control experiment.PD98059-mediated degradation of ABCG2 protein was not affected by MG132, an ubiquitin/endosome inhibitor, but was completely blocked by bafilomycin A1, an endosomal inhibitor. These data suggest that inhibition of p44/p42 MAPK pathway may accelerate endosomal degradation of ABCG2 protein and makes it possible to overcome ABCG2-mediated multidrug resistance. These data may also suggest that p44/p42 MAPK inhibitors may serve for establishment of safe and effective chemotherapeutic regimen. Less