Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

中文翻译：

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ERK5-IN-1逆转ABCB1相关的多药耐药性

抑制ABC转运蛋白被认为是规避多药耐药性（MDR）的最有效方法。在本研究中，我们评估了ERK5-IN-1（一种有效的细胞外信号调节激酶5（ERK5）抑制剂）的MDR调节潜力。通过MTT法评估ERK5-IN-1的细胞毒性和MDR逆转作用。接种KBv200的裸鼠异种移植模型用于体内研究。通过流式细胞术测量阿霉素的外排和积累。ABCB1活性的调制通过比色ATPase测定和[125I]-碘芳基叠氮吡唑嗪（IAAP）光标记测定进行测量。通过PCR和/或Western印迹测定ERK5-IN-1对ABCB1及其下游标志物表达的影响。通过流式细胞仪和免疫荧光测试了ABCB1的细胞表面表达和亚细胞定位。我们的结果表明，ERK5-IN-1分别显着提高了长春新碱，紫杉醇和阿霉素在KBv200，MCF7 / adr和HEK293 / ABCB1细胞中的敏感性。在相应的药物敏感性亲本细胞系中未发现这种作用。此外，体内联合研究表明，ERK5-IN-1可有效增强紫杉醇在KBv200异种移植物中的抗肿瘤活性，而不会引起其他毒性。从机理上讲，ERK5-IN-1通过直接抑制ABCB1的外排功能来依赖性地增加阿霉素的细胞内积累。ERK5-IN-1刺激ABCB1 ATPase活性，并以浓度依赖的方式抑制[125I]-碘芳基叠氮吡唑嗪（IAAP）掺入ABCB1中。此外，ERK5-IN-1处理既不会改变ABCB1的表达水平，也不会阻断下游Akt或Erk1 / 2的磷酸化。在ABCG2，ABCC1，MRP7和LRP介导的耐药性上未观察到明显的逆转作用。总体而言，这些结果表明，ERK5-IN-1通过竞争性抑制ABCB1药物外排功能，有效逆转了ABCB1介导的MDR。使用ERK5-IN-1恢复对化疗的敏感性或预防耐药可能是癌症患者的潜在治疗策略。