State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, 510060, People's Republic of China.
Department of Gastrointestinal surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, Guangdong, People's Republic of China.
J Exp Clin Cancer Res. 2020 Mar 12;39(1):50. doi: 10.1186/s13046-020-1537-9.
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 [I]-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 [I]-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.
抑制 ABC 转运蛋白被认为是克服多药耐药（MDR）最有效的方法。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，对 MDR 调节的潜力。
通过 MTT 测定法评估 ERK5-IN-1 的细胞毒性和 MDR 逆转作用。使用 KBv200 接种的裸鼠异种移植模型进行体内研究。通过流式细胞术测量阿霉素的外排和积累。通过比色 ATP 酶测定法和 [I]-碘代氮杂唑苯丙嗪（IAAP）光标记测定法测量 ABCB1 活性的调节。通过 PCR 和/或 Western blot 测量 ERK5-IN-1 对 ABCB1 及其下游标志物表达的影响。通过流式细胞术和免疫荧光法测试 ABCB1 的细胞表面表达和亚细胞定位。
我们的结果表明，ERK5-IN-1 显著增加了长春新碱、紫杉醇和阿霉素在 KBv200、MCF7/adr 和 HEK293/ABCB1 细胞中的敏感性，分别。在各自的药物敏感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 有效地增强了紫杉醇在 KBv200 异种移植物中的抗肿瘤活性，而没有引起额外的毒性。在机制上，ERK5-IN-1 直接抑制 ABCB1 的外排功能，剂量依赖性地增加阿霉素的细胞内积累。ERK5-IN-1 以浓度依赖的方式刺激 ABCB1 ATP 酶活性并抑制 [I]-碘代氮杂唑苯丙嗪（IAAP）掺入 ABCB1。此外，ERK5-IN-1 处理既不改变 ABCB1 的表达水平，也不阻断下游 Akt 或 Erk1/2 的磷酸化。在 ABCG2、ABCC1、MRP7 和 LRP 介导的耐药性方面未观察到明显的逆转作用。
总之，这些结果表明，ERK5-IN-1 通过竞争性抑制 ABCB1 药物外排功能有效地逆转了 ABCB1 介导的 MDR。使用 ERK5-IN-1 恢复对化疗的敏感性或预防耐药性可能是癌症患者的一种潜在治疗策略。
