State Key Laboratory of Oncology in South China, Guangdong Esophageal Cancer Institute, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China.
Hubei University of Medicine, Shiyan, Hubei, 442000, China.
J Exp Clin Cancer Res. 2018 Feb 20;37(1):31. doi: 10.1186/s13046-018-0690-x.
ATP-binding cassette subfamily G member 2 (ABCG2), a member of the ABC transporter superfamily proteins, mediates multidrug resistance (MDR) by transporting substrate anticancer drugs out of cancer cells and decreasing their intracellular accumulation. MDR is a major hurdle to successful chemotherapy. A logical approach to overcome MDR is to inhibit the transporter. However, no safe and effective MDR inhibitor has been approved in the clinic.
The MTT assay was used to evaluate cell cytotoxicity and MDR reversal effect. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The H460/MX20 cell xenograft model was established to evaluate the enhancement of anticancer efficacy of topotecan by dacomitinib in vivo. To ascertain the interaction of dacomitinib with the substrate binding sites of ABCG2, the competition of dacomitinib for photolabeling of ABCG2 with [I]- iodoarylazidoprazosin (IAAP) was performed. Vanadate-sensitive ATPase activity of ABCG2 was measured in the presence of a range of different concentrations of dacomitinib to evaluate the effect of dacomitinib on ATP hydrolysis as the energy source of the transporter. A flow cytometry-based assay and western blotting were employed to study whether dacomitininb could inhibit the expression level of ABCG2. The mRNA expression levels of ABCG2 were analyzed by real-time quantitative RT-PCR. The protein expression level of AKT, ERK and their phosphorylations were detected by Western blotting.
Here, we found that dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor (TKI) in phase III clinical trial, could enhance the efficacy of conventional chemotherapeutic agents specifically in ABCG2-overexpressing MDR cancer cells but not in the parental sensitive cells. Dacomitinib was found to significantly increase the accumulation of ABCG2 probe substrates [doxorubicin (DOX),Rhodamine 123 (Rho 123) and Hoechst 33342] by inhibiting the transporter efflux function. Moreover, dacomitinib stimulated ABCG2 ATPase activity and competed with [I]-IAAP photolabeling of ABCG2 in a concentration-dependent manner. However, dacomitinib did not alter ABCG2 expression at protein and mRNA levels or inhibit ErbB downstream signaling of AKT and ERK. Importantly, dacomitinib significantly enhanced the efficacy of topotecan in ABCG2-overexpressing H460/MX20 cell xenografts in nude mice without incurring additional toxicity.
These results suggest that dacomitinib reverses ABCG2-mediated MDR by inhibiting ABCG2 efflux function and increasing intracellular accumulation of anticancer agents. Our findings advocate further clinical investigation of combinations of dacomitinib and conventional chemotherapy in cancer patients with ABCG2-overexpressing MDR tumors.
三磷酸腺苷结合盒亚家族 G 成员 2（ABCG2）是 ABC 转运蛋白超家族蛋白的成员，通过将底物抗癌药物从癌细胞中转运出去并减少其细胞内积累来介导多药耐药（MDR）。MDR 是化疗成功的主要障碍。克服 MDR 的合理方法是抑制转运蛋白。然而，临床上尚未批准任何安全有效的 MDR 抑制剂。
MTT 测定法用于评估细胞细胞毒性和 MDR 逆转作用。通过流式细胞术测量药物外排和细胞内药物积累。建立 H460/MX20 细胞异种移植模型，以评估达克替尼在体内对拓扑替康的抗癌功效的增强作用。为了确定达克替尼与 ABCG2 的底物结合位点的相互作用，用 [I]-碘代氮杂唑嗪（IAAP）对达克替尼进行光标记竞争，以确定达克替尼与 ABCG2 的竞争。在一系列不同浓度的达克替尼存在下测量 ABCG2 的钒酸盐敏感 ATP 酶活性，以评估达克替尼对作为转运体能量来源的 ATP 水解的影响。采用流式细胞术测定和 Western blot 法研究达克替尼是否能抑制 ABCG2 的表达水平。通过实时定量 RT-PCR 分析 ABCG2 的 mRNA 表达水平。通过 Western blot 检测 AKT、ERK 及其磷酸化的蛋白表达水平。
在这里，我们发现达克替尼，一种正在进行 III 期临床试验的不可逆泛-ErbB 酪氨酸激酶抑制剂（TKI），可以特异性增强 ABCG2 过表达的 MDR 癌细胞中常规化疗药物的疗效，但不能增强亲本敏感细胞的疗效。发现达克替尼通过抑制转运体外排功能显著增加 ABCG2 探针底物 [阿霉素（DOX）、罗丹明 123（Rho 123）和 Hoechst 33342]的积累。此外，达克替尼以浓度依赖的方式刺激 ABCG2 ATP 酶活性并与 [I]-IAAP 光标记 ABCG2 竞争。然而，达克替尼并未改变 ABCG2 的蛋白和 mRNA 水平的表达，也未抑制 AKT 和 ERK 的 ErbB 下游信号。重要的是，达克替尼在裸鼠中显著增强了 ABCG2 过表达的 H460/MX20 细胞异种移植中拓扑替康的疗效，而没有增加额外的毒性。
这些结果表明，达克替尼通过抑制 ABCG2 外排功能并增加抗癌药物的细胞内积累来逆转 ABCG2 介导的 MDR。我们的研究结果主张在 ABCG2 过表达的 MDR 肿瘤患者中进一步进行达克替尼与常规化疗联合的临床研究。
