Dong Xing-Duo, Lu Qisi, Li Yi-Dong, Cai Chao-Yun, Teng Qiu-Xu, Lei Zi-Ning, Wei Zeng-Hui, Yin Fan, Zeng Leli, Chen Zhe-Sheng
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY 11439, USA.
Department of Hematology, Foresea Life Insurance Guangzhou General Hospital, Guangzhou 515500, Guangdong Province, China.
J Transl Int Med. 2024 Jul 27;12(3):288-298. doi: 10.2478/jtim-2024-0011. eCollection 2024 Jun.
Overcoming ATP-binding cassette subfamily G member 2 (ABCG2)-mediated multidrug resistance (MDR) has attracted the attention of scientists because one of the critical factors resulting in MDR in cancer is the overexpression of ABCG2. RN486, a Bruton's Tyrosine Kinase (BTK) inhibitor, was discovered to potentially reverse ABCB1-mediated MDR. However, there is still uncertainty about whether RN486 has a reversal off-target impact on ABCG2-mediated MDR.
MTT assay was used to detect the reversal effect of RN486 on ABCG2-overexpressing cancer cells. The ABCG2 expression level and subcellular localization were examined by Western blotting and immunofluorescence. Drug accumulation and eflux assay and ATPase assay were performed to analyze the ABCG2 transporter function and ATPase activity. Molecular modeling predicted the binding between RN486 and ABCG2 protein.
Non-toxic concentrations of RN486 remarkably increased the sensitivity of ABCG2-overexpressing cancer cells to conventional anticancer drugs mitoxantrone and topotecan. The reversal mechanistic studies showed that RN486 elevated the drug accumulation because of reducing the eflux of ABCG2 substrate drug in ABCG2-overexpressing cancer cells. In addition, the inhibitory efect of RN486 on ABCG2-associated ATPase activity was also verified. Molecular docking study implied a strong binding afinity between RN486 and ABCG2 transporter. Meanwhile, the ABCG2 subcellular localization was not altered by the treatment of RN486, but the expression level of ABCG2 was down-regulated.
Our studies propose that RN486 can antagonize ABCG2-mediated MDR in cancer cells via down-regulating the expression level of ABCG2 protein, reducing ATPase activity of ABCG2 transporter, and inhibiting the transporting function. RN486 could be potentially used in conjunction with chemotherapy to alleviate MDR mediated by ABCG2 in cancer.
克服ATP结合盒亚家族G成员2（ABCG2）介导的多药耐药（MDR）已引起科学家的关注，因为导致癌症多药耐药的关键因素之一是ABCG2的过表达。布鲁顿酪氨酸激酶（BTK）抑制剂RN486被发现可能逆转ABCB1介导的多药耐药。然而，关于RN486对ABCG2介导的多药耐药是否有逆转脱靶效应仍存在不确定性。
采用MTT法检测RN486对过表达ABCG2的癌细胞的逆转作用。通过蛋白质免疫印迹法和免疫荧光法检测ABCG2的表达水平和亚细胞定位。进行药物积累与外排试验以及ATP酶试验，以分析ABCG2转运蛋白功能和ATP酶活性。分子模拟预测RN486与ABCG2蛋白之间的结合。
无毒浓度的RN486显著提高了过表达ABCG2的癌细胞对传统抗癌药物米托蒽醌和拓扑替康的敏感性。逆转机制研究表明，RN486增加了药物积累，原因是减少了过表达ABCG2的癌细胞中ABCG2底物药物的外排。此外，还证实了RN486对ABCG2相关ATP酶活性的抑制作用。分子对接研究表明RN486与ABCG2转运蛋白之间具有很强的结合亲和力。同时，RN486处理未改变ABCG2的亚细胞定位，但下调了ABCG2的表达水平。
我们的研究表明，RN486可通过下调ABCG2蛋白的表达水平、降低ABCG2转运蛋白的ATP酶活性以及抑制转运功能来拮抗癌细胞中ABCG2介导的多药耐药。RN486可能潜在地与化疗联合使用，以缓解癌症中由ABCG2介导的多药耐药。
