Liu Wei, Meng Qiang, Sun Yaoting, Wang Changyuan, Huo Xiaokui, Liu Zhihao, Sun Pengyuan, Sun Huijun, Ma Xiaodong, Liu Kexin
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China.
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian,
Cell Physiol Biochem. 2018;51(4):1616-1631. doi: 10.1159/000495650. Epub 2018 Nov 29.
BACKGROUND/AIMS: The emergence of multidrug resistance (MDR) caused by P-glycoprotein (P-gp) overexpression is a serious obstacle to the treatment of chronic myelocytic leukemia. In recent years, some clinical trials have shown that nelfinavir (NFV), a traditional anti-HIV drug, has anti-cancer effects. Some researchers have also shown NFV might be a potential P-gp inhibitor. This study is aimed at investigating whether nelfinavir can act as an MDR-reversal drug and to clarify its molecular mechanism as well.
K562 and K562/ADR cell lines were applied in the study. Cytotoxicity was detected by CCK-8 reagents. Cell apoptosis was detected by flow cytometry and inverted fluorescence microscopy to detect the binding of apoptotic dyes to cells. Western blot was used to detect the expression of proteins. Drug-protein molecular docking simulation by using Sybyl-x 2.0 software.
Non-toxic concentrations of NFV (1.25-5 μM) could reverse Adriamycin (ADR), colchicine, paclitaxel, and imatinib resistance of K562/ADR cells, with reversal indexes of up to 10.8, 7.4, 57, and 9.3, respectively. NFV inhibited P-gp efflux function, as evidenced by the significant increase in the intracellular accumulation of ADR and Rho-123, without affecting P-gp protein and mRNA expression levels. Further ATP content detection and molecular docking simulations showed that NFV could decrease intracellular ATP content and has a high affinity with the active functional regions of P-gp, respectively. When co-administered with ADR, NFV increased intracellular reactive oxygen species as well as blocked the ERK/Akt signaling pathway, leading to cell apoptosis.
NFV inhibited P-gp function, decreased intracellular ATP content, and promoted cell apoptosis in K562/ADR cells, thereby reversing MDR. These findings encourage further animal and clinical MDR studies with a combination therapy consisting of NFV and chemotherapeutic drugs.
背景/目的：由P-糖蛋白（P-gp）过表达引起的多药耐药（MDR）的出现是慢性粒细胞白血病治疗的严重障碍。近年来，一些临床试验表明，传统抗HIV药物奈非那韦（NFV）具有抗癌作用。一些研究人员还表明，NFV可能是一种潜在的P-gp抑制剂。本研究旨在探讨奈非那韦是否可作为MDR逆转药物，并阐明其分子机制。
本研究采用K562和K562/ADR细胞系。用CCK-8试剂检测细胞毒性。通过流式细胞术检测细胞凋亡，并用倒置荧光显微镜检测凋亡染料与细胞的结合情况。用蛋白质免疫印迹法检测蛋白质表达。使用Sybyl-x 2.0软件进行药物-蛋白质分子对接模拟。
无毒浓度的NFV（1.25 - 5 μM）可逆转K562/ADR细胞对阿霉素（ADR）、秋水仙碱、紫杉醇和伊马替尼的耐药性，逆转指数分别高达10.8、7.4、57和9.3。NFV抑制P-gp外排功能，ADR和罗丹明-123细胞内蓄积量显著增加证明了这一点，且不影响P-gp蛋白和mRNA表达水平。进一步的ATP含量检测和分子对接模拟表明，NFV可降低细胞内ATP含量，且与P-gp的活性功能区具有高亲和力。与ADR联合使用时，NFV增加细胞内活性氧物种并阻断ERK/Akt信号通路导致细胞凋亡。
NFV抑制K562/ADR细胞中P-gp功能，降低细胞内ATP含量并促进细胞凋亡，从而逆转MDR。这些发现鼓励进一步开展动物和临床研究，探索NFV与化疗药物联合治疗MDR的方案。
