β-胡萝卜素通过选择性调节人 P-糖蛋白功能逆转多药耐药癌细胞。
β-carotene reverses multidrug resistant cancer cells by selectively modulating human P-glycoprotein function.
机构信息
Department of Pharmacy, College of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan 40402, R.O.C.
Department of Pharmacy, College of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan 40402, R.O.C.; Department of Pharmacy, China Medical University Hospital, 2 Yude Road, Taichung, 40447, Taiwan, R.O.C.
出版信息
Phytomedicine. 2016 Mar 15;23(3):316-23. doi: 10.1016/j.phymed.2016.01.008. Epub 2016 Feb 6.
BACKGROUND
The issue of multidrug resistance (MDR) cancer is one of the major barriers to successful chemotherapy treatment. The ATP-binding cassette (ABC) efflux transporters play an important role in the chemotherapeutic failure. Several generations of ABC efflux transporter inhibitors have been developed, however, none of them could provide better clinical outcome due to systemic toxicities and significant drug-drug interactions. Therefore, the present study focused on identifying the effect of the natural carotenoid on ABC transporters and may provide a safer choice to defeat MDR cancer.
PURPOSE
The aim of the present study was to evaluate the inhibitory potency of β-carotene on the ABC efflux transporters, as well as the reversal effect of β-carotene toward MDR cancers. The underlying molecular mechanisms and inhibitory kinetics of β-carotene on the major ABC efflux transporter, P-glycoprotein, were further investigated.
METHODS
The human P-gp (ABCB1/Flp-In(TM)-293), MRP1 (ABCC1/Flp-In(TM)-293) and BCRP (ABCG2/Flp-In(TM)-293) stable expression cells were established by using the Flp-In(TM) system. The cytotoxicity of β-carotene was evaluated by MTT assay in the established cell lines, sensitive cancer cell lines (HeLaS3 and NCI-H460) and resistant cancer cell lines (KB-vin and NCI-H460/MX20). Surface protein detection assay and eFluxx-ID Green Dye assay were applied for confirmation of surface expression and function of the transporters. The transporter inhibition potency of β-carotene was evaluated by calcein-AM uptake assay and mitoxantrone accumulation assay. Further interaction kinetics between β-carotene and P-gp were analyzed by rhodamine123 and doxorubicin efflux assay. The influence of β-carotene on ATPase activity was evaluated by Pgp-Glo(TM) Assay System.
RESULTS
Among the tested ABC efflux transporters, β-carotene significantly inhibited human P-gp efflux function without altering ABCB1 mRNA expression. Furthermore, β-carotene stimulated both P-gp basal ATPase activity and the verapamil-stimulated P-gp ATPase activity. In addition, β-carotene exerted partially inhibitory effect on BCRP efflux function. The combination of β-carotene and chemotherapeutic agents significantly potentiated their cytotoxicity in both cell stably expressed human P-gp (ABCB1/Flp-In(TM)-293) and MDR cancer cells (KB-vin and NCI-H460/MX20).
CONCLUSION
The present study indicated that β-carotene may be considered as a chemo-sensitizer and regarded as an adjuvant therapy in MDR cancer treatment.
背景
多药耐药性（MDR）癌症是化疗治疗成功的主要障碍之一。ATP 结合盒（ABC）外排转运体在化疗失败中起着重要作用。已经开发了几代 ABC 外排转运体抑制剂，但是由于全身毒性和明显的药物相互作用，它们都没有提供更好的临床结果。因此，本研究集中于鉴定天然类胡萝卜素对 ABC 转运体的作用，并可能提供更安全的选择来战胜 MDR 癌症。
目的
本研究旨在评估 β-胡萝卜素对 ABC 外排转运体的抑制能力，以及 β-胡萝卜素对 MDR 癌症的逆转作用。还进一步研究了 β-胡萝卜素对主要 ABC 外排转运蛋白 P-糖蛋白的潜在分子机制和抑制动力学。
方法
使用 Flp-In（TM）系统建立了人 P-糖蛋白（ABCB1/Flp-In（TM）-293），MRP1（ABCC1/Flp-In（TM）-293）和 BCRP（ABCG2/Flp-In（TM）-293）稳定表达细胞。通过 MTT 测定法在建立的细胞系，敏感癌细胞系（HeLaS3 和 NCI-H460）和耐药癌细胞系（KB-vin 和 NCI-H460/MX20）中评估了 β-胡萝卜素的细胞毒性。表面蛋白检测测定法和 eFluxx-ID 绿色染料测定法用于确认转运体的表面表达和功能。通过 calcein-AM 摄取测定法和米托蒽醌积累测定法评估 β-胡萝卜素的转运体抑制能力。通过罗丹明 123 和阿霉素外排测定法进一步分析 β-胡萝卜素与 P-糖蛋白之间的相互作用动力学。通过 Pgp-Glo（TM）测定系统评估了 β-胡萝卜素对 ATP 酶活性的影响。
结果
在所测试的 ABC 外排转运体中，β-胡萝卜素显著抑制了人 P-糖蛋白的外排功能，而不改变 ABCB1 mRNA 表达。此外，β-胡萝卜素刺激了 P-糖蛋白的基础 ATP 酶活性和维拉帕米刺激的 P-糖蛋白 ATP 酶活性。另外，β-胡萝卜素对 BCRP 的外排功能表现出部分抑制作用。β-胡萝卜素与化学治疗药物联合使用可显著增强它们在稳定表达人 P-糖蛋白（ABCB1/Flp-In（TM）-293）和 MDR 癌细胞（KB-vin 和 NCI-H460/MX20）中的细胞毒性。
结论
本研究表明，β-胡萝卜素可被视为化疗增敏剂，并可作为 MDR 癌症治疗的辅助治疗。
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