背景:多药耐药性是非小细胞肺癌化疗药物的固有弱点,在全世界范围内频繁发生。临床上,人参和中药处方,包括人参,由于其补气的特点,通常用作抗肿瘤的辅助剂,能提高化疗药物的疗效,减少其毒副作用。三萜皂苷是人参中的关键活性成分,人参皂苷Rb1的含量最高。然而,人参皂苷Rb1逆转肿瘤耐药的作用及机制研究尚不明确。
目的:本研究旨在系统评价人参皂苷Rb1对A549/DDP细胞顺铂不敏感的逆转活性,并揭示其前瞻性分子机制。
方法:MTT法评价人参皂苷Rb1对A549/DDP细胞顺铂不敏感的逆转作用,并通过建立BALB/c-nu小鼠A549/DDP皮下移植瘤模型研究了其行为。此外,P-gpATP酶活性测定,顺铂积累试验,膜联蛋白V-FITC凋亡检测,实时qPCR分析和蛋白质印迹分析用于阐明潜在的机制。
结果:人参皂苷Rb1可在体内外有效逆转A549/DDP的顺铂耐药。人参皂苷Rb1联合顺铂治疗后,顺铂在A549/DDP细胞中的积累增加,伴随着ABCB1,SHH的mRNA和蛋白表达水平的下调,PTCH1和GLI2。此外,通过对Bax和Bcl-2蛋白表达水平的相对调节,提高了顺铂诱导细胞凋亡的能力。更重要的是,CYP3A4mRNA和蛋白水平变化不显著。
结论:人参皂苷Rb1可增加细胞内顺铂的浓度,改善A549/DDP细胞对顺铂的不敏感性。甚至更好,人参皂苷Rb1+顺铂联合用药后,CYP3A4介导的药代动力学相互作用可能没有预见性.人参皂苷Rb1可能是A549/DDP细胞顺铂不敏感的逆转剂,具有通过靶向ABCB1和Hedgehog(Hh)途径抑制两种药物泵(P-gp和PTCH1)的外排的双重功能。总的来说,这项研究为开发NSCLC顺铂不敏感的新逆转剂奠定了基础.
BACKGROUND: The multi-drug resistance is an inherent weakness in the chemotherapeutics of non-small cell lung cancer occurring frequently all over the world. Clinically, ginseng and Chinese medicinal prescriptions including ginseng usually used as anti-tumor adjuncts due to its characteristic of qi-invigorating, which could improve the curative effect of chemotherapy drugs and reduce their toxic side effects. Triterpenoid saponins are the crucial active ingredients in Panax ginseng, and Ginsenoside Rb1 is of the highest quantities. However, the research on the tumor drug-resistance reversal effect and mechanism of ginsenoside Rb1 is still not clear.
OBJECTIVE: This study aimed to systematically estimate the reversal activity of Ginsenoside Rb1 on cisplatin-insensitivity of A549/DDP cells and to reveal its prospective molecular mechanism.
METHODS: MTT assay were conducted to evaluate the reversal activity on cisplatin-insensitivity of A549/DDP cells of Ginsenoside Rb1in vitro, and the behavior was also studied by establishing a subcutaneous transplanted tumor model of A549/DDP in BALB/c-nu mice. In addition, P-gp ATPase activity assay, cisplatin accumulation assay, Annexin V-FITC apoptosis assay, real-time qPCR analysis and western blotting analysis were used to clarify the potential mechanism.
RESULTS: Ginsenoside Rb1 could effectively reverse the cisplatin-resistance of A549/DDP in vitro and vivo. And after the co-treatment of Ginsenoside Rb1 plus cisplatin, the accumulation of cisplatin increased in A549/DDP cells, which was accompanied with the down-regulation of the mRNA and protein expression levels of ABCB1, SHH, PTCH1 and GLI2. Besides, the apoptosis-inducing ability of cisplatin improved by the relative regulation on the protein expression level of Bax and Bcl-2. Far more importantly, the changes of CYP3A4 mRNA and protein levels were not significant.
CONCLUSIONS: Ginsenoside Rb1 could increase the concentration of intracellular cisplatin and improve the insensitivity for cisplatin on A549/DDP cells. Even better, there was perhaps no unpredictable CYP3A4-mediated pharmacokinetic interactions after the combination of Ginsenoside Rb1 plus cisplatin. Ginsenoside Rb1 was a probable reversal agent for the cisplatin-insensitivity of A549/DDP cells, with a bifunction of inhibiting the efflux of two drug pumps (P-gp and PTCH1) by targeting ABCB1 and Hedgehog (Hh) pathway. In general, this research laid the groundwork for the development of a new reversal agent for the cisplatin-insensitivity of NSCLC.