背景:多药耐药(MDR)限制了成功的癌症化疗。P-糖蛋白(P-gp),BCRP和MRP1是MDR的关键触发因素。不幸的是,迄今为止,FDA尚未批准MDR调节剂.这里,我们将研究BI-2865,一种泛KRAS抑制剂,逆转P-gp诱导的MDR,BCRP和MRP1在体外和体内,及其逆转机制将被探索。
方法:通过MTT法检测BI-2865的细胞毒性及其体外MDR去除效果,并通过P-gp介导的小鼠KBv200异种移植物评估体内相应的逆转功能。通过荧光多柔比星流式细胞术实验估计BI-2865诱导的细胞内药物释放和保留的改变,并通过LC-MS分析异种移植物肿瘤中的化疗药物积累。BI-2865抑制P-gp底物流出的机制通过钒酸敏感的ATP酶测定进行分析,[125I]-IAAP-光标记测定和计算机分子对接。通过蛋白质印迹法检测BI-2865对P-gp表达和KRAS下游信号的影响。流式细胞术和/或qRT-PCR。通过免疫荧光观察P-gp的亚细胞定位。
结果:我们发现BI-2865显著增强了P-gp驱动的MDR癌细胞对包括紫杉醇在内的化疗药物的反应,长春新碱和阿霉素,而在其亲代敏感细胞和BCRP或MRP1驱动的MDR细胞中未观察到这种作用。重要的是,小鼠体内联合研究证实,BI-2865可有效提高紫杉醇的抗肿瘤作用,且无毒性损伤。在机制上,BI-2865通过直接阻断P-gp的外排功能促使阿霉素在癌细胞中积累,更具体地说,通过BI-2865竞争性结合P-gp的药物结合位点来实现。更重要的是,在有效的MDR逆转浓度下,BI-2865既不改变P-gp的表达和位置,也不降低其下游AKT或ERK1/2信号活性。
结论:本研究揭示了BI-2865作为MDR调节剂的新应用,这可能被用来有效地,安全和特异性地提高临床P-gp介导的MDR难治性癌症的化疗疗效。
BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored.
METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts\' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate\'s efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence.
RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What\'s more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity.
CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.