中文摘要：

目的：观察新型激酶抑制剂类抗癌药瓦他拉尼（ vata-lanib）逆转肿瘤细胞多药耐药（ MDR）的效果,研究其对乳腺癌耐药蛋白（ BCRP）、P-糖蛋白（ P-gp）、多药耐药相关蛋白1（MRP1）的作用,探讨其作用机制。方法应用 MTS 法或SRB法检测瓦他拉尼对耐药细胞与敏感细胞的细胞毒性和逆转 MDR 的效果；分别以罗丹明（ Rh-123）、米托蒽醌（ MX）、阿霉素（ ADR）为P-gp、BCRP、MRP1的荧光底物,应用流式细胞术检测该药对P-gp、BCRP、MRP1外排功能的影响；应用Western blot检测该药对耐药细胞中BCRP蛋白表达水平的影响,并研究其对细胞增殖相关信号分子的影响；应用qRT-PCR检测其对耐药细胞中BCRP基因表达水平的影响；通过超速离心法制备BCRP转运蛋白的粗膜,检测瓦他拉尼对BCRP的ATPase活性的影响。结果无毒剂量的瓦他拉尼（5μmol·L-1）可有效逆转BCRP高表达的肿瘤细胞株HEK293/ABCG2对MX、ADR和托泊替康（ TOPT）的耐药,而对P-gp高表达的肿瘤细胞株K562/A02和MRP1高表达的肿瘤细胞株HEK293/ABCC1的耐药无逆转作用。该药能够下调耐药肿瘤细胞的BCRP基因和蛋白的表达水平,并具有时间和剂量依赖效应,但对BCRP外排MX的功能无明显抑制作用。该药可以激活BCRP的ATPase活性,但并不改变BCRP 的构象,对耐药和敏感细胞中AKT 和ERK 的表达及其磷酸化水平也均无明显影响。结论摇瓦他拉尼可有效、特异地逆转BCRP 介导的肿瘤MDR,其机制可能是通过下调耐药肿瘤细胞中BCRP 基因和蛋白的表达来达到逆转 MDR 的效果。

英文摘要：

Aim To investigate the reversal effect of vatalanib, a novel kinase inhibitor, on multidrug re-sistance in cancer cells and its mechanism. Methods The cytotoxicity and reversal effects of vatalanib were evaluated in both resistant and sensitive tumor cell lines by MTS or SRB assays. The intracellular accumu-lation of fluorescence substrates （ Rh-123 , MX and ADR for P-gp, BCRP, MRP1, respectively） were ana-lysed by flow cytometry. Western blot or qRT-PCR was used to determine the protein or mRNA expression level of BCRP. The effect of vatalanib on ATPase activity of BCRP was determined using crude membranes pre-pared from HEK293/ABCG2 cells. Results Vata-lanib at the nontoxic dose （ 5 μmol · L-1 ） potentially reversed BCRP-mediated MDR in cancer cells, howev-er it had no effect on P-gp or MRP1 mediated MDR. Vatalanib did not alter the intracellular accumulation of MX in HEK 2 9 3 / ABCG 2 , and had no influence on the BCRP-mediated drug efflux. The ATPase assay indica-ted that vatalanib may serve as a substrate of BCRP. Furthermore, vatalanib dramatically suppressed levels of both the protein and mRNA expression of BCRP in concentration-and time-dependent manners. However, reversal concentration of vatalanib had no influence on the total and phosphorylated forms of AKT and ERK1/ 2 in resistant cancer cells. Conclusion Vatalanib could significantly reverse BCRP-mediated MDR with specificity, and its mechanism may correlate with the down-regulation levels of BCRP both mRNA and pro-tein in resistant cancer cells.