瓦他拉尼对乳腺癌耐药蛋白介导的肿瘤多药耐药的逆转研究  

Reversal effect of vatalanib on BCRP-mediated multidrug resistance

作　　者：张志强[1] 魏寅祥[2] 赵青[2] 任志广[2] 彭晖[2] 李鹏[1] 夏笠 许建华[1] 

机构地区：[1]福建医科大学药学院,福建医科大学新药研究所,福建省天然药物药理学重点实验室,福建福州350004 [2]军事医学科学院卫生学环境医学研究所军事环境药学研究室,天津300050

出　　处：《中国药理学通报》2014年第6期774-782,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 30873083,81173082,81173096,81202560);国家科技部“重大新药创制”科技重大专项“十二五”计划基金资助项目(No 2012ZX09103-101-028);福建省中青年教师教育科研基金资助项目(No JA13151);福建医科大学重大科研基金资助项目(No 09ZD006)

摘　　要：目的观察新型激酶抑制剂类抗癌药瓦他拉尼(vatalanib)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。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.

关 键 词：激酶抑制剂 瓦他拉尼 多药耐药 ABC转运蛋白 乳腺癌耐药蛋白 逆转剂 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R394.2[医药卫生—基础医学]