肠胃清对结肠癌细胞草酸铂药代动力学的影响

目的：研究肠胃清逆转人结肠癌耐草酸铂（oxaliplatin,L—OHP）细胞（HCT116／L—OHP）的耐L—OHP作用,并从药代动力学角度探讨其逆转耐药作用与细胞内铂含量,细胞摄入、排出L—OHP以及铜转运蛋白等的关系。方法：以四甲基偶氮唑盐比色法确定人结肠癌耐药细胞株的耐药性,明确药物血清对HCT116／L-0HP细胞耐L-OHP的逆转作用;采用原子吸收分光光度法测定细胞中铂的浓度,并检测加入药物血清后细胞内铂含量以及L—OHP摄入和排出的变化;采用蛋白质印迹法检测铜转运相关蛋白人铜转运蛋白1（humancoppertransporter1,hCTRl）、铜转运蛋白0t链（ATPaseCu”transportingalphapolypeptide,ATP7A＇）、P型铜转运ATP酶（copper—transportingP—typeadenosinetriphosphatase,ATP7B）、谷胱甘肽S一转移酶n（glutathioneS—transferase-π,GST-π）和多药耐药相关蛋白MRP2）的表达。结果：7．5％药物血清联合L—OHP对HCTll6／L—OHP细胞作用后,HCT116／L-OHP细胞对L—OHP的敏感性增加,半数抑制浓度由89μg／mL下降到32．45μg／mL,逆转指数达到2．74。HCTll6／L—OHP细胞内铂含量比HCT116细胞株降低;HCT116和HCT116／L—OHP细胞株经过7．50A中药血清联合L—OHP处理后,胞内铂-DNA含量比单独L—OHP处理细胞增加。肠胃清药物血清可剂量依赖性地增加人结肠癌细胞HCT116及HCT116／L—OHP对L—OHP的吸收,同时抑制其排出。肠胃清药物血清联合L—OHP处理后HCT116／L-0HP细胞中ATP7A、ATP7B表达量相对于单独L—OHP处理者减少,hCTR1蛋白表达增多,但对GST-π、MRP2蛋白的表达无明显影响。结论：肠胃清通过上调hCTR1蛋白的表达,下调ATP7A、ATP7B蛋白的表达,促进HCT116和HCT116／L—OHP细胞对L—OHP的摄入,抑制其对L—OHP的排出,增加细胞内铂-DNA加合物含量,从而逆转其耐药株HCT116／L—OHP对L-OHP的耐药性。

Effects of medicated serum prepared with Chinese herbal medicine Changweiqing on pharmacokinetics of oxaliplatin in colon cancer cells

OBJECTIVE： To investigate the effects of Changweiqing-medicated serum, which was prepared with a compound traditional Chinese herbal medicine, on the reversal of oxaliplatin （L-OHP） resistance and the relationshipbetween the reversal and cellular accumulation of platinum and proteins associated with copper transporter in HCT116/L-OHP cells. METHODS. For clarifying the reversal effect of Changweiqing, methyl thiazolyl tetrazolium was applied to determine the L-OHP resistance of HCT]16/L-OHP cell line. The relationship between the cellular accumula- tion of platinum and the L-OHP resistance in HCTll6/L-OHP cells, and the effects of drug-medicated serum on intracellular contents of platinum were detected by atomic absorption spectrophotometry. Western blot method was used to determine the expressions of human copper transporter ] （hCTR]）, ATPase Cu2＋ transporting alpha polypeptide （ATP7A）, copper-transporting P-type adenosine triphosphatase （ATP7B）, giutathione S- transferase-n （GST-n） and multidrug resistance-associated protein 2 （MRP2）. RESULTS= The inhibitory concentration 50% values of different pairs of L-OHP-sensitive and -resistant cells were 7.2 and 89.00. The resistance index of HCT116/L-OHP cells was 12.36. The reverse index of drug serum on HCT116/L-OHP cells was 2.74. The platinum content in HCT116/L-OHP cells was decreased com- pared with HCT1]6 cells in condition of 7.2 μg/mL L-OHP. After treating by 7.5% Changweiqing-medicated serum, the intracellular platinum contents in L-OHP-sensitive and -resistant cells were increased. It was dose-dependent that drug-medicated serum promoted the uptake of L-OHP by HCT116 or HCT116/L-OHP cells and inhibited the discharge. The 7. 5% Changweiqing-medicated serum increased the expression of hCTR1 and decreased the expressions of ATP7A and ATP7B in HCT116/L-OHP cells, but had no effects on GST-n and MRP2 protein expressions. CONCLUSION. Changweiqing can reverse the L-OHP resistance of HCT116/L-OHP by increasing the cellular piatinum-DNA accumulation. Down-regulation of expression of ATPTB and ATP7A, and up-regulation of hCTR1 may cause the increase of intracellular platinum content in HCT116/L-OHP cells.