乌苏烷三萜类化合物RA逆转的K562/ADM细胞耐药作用及其机制研究

Reversal of Drug Resistance in K562/ADM Cells Caused by RA and the Related Mechanisms

目的:研究乌苏烷三萜类化合物3β,19α-二羟基乌苏-12-烯-23,28-二羧酸(Rotundioic acid,RA)对耐阿霉素的K562细胞(K562/ADM细胞)抗肿瘤药物敏感性的影响,并探讨RA对K562/ADM细胞多药耐药作用的影响及相关机制。方法:采用CCK-8法检测RA对K562细胞及K562/ADM细胞的抗肿瘤药物敏感性的影响;逆转录-聚合酶链式反应(RT-PCR)和Western blot检测K562细胞和K562/ADM细胞MDR1 mRNA和蛋白表达水平,并检测RA对K562/ADM细胞MDR1 mRNA及P-gp表达的影响;Western blot检测K562/ADM细胞p-JNK、p-p38、pERK1/2的表达。结果:RA增加K562/ADM细胞对阿霉素的敏感性,逆转倍数为1.61倍,差异有统计学意义(P<0.05);K562/ADM对ADM的耐药倍数为41.76倍。K562细胞内MDR1 mRNA表达极低,其蛋白产物P-糖蛋白(Pglycoprotein,P-gp)几乎不表达;K562/ADM细胞内的MDR1 mRNA及P-gp高表达;RA能够下调K562/ADM细胞MDR1及P-gp的表达水平。此外,RA可以上调K562/ADM细胞p38、ERK1/2的磷酸化水平,不影响p-JNK的表达。结论:RA可能通过上调K562/ADM细胞p-p38、p-ERK1/2的表达水平参与MAPK信号通路的调节,从而抑制MDR1的转录和翻译水平,最终逆转白血病细胞多药耐药作用。

Objective: To investigate the effect of ursane triterpenoids 3β,19α-dihydroxyursu-12-ene-23,28-dicarboxylic acid( Rotundioic acid,RA) on the sensitivity of adriamycin-resistant K562 cells( K562/ADM Cell) antitumor drug,and to explore the effect and mechanism of RA on the multidrug resistance of K562/ADM cells. Methods:CCK-8 method was used to detect the effect of RA on the sensitivity of K562 cells and K562/ADM cells to anti-tumor drug. Reverse transcription-polymerase chain reaction( RT-PCR) and Western blot were used to detect the expression level of mRNA and the protein in K562 and K562/ADM cells,and the effect of RA on the expression of MDR1 mRNA and P-gp in K562/ADM cells was also detected;Western blot was used to detect the expression of p-JNK,p-p38 and pERK1/2 in K562/ADM cells. Results: RA could increased the sensitivity of K562/ADM cells to adriamycin( the reversal factor was 1. 61 times),the difference showed statistically significantly( P < 0. 05);the resistance factor of K562/ADM to ADM was 41. 76 times. The expression of MDR1 mRNA in K562 cells was extremely low,and the protein product P-glycoprotein( P-gp) was almost not expressed;MDR1 mRNA and P-gp in K562/ADM cells were highly expressed;RA could down-regulate the expression levels of MDR1 and P-gp in K562/ADM cells. In addition,RA could upregulate the phosphorylation levels of p38 and ERK1/2 in K562/ADM cells,but it has no effect on the expression of p-JNK. Conclusion: RA may participate in the regulation of MAPK signaling pathway by upregulating the expression levels of p-p38 and p-ERK1/2 in K562/ADM cells,and thus inhibit the transcription and translation levels of MDR1,and finally reverse the multidrug resistance of leukemia cells.