白桦脂酸增强K562/A02细胞株对阿霉素敏感性的研究

A Study on the Effects and Mechanism of Betulinic Acid on Sensitizing K562/A02 to Adriamycin

目的探讨低剂量白桦脂酸对K562/A02细胞对阿霉素敏感性的影响及其相关机制。方法单用阿霉素和低剂量白桦脂酸联用阿霉素处理K562/A02细胞,分别采用MTT法和流式细胞术检测低浓度白桦脂酸对阿霉素诱导的K562/A02细胞增殖、凋亡及胞内p H值的影响;通过real-time PCR和Western Blot分别检测低浓度白桦脂酸对阿霉素诱导的K562/A02细胞NHE1及MDR1 m RNA及蛋白表达的影响。结果低浓度的白桦脂酸可以显著提高阿霉素诱导的K562/A02细胞的增殖抑制效应(P<0.05),使其凋亡显著增强(P<0.05)。低浓度白桦脂酸联合阿霉素作用K562/A02细胞后,胞内p H值降低(P<0.05),同时NHE1和MDR1 m RNA的表达减少(P<0.05),NHE1和P-gp蛋白表达下降(P<0.05)。给予NHE1抑制剂Amiloride处理K562/A02细胞后,再用白桦脂酸联合阿霉素处理,P-gp的表达与单用Amiloride处理K562/A02细胞相比无明显变化,而BA处理组、Amiloride处理组、BA联合Amiloride处理组的NHE1和P-gp的表达均较对照组显著下降(P<0.05)。结论低浓度白桦脂酸可以增强K562/A02细胞株对阿霉素的敏感性,这可能与其降低NHE1表达,进而减少P-gp的表达,改变细胞内p H值有关。

Objective To explore the effects and mechanism of low dose of Betulinic on the sensitivity to Adriamycin in Adriamycin-resistant cells line K562/A02. Methods The chronic myeloid leukemia cells line K562 and Adriamycin-resistant cells line K562/A02 treated with low dose of Betulinic acid, NHE1 inhibitor（Amiloride） alone or plus Adriamycin. The effects of low dose of Betulinic acid on proliferation inhibition, apoptosis and intracellular p H values induced by Adriamycin were tested by MTT assay and flow cytometry separately in K562 and K562/A02 cells, and the m RNA and protein levels of NHE1 and MDR1 expression were detected by real-time PCR, and western blot respectively. Results The effects on proliferation inhibition induced by Adriamycin can be significantly enhanced when it was combined with low dose of Betulinic acid in K562/A02 cells（P〈0.05）. In addition, the apoptotic rate of K562/A02 cells treated with Betulinic acid plus Amiloride was also significantly increased（P〈0.05）. After treatment of Adriamycin combined with low dose of Betulinic acid in K562/A02 cells, the intracellular p H value significantly decreased（P〈0.05）, the m RNA levels of NHE1 and MDR1 expression and the protein levels of NHE1 and P-gp expression all were significantly downregulated（P〈0.05）. After K562/A02 cells treated with NHE1 inhibitor（Amiloride）, and then treated with low dose of Betulinic acid with Adriamycin again, the P-gp expression showed no obvious changes when compared with that after treatment of Adriamycin alone. However, when compared with control group, both NHE1 and P-gp expression levels decreased significantly in BA, Amiloride, and BA plus Amiloride group（P〈0.05）. Conclusion Low dose of Betulinic acid can significantly enhance the sensitivity to Amiloride in K562/A02 cells. This may be associated with down-regulation of NHE1 expression,and further decrease of P-gp expression and p H value.