槲皮素抑制HL-60细胞增殖与诱导凋亡机制探讨

Mechanism of quercetin in inducing apoptosis and inhibiting proliferation on HL-60 cells

目的观察槲皮素孵育后HL-60细胞腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)、Cox-2表达和线粒体凋亡信号转导通路Bcl-2、Bax和Caspase-3表达,探讨槲皮素抑制HL-60细胞增殖及诱导凋亡可能机制。方法Hoechst33342染色及流式细胞仪检测槲皮素孵育HL-60细胞48h后细胞凋亡情况。蛋白质印迹法检测HL-60细胞及人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)LKB1、p-AMPK和Cox-2蛋白的表达。不同浓度槲皮素与HL-60细胞共同孵育48h后,蛋白质印迹法检测细胞LKB1、p-AMPK、Cox-2、Bcl-2、Bax和Caspase-3的表达。不同浓度AMPK抑制剂Compound c孵育HL-60细胞48h后蛋白质印迹法检测p-AMPK的表达。50和100μmol/L槲皮素与25μmol/L Compound c共同孵育HL-60 48h后检测LKB1、p-AMPK和Cox-2蛋白表达。结果HL-60细胞凋亡率检测结果显示,槲皮素0μmol/L凋亡率为(2.40±2.31)%,25μmol/L为(8.20±0.92)%,50μmol/L为(15.06±1.87)%,100μmol/L为(19.29±0.91)%,F=8.48,P=0.01。人PBMC中p-AMPK的表达高于HL-60,P<0.001;人PBMC中Cox-2的表达低于HL-60,P<0.001。50和100μmol/L槲皮素组p-AMPK和Caspase-3表达增加,P值均<0.05;50和100μmol/L槲皮素组Cox-2和Bcl-2表达下降,P值均<0.01;25、50和100μmol/L槲皮素组Bax的表达水平上升,P<0.05。25μmol/L Compound c能够显著抑制HL-60细胞p-AMPK的表达。与单用槲皮素(50和100μmol/L)相比,槲皮素与25μmol/L Compound c联合组p-AMPK表达下降,P值均<0.05;100μmol/L槲皮素与Compound c(25μmol/L)共同孵育HL-60细胞Cox-2的表达为0.51±0.03,与单用槲皮素(100μmol/L)相比,表达上升,P<0.001。结论槲皮素能够激活HL-60细胞中AMPK,导致AMPK磷酸化增加,抑制Cox-2的表达进而调控Bcl-2依赖的细胞凋亡途径诱导HL-60细胞的凋亡,从而发挥其抗白血病作用。槲皮素对HL-60细胞中AMPK的激活不依赖于LKB1的活化。

OBJECTIVE To explore the mechanism of quercetin in inducing apoptosis and inhibiting proliferation on HL-60 ceils by examining the expressions of AMP-activated protein kinase（AMPK） and Cox-2 protein, and the apoptosis related genes such as Bcl-2, Bax, Caspase-3 in HL-60 cells after culture with quercetin. METHODS The cell apoptosis after exposed to quercetin was determined by Hoechst33342 staining and the flow cytometry. The expressions of LKB1, phosphorylated AMPK（p-AMPK） and Cox-2 protein were detected in HL-60 cells, and normal peripheral blood mononu- clear cells（PBMC） by Western-blot. The expressions of LKB1, p-AMPK, Cox-2,Bcl-2, Bax, Caspase-3 were assayed in HL-60 cells after 48h culture with different concentrations of quercetin. The expressions of p-AMPK were detected in HL-60 cells after 48h culture with AMPK inhibitor Compound c. The expressions of LKB1, p-AMPK and Cox-2 were de- tected in HL-60 cells after 48h culture with quercetin-alone and quercetin ＋ Compound c. RESULTS Quercetin induced HL-60 cell apoptosis. The apoptosis rates of HL-60 cells cultured with 0, 25, 50 and 100 vmol/L of quercetin were （2.40±2.31）%, （8.20±0. 92）%, （15. 06±1. 8？）% and （19. 29±0. 91）%, respectively （F=8. 48,P〈0.01）. p-AMPK in PBMC was higher than that in HL-60 cells,P〈0. 001. But Cox-2 in PBMC was lower than that in HL-60 cells,P〈0. 001. p-AMPK and Caspase-3 were increased in HL-60 cells which were cultured with quercetin （50 and 100 μmol/L） for 48 h,P〈0.05. But the expressions of Cox-2 and Bel-2 were decreased, P〈0.01. When the cells were trea- ted with 25, 50 and 100 μmol/L quercetin, the expressions of Bax were higher than that in the control group, P〈0. 05. After culture of HL-60 with 25 μmol/L Compound c, p-AMPK expression was significantly decreased. Comparing with the expression of p-AMPK in quercetin-alone groups （50 and 100 μmol/L）, p-AMPK decreased significantly in quercetin （50 and 100 μmol/L） ＋ Compound c （25 μmol/L） groups （P〈0. 05）. Cox-2 increased significantly in quercetin（100 μmol/L）＋ Compound c（25 μmol/L） group than that in quercetin（100 μmol/L） groups, P〈0. 001. CONCLUCION Quercetin activated AMPK expression in HL-60 cells independently of LKB1 activation, inhibited Cox-2 expression by ac- tivating AMPK, and further regulated the Bcl-2 dependent Dathwavs of apoptosis to exert its anti-leukemia effect.