榭皮黄酮通过抑制Traf6/NF-κB信号转导通路增强5-氟尿嘧啶对HepG2的化疗作用

Quercetin enhances the chemotherapeutic effect of 5-fluorouracil on HepG2 by suppressing Traf6/NF-κB signaling transduction pathway

目的研究榭皮黄酮(quercetin,Qu)影响肝癌细胞HepG2对5-氟尿嘧啶(5-fluorouracil,5-FU)的敏感性及Traf6/NF-κB信号转导通路。方法体外培养HepG2细胞,取对数期细胞进行后续实验,将细胞分为对照组(不含任何药物的培养基孵育细胞24 h)、单独Qu组(含40μg/ml Qu的培养基孵育细胞24 h)、单独5-FU组(含50μmol/L 5-FU的培养基孵育细胞24 h)、Qu+5-FU联合处理组(含40μg/ml Qu和50μmol/L 5-FU的培养基共同孵育细胞24 h)、单独Traf6抑制剂C25-140组(2μmol/L C25-140的培养基孵育细胞8 h)、C25-140+Qu+5-FU组(C25-140预处理细胞8 h,然后含40μg/ml Qu和50μmol/L 5-FU的培养基共同处理细胞24 h)。采用CCK8法检测细胞活力,采用倒置显微镜记录细胞克隆数,采用流式细胞术检测细胞凋亡率,采用Western blot检测剪切的半胱氨酸蛋白酶-7(cleaved-caspase 7,Cle-caspase 7)、Clecaspase 3、剪切的聚腺苷二磷酸-核糖聚合酶(cleaved poly ADP-ribose polymerase,Cle-PARP)、肿瘤坏死因子受体相关蛋白6(tumor necrosis factor receptor-associated factor-6,Traf6)、磷酸化转化生长因子-β活化激酶1(phosphorylation transforming growth factor-β-activated kinase 1,p-TAK1)和磷酸化核转录因子(phosphorylation nuclear factor kappa-B,p-NF-κB)蛋白的表达水平。结果Qu(40μg/ml)、5-FU(50μmol/L)和Qu+5-FU组细胞相对活力分别为(82.3±3.1)%、(53.7±4.1)%和(42.4±4.4)%,均显著低于对照组的(100.0±3.4)%,差异有统计学意义(t=5.83、9.54、14.65,P均<0.05);Qu(40μg/ml)组、5-FU(50μmol/L)组和Qu+5-FU组HepG2细胞克隆数分别为534±26、236±25、115±42,均显著低于对照组的701±32(P均<0.05),且Qu+5-FU组显著低于Qu(40μg/ml)组和5-FU(50μmol/L)组(t=31.74,P<0.001;t=11.34,P=0.008)。Qu(40μg/ml)组、5-FU(50μmol/L)组和Qu+5-FU组HepG2细胞凋亡率[(18.9±4.2)%vs(21.4±4.1)%vs(35.7±3.6)%vs(4.6±1.5)%]、Cle-caspase 7(0.11±0.02 vs 0.22±0.03 vs 0.32±0.03 vs 0.05±0.02)、Cle-caspase 3(0.13±0.02 vs 0.18±0.03 vs 0.28±0.03)和Cle-PARP(0.15±0.02 vs 0.24±0.03 vs 0.41±0.03 vs 0.08±0.02)表达水平均显著高于对照组,且Qu+5-FU组显著高于Qu(40μg/ml)组和5-FU(50μmol/L)组(P均<0.05)。Qu(40μg/ml)组、5-FU(50μmol/L)组、Qu+5-FU组HepG2细胞Traf6(0.28±0.02 vs 0.19±0.03 vs 0.11±0.03 vs 0.38±0.02)、p-TAK1(0.23±0.02 vs 0.11±0.03 vs 0.04±0.03 vs 0.38±0.02)和p-NF-κB(0.28±0.02 vs 0.13±0.03 vs 0.05±0.02 vs 0.44±0.03)蛋白相对表达量均显著低于对照组,且Qu+5-FU组均显著低于Qu(40μg/ml)组和5-FU(50μmol/L)组(P均<0.05)。与对照组相比,C25-140组、Qu+5-FU组和Qu+5-FU+C25-140组HepG2细胞相对活力[(73.4±4.1)%vs(65.8±3.6)%vs(47.7±3.9)%vs(100±3.1)%]和细胞克隆数(456±26 vs 413±25 vs 305±42 vs 763±32)显著降低,细胞凋亡率[(24.4±4.1)%vs(29.9±3.7)%vs(51.2±3.7)%vs(3.9±5.2)%]显著升高(P均<0.05);与Qu+5-FU组比较,Qu+5-FU+C25-140组HepG2细胞活力和克隆数显著降低,细胞凋亡率显著升高(P均<0.05)。与对照组相比,Traf6 mimics组HepG2细胞相对活力[(152.4±6.3)%vs(100±3.5)%]、细胞凋亡率[(5.3±3.2)%vs(3.8±2.1)%]和细胞克隆数(978±26 vs 783±32)均显著升高(P均<0.05),Qu+5-FU组和Traf6 mimics+Qu+5-FU组HepG2细胞相对活力[(65.8±4.3)%vs(100±3.5)%;(79.4±4.9)%vs(100±3.5)%]和细胞克隆数(454±25 vs 783±32;623±42 vs 783±32)显著降低,细胞凋亡率[(34.7±4.4)%vs(3.8±2.1)%;(24.4±3.5)%vs(3.8±2.1)%]显著升高(P均<0.05)。与Qu+5-FU组相比,Traf6 mimics+Qu+5-FU组HepG2细胞活力和克隆数显著升高,细胞凋亡率显著降低(P均<0.05)。结论Qu通过抑制Traf6/NF-κB信号转导通路协同诱导5-FU对肝细胞癌的化学治疗作用。

Objective To observe the effects of quercetin(Qu)on the chemotherapeutic sensitivity of 5-fluorouracil(5-FU)and Traf6/NF-κB signaling transduction pathway in HepG2.Methods HepG2 cells were cultured in vitro and log-phase cells were harvested for subsequent experiments.Cells were divided into control group(cells were incubated in a drug-free medium for 24 h),Qu group(cells were incubated in a medium containing 40μg/ml of Qu for 24 h),5-FU group(cells were incubated in a medium containing 50μmol/L of 5-FU for 24 h),Qu+5-FU group(cells were incubated in a medium containing 40μg/ml of Qu and 50μmol/L of 5-FU for 24 h),C25-140 group(cells were incubated in a medium containing 2μmol/L of C25-140 for 8 h)and C25-140+Qu+5-FU group(cells were firstly incubated in a medium containing 2μmol/L of C25-140 for 8 h,then treated with 40μg/ml of Qu and 50μmol/L of 5-FU for 24 h).CCK8 assay was performed to detect the cell viability.Cell colonies were measured by inverted microscope.HepG2 cell apoptotic rate was determined by flow cytometry.The expression levels of cleaved-caspase 7(Cle-caspase 7),Cle-caspase 3,cleaved poly ADP-ribose polymerase(Cle-PARP),tumor necrosis factor receptor-associated factor-6(Traf6),phosphorylation transforming growth factor-β-activated kinase 1(p-TAK1)and phosphorylation nuclear factor kappa-B(p-NF-κB)protein were detected by Western blot.Results The relative cell viability in Qu(40μg/ml)group,5-FU(50μmol/L)group and Qu+5-FU group were(82.3±3.1)%,(53.7±4.1)%and(42.4±4.4)%,respectively,which were significantly lower than that in control group[(100.0±3.4)%,t=5.83,9.54,14.65,all P<0.05].The HepG2 cell colonies number in Qu(40μg/ml)group,5-FU(50μmol/L)group and Qu+5-FU group were 534±26,236±25 and 115±42,respectively,which were significantly lower than that in control group(701±32;all P<0.05).The HepG2 cell colonies number in Qu+5-FU group was significantly lower than that in Qu(40μg/ml)group and 5-FU(50μmol/L)group(t=31.74,P<0.001;t=11.34,P=0.008).The HepG2 cell apoptotic rates[(18.9±4.2)%vs(21.4±4.1)%vs(35.7±3.6)%vs(4.6±1.5)%],Cle-caspase 7(0.11±0.02 vs 0.22±0.03 vs 0.32±0.03 vs 0.05±0.02),Cle-caspase 3(0.13±0.02 vs 0.18±0.03 vs 0.28±0.03)and Cle-PARP(0.15±0.02 vs 0.24±0.03 vs 0.41±0.03 vs 0.08±0.02)in Qu(40μg/ml)group,5-FU(50μmol/L)group and Qu+5-FU group were significantly higher than those in control group,and the above indexes in Qu+5-FU group were significantly higher than those in Qu(40μg/ml)group and 5-FU(50μmol/L)group(all P<0.05).The relative expression levels of Traf6(0.28±0.02 vs 0.19±0.03 vs 0.11±0.03 vs 0.38±0.02),p-TAK1(0.23±0.02 vs 0.11±0.03 vs 0.04±0.03 vs 0.38±0.02)and p-NF-κB(0.28±0.02 vs 0.13±0.03 vs 0.05±0.02 vs 0.44±0.03)in Qu(40μg/ml)group,5-FU(50μmol/L)group and Qu+5-FU group were significantly lower than those in control group,and the above indexes in Qu+5-FU group were significantly lower than those in Qu(40μg/ml)group and 5-FU(50μmol/L)group(all P<0.05).Compared with control group,the cell viability[(73.4±4.1)%vs(65.8±3.6)%vs(47.7±3.9)%vs(100±3.1)%]and colonies number(456±26 vs 413±25 vs 305±42 vs 763±32)in C25-140 group,Qu+5-FU group and Qu+5-FU+C25-140 group decreased significantly and the apoptosis rate[(24.4±4.1)%vs(29.9±3.7)%vs(51.2±3.7)%vs(3.9±5.2)%]increased significantly(all P<0.05);compared with Qu+5-FU group,the cell viability and colonies number in Qu+5-FU+C25-140 group decreased significantly and the apoptosis rate increased significantly(all P<0.05).Compared with control group,the cell viability[(152.4±6.3)%vs(100±3.5)%],apoptosis rate[(5.3±3.2)%vs(3.8±2.1)%]and colonies number(978±26 vs 783±32)in Traf6 mimics group increased significantly,the cell viability[(65.8±4.3)%vs(100±3.5)%;(79.4±4.9)%vs(100±3.5)%]and colonies number(454±25 vs 783±32;623±42 vs 783±32)in Qu+5-FU group and Traf6 mimics+Qu+5-FU group decreased significantly and the apoptosis rate[(34.7±4.4)%vs(3.8±2.1)%;(24.4±3.5)%vs(3.8±2.1)%]increased significantly(all P<0.05).Compared with Qu+5-FU group,the cell viability and colonies number in Traf6 mimics+Qu+5-FU group increased significantly,and the apoptosis rate decreased significantly(all P<0.05).Conclusions Qu synergized the chemotherapeutic effect of 5-FU on hepatocellular carcinoma through the suppression of Traf6/NF-κB signaling transduction pathway.