甘草查尔酮A对结肠癌细胞中氧化还原稳态调控的影响

Effects of licochalcone A on redox homeostasis in colon cancer cells

目的研究甘草查尔酮A对结肠癌细胞氧化还原调控的作用机制。方法噻唑蓝(MTT)法检测甘草查尔酮A对结肠癌细胞活性的影响;经甘草查尔酮A或活性氧(ROS)抑制剂处理后,流式细胞术和Western blot法检测结肠癌细胞凋亡率、凋亡蛋白和ROS水平;酶学实验和试剂盒检测甘草查尔酮A对硫氧还蛋白还原酶1(TrxR1)和谷胱甘肽还原酶(GR)活性的影响;分子对接和Western blot法检测甘草查尔酮A对TrxR1蛋白的作用;沉默结肠癌细胞中TrxR1蛋白,经甘草查尔酮A处理后,检测ROS水平及凋亡率变化;经甘草查尔酮A或还原型谷胱甘肽(GSH)抑制剂处理后,检测细胞GSH水平及凋亡率。结果甘草查尔酮A能抑制结肠癌细胞活性(P<0.05,P<0.01),诱导结肠癌细胞发生凋亡(P<0.05,P<0.01),并上调其ROS水平;ROS抑制剂能逆转甘草查尔酮A引起的ROS水平上升和促凋亡作用(P<0.01);甘草查尔酮A能选择性抑制TrxR1活性(P<0.05,P<0.01),对TrxR1蛋白表达则几乎无影响(P>0.05);沉默TrxR1和GSH抑制剂均能协同甘草查尔酮A的促凋亡作用(P<0.01),甘草查尔酮A对GSH水平几乎无影响(P>0.05)。结论甘草查尔酮A可能通过抑制TrxR1蛋白活性、上调ROS水平的方式诱导结肠癌细胞凋亡。

AIM To investigate the mechanism of licochalcone A on redox regulation of colon cancer cells.METHODS Methyl thiazolyl tetrazolium(MTT)assay was used to detect the effects of licochalcone A on the activity of colon cancer cells.After treatment with licochalcone A or reactive oxygen species(ROS)inhibitor,the colon cancer cells underwent detection of apoptosis rate,apoptotic protein and ROS levels by flow cytometry and Western blot.Licochalcone A had its effects on thioredoxin reductase 1(TrxR1)and glutathione reductase(GR)activities detected by enzyme assay and kit;and its effect on TrxR1 protein detected by molecular docking and Western blot.With TrxR1 protein silencing followed by licochalcone A treatment,the colon cancer cells had their ROS level and apoptosis rate detected.The impacts of licochalcone A or reduced glutathione(GSH)inhibitor on the colon cancer cells,in terms of GSH level and apoptosis rate,were detected as well.RESULTS Upon the colon cancer cells,licochalcone A inhibited their activity(P<0.05,P<0.01),induced their apoptosis(P<0.05,P<0.01),and up-regulated their ROS level.ROS inhibitor offset the licochalcone A-induced increase of ROS level and pro-apoptosis effect(P<0.01).With little influence on TrxR1 protein expression(P>0.05),licochalcone A selectively inhibited TrxR1 activity(P<0.05,P<0.01).Barely being influential to GSH level(P>0.05),licochalcone A synergistically promoted apoptosis of colon cancer cells with GSH inhibitors and TrxR1 silencing(P<0.01).CONCLUSION Licochalcone A may induce apoptosis of colon cancer cells by inhibiting TrxR1 protein activity and upregulating ROS level.