铁死亡诱导剂Erastin促进紫草素诱导的结直肠癌细胞凋亡的作用

Effect of ferroptosis inducer Erastin on apoptosis of colorectal cancer induced by Shikonin

目的探究铁死亡诱导剂Erastin联合紫草素(shikonin,Shi)对结直肠癌细胞增殖及凋亡的影响及其机制。方法采用Erastin(0、4、8、16、32、64μmol·L^(-1))和Shi(SW-480:0、0.5、1、2、4、8μmol·L^(-1)与SW-620:0、0.2、0.4、0.8、1.6、3.2μmol·L^(-1))单独作用以及10μmol·L^(-1) Erastin与各浓度Shi联合作用于结直肠癌SW480、SW620细胞;使用CCK-8法检测细胞活力,AnnexinV/PI双染法检测细胞凋亡;活性氧检测试剂盒测定结直肠癌细胞内活性氧含量的变化;10μmol·L^(-1) Erastin与2、1μmol·L^(-1) Shi分别单独或联合作用于SW480、SW620细胞,测定细胞内乳酸含量的变化;Western blot检测SW480和SW620细胞Bax、Bcl-2、PARP1、Caspase3、Caspase8、AKT和p-AKT蛋白表达情况。结果CCK-8结果表明,联合用药组能够明显抑制结直肠癌细胞活力,增加细胞凋亡率,乳酸水平被明显抑制,细胞内活性氧含量明显增高以及凋亡蛋白和p-AKT蛋白表达也发生明显变化。结论Erastin联合Shi可以协同诱导结直肠癌细胞凋亡,其机制可能与抑制肿瘤细胞乳酸产生、增加肿瘤细胞内活性氧含量、抑制AKT信号通路以及活化促凋亡蛋白有关。

Aim To explore the effect of ferroptosis inducer Erastin combined with Shikonin on the anti-tumor activity of colorectal cancer cells and its mechanism.Methods Erastin(0,4,8,16,32,64μmol·L^(-1)) and Shikonin(SW-480:0,0.5,1,2,4,8μmol·L^(-1) with SW-620:(0,0.2,0.4,0.8,1.6,3.2μmol·L^(-1)) alone and 10μmol·L^(-1) Erastin combined with various concentrations of Shikonin were used to treat colorectal cancer cells SW480 and SW620;Cell viability was detected by CCK-8 method and the apoptosis was detected by AnnexinV/PI double staining.The changes of active oxygen content in colorectal cancer cells were measured by ROS detection kit,and the changes of intracellular lactic acid content in SW480 and SW620 were measured by 10μmol·L^(-1) Erastin alone or in combination with 2μmol·L^(-1) and 1μmol·L^(-1) Shikonin,respectively.The protein expressions of Bax,Bcl-2,PARP1,Caspase3,Caspase8,AKT and P-akt in SW480 and SW620 cells were detected by Western blot.Results The results of CCK-8 showed that the combination group could significantly inhibit the viability of colorectal cancer cells and the apoptotic rate was the highest.At the same time,lactic acid was inhibited most obviously.The content of intracellular reactive oxygen species and apoptosis-related proteins also changed significantly.Conclusions Erastin combined with Shikonin can synergistically induce the apoptosis of colorectal cancer cells.The mechanism may be inhibiting the production of lactic acid in tumor cells,increasing the content of reactive oxygen species in tumor cells,inhibiting the AKT signaling pathway,and activating pro-apoptotic proteins to induce colorectal cancer cell apoptosis.