法卡林二醇通过线粒体途径和内质网应激途径诱导肺癌细胞凋亡的机制研究

Mechanism of fakarinediol inducing apoptosis in lung cancer cells through mitochondrial pathway and endoplasmic reticulum stress pathway

目的研究法卡林二醇(FAD)通过线粒体途径和内质网应激途径诱导肺癌细胞凋亡的机制研究。方法将A549细胞分为对照组(不添加任何药物)和低、中、高剂量实验组(2.5,5.0,10.0μmol·L^(-1)法卡林二醇)。用噻唑蓝(MTT)法检测法卡林二醇对A549细胞增殖的影响;用Hoechst33342染色和流式细胞术检测细胞凋亡情况;用罗丹明123染色法检测细胞的线粒体膜电位变化;用蛋白质印迹(Western blot)法检测内质网应激及凋亡相关蛋白表达的影响。结果对照组和低、中、高剂量实验组细胞的生长抑制率分别为0,(14.42±1.30)%,(23.38±4.36)%和(53.52±5.43)%;细胞凋亡率分别为(4.73±1.08)%,(9.09±0.91)%,(15.76±1.54)%和(22.18±1.89)%;各组细胞的线粒体膜电位分别为404.88±12.52,317.75±25.76,269.39±19.36和217.53±29.11。低、中、高剂量实验组与对照组比较,差异均有统计学意义(均P<0.05)。与对照组相比,随着法卡林二醇剂量升高A549细胞内质网应激相关蛋白C/EBP同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)、磷酸化的肌醇需求激酶-1α(p-IRE1α)及凋亡相关蛋白B细胞淋巴瘤-2相关X蛋白(Bax)、裂解的胱天蛋白酶3(Cl-caspase-3)、细胞色素-C的表达水平逐渐升高;B细胞淋巴瘤-2表达水平逐渐降低,差异均有统计学意义(均P<0.05)。结论法卡林二醇通过线粒体途径和内质网应激途径,抑制A549细胞增殖,诱导肺癌细胞凋亡。

Objective To investigate the mechanism of apoptosis in lung cancer cells induced by falcarindiol through mitochondrial pathway and endoplasmic reticulum stress pathway.Methods A549 cells were divided into control group(no drug was added)and low,medium,high dose experimental groups(2.5,5.0,10.0μmol·L^(-1) falcarindiol).The proliferation of A549 cells was detected by MTT assay;Hoechst33342 staining and flow cytometry were used to detect apoptosis;the changes of mitochondrial membrane potential were detected by rhodamine 123 staining;the expression of endoplasmic reticulum stress and apoptosis related protein were detected by Western blot.Results The cell growth inhibition rates in control and low,medium,high dose experimental groups were 0%,(14.42±1.30)%,(23.38±4.36)%and(53.52±5.43)%;the apoptosis rates were(4.73±1.08)%,(9.09±0.91)%,(15.76±1.54)%and(22.18±1.89)%;the mitochondrial membrane potentials of the cells were 404.88±12.52,317.75±25.76,269.39±19.36 and 217.53±29.11 respectively.Low,medium,high dose experimental groups compared with control group,the difference were all statistically significant(all P<0.05).Western blot results showed that,compared with control group,the expression levels of endoplasmic reticulum stress-related proteins C/EBP homologous protein(CHOP),glucose regulated protein 78(GRP78),phosphorylated inositol requiring enzyme 1α(p-IRE1α)and apoptosis-related proteins B-cell lymphoma-2 associated X(Bax),Cl-caspase-3 and cytochrome-C in A549 cells were gradually increased with the increase of the dose of falcarindiol,and the expression level of B-cell lymphoma-2(Bcl-2)was gradually decreased(all P<0.05).Conclusion Falcarindiol inhibits the proliferation of A549 cells and induces apoptosis of lung cancer cells through mitochondrial pathway and endoplasmic reticulum stress pathway.