斑蝥酸钠诱导非小细胞肺癌H1975细胞凋亡的作用及机制研究

Effect and Mechanism of Sodium Cantharidate on Apoptosis of Non-Small Cell Lung Cancer H1975 Cells

目的:探究斑蝥酸钠(Sodium cantharidate,SCA)对非小细胞肺癌H1975细胞增殖和凋亡的影响并探究其相关机制。方法:人非小细胞肺癌H1975细胞分为空白对照组(生理盐水)、斑蝥酸钠4、8、16μmol/L组。药物处理24 h和48 h后,使用细胞计数试剂盒-8(CCK-8)检测各组细胞存活率;集落克隆试验检测细胞增殖能力;Transwell试验检测细胞迁移和侵袭能力的变化。AnnexinⅤ-FITC/PI双染法检测细胞凋亡率;4’,6-二脒基-2-苯基吲哚(DAPI)染色检测细胞核变化;Western Blot法检测斑蝥酸钠对H1975细胞蛋白激酶B(AKT)通路磷酸化和哺乳动物雷帕霉素靶蛋白(mTOR)蛋白及抑凋亡蛋白B淋巴细胞瘤-2(BCL-2)、促凋亡蛋白BCL-2相关X蛋白(BAX)和活化半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达的影响。结果:与空白对照组相比,4、8、16μmol/L的斑蝥酸钠组H1975细胞的增殖能力受到显著抑制,细胞存活率、侵袭、迁移能力明显降低,细胞凋亡率明显升高(P<0.05)。磷酸化AKT、mTOR蛋白的表达下调,BCL-2/BAX比值明显降低,cleaved-Caspase-3蛋白的表达明显上调(P<0.05)。结论:斑蝥酸钠能抑制人非小细胞肺癌H1975细胞增殖,侵袭迁移,诱导细胞凋亡,其机制可能与抑制AKT/mTOR通路,调控BCL-2和BAX的表达有关。

Objective:To investigate the effect of sodium cantharidate(SCA)on the proliferation and apoptosis of non-small cell lung cancer H1975 cells and explore its underlying mechanism.Methods:Human non-small cell lung cancer H1975 cells were divided into a blank group(normal saline)and low-,medium-,and high-dose SCA groups(4,8,and 16μmol/L).After 24 h and 48 h of drug treatment,the cell survival rate of each group was detected by Cell Counting Kit-8(CCK-8).Colony cloning assay was used to detect cell proliferation.The cell migration and invasion abilities were detected by the Transwell assay.Cell apoptosis rate was detected by AnnexinⅤ-FITC/PI double staining.Nuclear changes were detected by 4’,6-diamidino-2-phenylindole(DAPI)staining.Western blot was used to detect the effect of SCA on the phosphorylation of protein kinase B(Akt)pathway and protein expression of mammalian target of rapamycin(mTOR),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and cysteinyl aspartate-specific protease 3(Caspase-3)in H1975 cells.Results:Compared with the blank group,all SCA groups showed inhibited cell proliferation,reduced cell survival rate,blunted invasion and migration activities,increased apoptosis of H1975 cells(P<0.05),down-regulated expression of phosphorylated Akt and mTOR,decreased Bcl-2/Bax ratio,and up regulated protein expression of cleaved-Caspase-3(P<0.05).Conclusion:SCA can inhibit the proliferation,invasion,and migration and induce cell apoptosis of human non-small cell lung cancer H1975 cells.The mechanism may be related to the inhibition of the Akt/mTOR pathway and the regulation of the expression of Bcl-2 and Bax.