硫利达嗪通过线粒体应激信号通路诱导肺腺癌细胞发生免疫原性死亡

Immunogenic Cell Death of Lung Adenocarcinoma Cells Induced by Thioridazine Through Mitochondrial Stress Signaling Pathway

硫利达嗪作为吩噻嗪类抗精神病类药物,具有诱导肿瘤发生免疫原性死亡(ICD)、激活特异性免疫反应的潜力。本研究利用A549和H1299细胞探讨了硫利达嗪诱导肺腺癌细胞免疫原性死亡及其分子机制。将不同浓度的硫利达嗪与A549和H1299细胞共孵育24 h后,利用四甲基偶氮唑蓝(MTT)法检测细胞的存活及生长情况,利用流式细胞术分析细胞凋亡率,利用ATP检测试剂盒检测细胞上清中的ATP含量,利用免疫荧光法检测细胞表面钙网蛋白(CRT)的表达,利用蛋白质印迹(Western blot)法检测凋亡相关蛋白裂解的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase 3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)的表达水平。结果表明,硫利达嗪能够显著抑制A549和H1299细胞的增殖,促进细胞凋亡,细胞增殖抑制及凋亡呈浓度依赖性。ATP分泌量增加及细胞表面CRT的表达水平上调,表明上述细胞发生了免疫原性死亡。而Bcl-2表达水平下降,Bax和Cyt C以及Cleaved caspase 3表达水平上调,进一步证明了硫利达嗪可诱导肿瘤细胞凋亡。上述结果表明,硫利达嗪可通过线粒体应激信号通路诱导肺腺癌细胞发生免疫原性死亡,从而抑制肿瘤细胞的增殖。

Thioridazine,a phenothiazine drug,has a potential to induce tumor immunogenic cell death(ICD)and activate a tumor-specific immune response.In this study,we examined the ICD effect induced by Thioridazine and elucidated its underlying mechanisms using lung adenocarcinoma A549 and H1299 cells.Various concentrations of Thioridazine were employed to treat A549 and H1299 cells.The cell inhibition rate was determined using the methyl thiazol tetrazolium(MTT)assay,apoptosis rate was assessed by Flow Cytometry,extracellular ATP release was measured by using an ATP Assay Kit,and the presence of calreticulin(CRT)on the cell surface was evaluated via immunofluorescence.Furthermore,Western blot analysis was conducted to assess the expression levels of apoptosis-related proteins,including cleaved caspase 3,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cytochrome C(Cyt C).Our findings revealed a significant,dose-dependent inhibition of A549 and H1299 cell proliferation and a marked increase in the apoptosis rate upon treatment with Thioridazine.Additionally,there was an elevation in extracellular ATP secretion and CRT expression on the cell surface,indicative of ICD occurrence.Consistently,the expression level of Bcl-2 decreased,while Bax,Cyt C,and cleaved caspase 3 were up-regulated that further indicate the induction apoptosis of tumor cell by Thiolidazine.Collectively,our results confirm that Thioridazine inhibits the proliferation of lung adenocarcinoma cells by inducing ICD through the mitochondrial stress signaling pathway.