三氧化二砷与重组人肿瘤坏死因子相关细胞凋亡诱导配体协同诱导胃癌细胞凋亡的机制

Mechanism Underlying Synergistic Apoptosis-inducing Activity of As_2O_3 Combined with rhTRAIL on Gastric Cancer Cells

目的探讨三氧化二砷(As2O3)和重组人肿瘤坏死因子相关细胞凋亡诱导配体(rhTRAIL)协同诱导胃癌细胞SGC7901凋亡的机制。方法分别以As2O3、rhTRAIL及两者联合处理人胃腺癌细胞株SGC7901,用AnnexinV-FITC/PI双染色流式细胞术检测细胞凋亡,用DiOC6荧光染色流式细胞术检测细胞线粒体跨膜电位(ΔΨm),比色法测定细胞Caspase-8、9活性的变化,观察Caspase-8阻断剂(Z-IETD-fmk)与二硫基还原剂(DTT)对SGC7901细胞凋亡、ΔΨm和Caspase-8、9活性变化的影响。结果As2O3和rhTRAIL均可以导致SGC7901细胞ΔΨm随时间延长逐渐下降,两者联用可增加SGC7901细胞ΔΨm下降。Caspase-8阻断剂Z-IETD-fmk可减少rhTRAIL诱导的SGC7901细胞发生凋亡与ΔΨm下降,但不影响As2O3的上述作用。二硫基还原剂(DTT)可减少As2O3诱导的SGC7901细胞凋亡与ΔΨm下降,但不影响rhTRAIL的上述作用。As2O3可导致SGC7901细胞Caspase-9活性升高,这种作用可被二硫基还原剂部分取消,但不受Caspase-8阻断剂的影响;rhTRAIL可导致SGC7901细胞Caspase-8、9活性升高,这种作用可被Caspase-8阻断剂部分取消,但不受二硫基还原剂的影响。结论As2O3与rhTRAIL通过不同机制激活SGC7901细胞凋亡的线粒体途径,两者联合增强了线粒体途径的激活,从而在诱导胃癌细胞凋亡方面形成协同作用。

Objective To investigate the mechanism of synergistic activity of the combined treatment of arsenic trioxide（ As2O3 ） and recombinant human tumor necrosis factor-related apoptosis-inducing ligand（rhTRAIL） in inducing apoptosis on gastric cancer cell line SGC7901. Methods The human gastric cancer cell line SGC7901 was treated with As2O3 and rhTRAIL, respectively, or in combination; apoptosis was detected by flow cytometry（ AnnexinV-FITC/PI assay） ; FCM （ DIOC6 staining assay） was used to assess the mitochondrial transmembrane potential （△Ψm） ; relative Caspase-8/9 activity was measured by colorimetric assay; effects of Caspase-8 inhibitor（Z-IETD-fmk） and disulfide-bond reducing agent（DTT） on the As2O3-or rhTRAIL-induced apoptosis, chang of △Ψm and activity of Caspase-8/9 were observed. Results As203 and rhTRAIL could induce collapse of △Ψm on SGC7901 cells in time-dependent manner and the △Ψm of the cells treated by the combination were lower than those in treatment respectively. Z-IETD-fmk showed some degree of inhibitory effect on the disruption of △Ψm and apoptosis induced by rhTRAIL, but did not influence the changes induced by As203 ; DTT showed some degree of inhibitory effect on As203-induced △Ψm collapse and apoptosis, but did not influence the changes induced by rhTRAIL. As2O3 could induce activation of Caspase-9, such effect could be partially antagonized by DTT, not by Z-IETD-fmk; rhTRAIL could induce activation of both Caspase-8 and Caspase-9, such effect could be partially antagonized through different mechanisms, can activate mitochoudrial pathway and by Z-IETD-fmk, not by DTT. Conclusion As2O3 and rhTRAIL, can result in apoptosis; the synergism of them is in association with mitochondrial amplification of apoptotic signals.