Caspases在ω-3脂肪酸联合跨膜型TNFα诱导HL-60细胞凋亡中的作用

Roles of caspases in apoptosis of HL-60 cells induced by ω-3PUFA and the transmembrane TNFα

目的 探讨ω3脂肪酸联合跨膜型TNFα诱导HL 6 0细胞凋亡与半胱氨酸蛋白酶(Caspases)之间的关系。方法 已证实转染带有脂肪酸受体反应元件的跨膜型TNFα表达载体(pPPRE tmTNFα)后,HL 6 0细胞可在ω3脂肪酸诱导下高效表达跨膜型TNFα。运用MTT比色法、DNA梯形带检测、RT PCR和免疫印迹技术分析ω3脂肪酸对转染细胞增殖能力和凋亡的影响,以及细胞Caspase 1、3、8和9基因的表达变化,检测Caspase 8活性以及Caspases特异性抑制剂对肿瘤细胞凋亡效应的拮抗作用。结果 HL 6 0细胞经ω3脂肪酸处理后,细胞增殖能力减弱,可检测到DNA梯形带形成,Caspase 1、3、8和9基因表达增加,Caspase 8活性增高,上述变化在转染细胞中更为显著,Caspases抑制剂可以部分拮抗ω3脂肪酸联合跨膜型TNFα对HL 6 0诱导凋亡和抑制增殖的作用。结论 ω3脂肪酸可以联合跨膜型TNFα更有效的诱导细胞凋亡,抑制肿瘤细胞增殖,而上调Caspases的表达和 或活性可能在此过程中发挥重要作用。

Objective To study the relationship between caspases and apoptosis of HL-60 cells induced by ω-3 polyunsaturated fatty acids (PUFA) and transmembrane TNFα. Methods After transfection with tmTNFα expression vector containing PPRE-tk promoter (pPPRE-tmTNFα), the HL-60 cells were treated with ω-3PUFA. The cellular proliferation capacity and the apoptosis of the HL-60 cells were analyzed by MTT assay and DNA ladder assay, respectively. The expression of caspase-1, 3, 8, and 9 was analyzed by RT-PCR and Western blotting. The activity of caspase-8 was analyzed with Ac-IETD-AMC. The inhibitory effects of Ac-IETD-CHO (an inhibitor of caspase) on apoptosis of HL-60 cells were measured. Results In HL-60 cells treated with ω-3PUFA, the DNA ladder was found and the proliferation capacity was decreased. The expression of caspase (1, 3, 8, and 9) and activity of caspase-8 were increased. those changes were more apparent in pPPRE-tmTNFα transfected cells. Growth inhibition and apoptosis induced by ω-3PUFA and/or tmTNFα were partly prevented by the special caspase inhibitor. Conclusion The ω-3PUFA and the product of exogenous tmTNFα gene could synergistically induce the tumor cell apoptosis and inhibit the cell proliferation, while the caspases network may play a key role in these processes.