Caspases在视黄酸诱导pRARE4-IFNα转染肿瘤细胞凋亡中的作用

Roles of caspases in the apoptosis of pRARE4-IFNα transfected tumor cells induced by retinoic acid

目的探讨视黄酸(RA)及其诱导的外源性α-干扰素(IFNα)抗肿瘤作用与半胱氨酸蛋白酶(caspase)的关系。方法含视黄酸反应元件(RARE)的IFNα表达载体(pRARE4-IFNα),转染肿瘤细胞并证实外源性IFNα基因的表达受RA的诱导后,分析了细胞增殖功能、细胞DNA梯形带,caspase-1、3基因表达,caspase-3活性以及caspase-3特异性抑制剂(DEVD-CHO)对RA和/或IFNα诱导细胞凋亡的抑制作用。结果对RA敏感的HL-60细胞和caspase-3基因缺失的MCF-7细胞经RA处理后,增殖能力减弱,出现明显的DNA梯形带,caspase-1基因表达上调节,HL-60细胞caspase-3基因表达上调节,活性升高,DEVD-CHO可拮抗RA引起的HL-60细胞caspase-3活性升高,并部分削弱RA引起的细胞增殖抑制和凋亡。结论Caspase-3在RA诱导的细胞凋亡中有重要作用,同时,RA可能通过其它途径诱导caspase-3基因缺失细胞的凋亡。

Objective To study the roles of caspases in retinoic acid (RA) induced apoptosis of tumor cells transfected with the IFNα expression vector containing retinoic acid response element. Methods The IFNα gene eukaryotic expression vector containing four copies of RARE (pRARE4-IFNα) was constructed with recombinant DNA technique, and transfected into the cells of HL-60 and MCF-7 with liposome DOTAP. The IFNα expression induced by RA was detected by RT-PCR and ELISA. Then, the cellular proliferation capacity and the cell apoptosis were measured by MTT assay and DNA laddering assay, respectively; the expressions of caspase-1 and caspase-3 mRNA were analyzed by RT-PCR. Meanwhile, the activity and the protein expression of caspase-3 were analyzed. Results The proliferation capacity was decreased but the caspase-1 expression was increased in RA-treated cells, in which the DNA ladder was detectable. The activity and expression of caspase-3 were up-regulated in RA-treated HL-60 cells. These changes in transfected cells were more obvious than those in nontransfected cells. DEVD-CHO, the specific caspase-3 inhibitor, could inhibit the activity of caspase-3 and impair the growth inhibition and apoptosis induced by RA. Conclusion RA and IFNα can cooperate in inhibiting cell growth and inducing apoptosis. the up-regulated expression and activity of caspase-3 may be play a partial role in cell apoptosis induced by RA and IFNα.