雷公藤内酯醇下调凋亡抑制蛋白、活化半胱天冬酶诱导骨髓增生异常综合征细胞株细胞凋亡的研究

Triptolide induces apoptosis in myelodysplastic syndromes MUTZ-1 cells through activation of caspases and down-regulation of IAPs

目的探讨雷公藤内酯醇诱导骨髓增生异常综合征细胞株MUTZ-1细胞凋亡与半胱天冬酶(caspase)激活及凋亡抑制蛋白(IAP s)表达的关系。方法不同浓度雷公藤内酯醇作用于MUTZ-1细胞12小时,采用A nnex-in V/P I双标流式细胞仪检测凋亡的细胞量。W estern b lot检测MUTZ-1细胞caspase-3、caspase-8、caspase-9及X I-AP、c-IAP 1、c-IAP 2的蛋白表达水平。结果0、20、40、60 ng/m l雷公藤内酯醇作用MUTZ-1细胞12小时,凋亡细胞百分比(%)分别为2.66±0.31、9.87±0.99、30.28±4.35、51.89±5.38。凋亡率与药物浓度呈正相关(r=0.980,P=0.020)。凋亡关键酶caspase-3、caspase-8、caspase-9被激活,且cleaved caspase-3、cleaved caspase-9、procaspase-8的表达水平与凋亡率相关(r1=0.957,P 1=0.043;r2=0.994,P 2=0.006;r3=-0.994,P 3=0.006)。随着药物浓度的增加,c-IAP 1、c-IAP 2蛋白表达量呈逐渐下降趋势,c-IAP 1、c-IAP 2蛋白表达与凋亡率之间呈负相关(r1=-0.961,P 1=0.039;r2=-0.963,P 2=0.037)。结论雷公藤内酯醇通过激活caspase诱导骨髓增生异常综合征细胞株MUTZ-1细胞发生凋亡,内源性及外源性途径皆参与其中。下调凋亡抑制蛋白IAP s的表达可能是雷公藤内酯醇诱导细胞凋亡的机制之一。

Objective To explore the relationship of triptolide （TPL）-induced apoptosis of myelodysplastic syndromes （MDS）MUTZ-1 cells with the protein expression of caspases and inhibitor of apoptosis （IAPs）. Methods MUTZ-1 cells were incubated with indicated concentrations of triptolide for 12 hours. The apoptotic rate was detected by flow cytometry using Annexin V-FITC/PI staining. The protein expression of caspase-3,caspase-8,caspase-9,XIAP,c-IAP1 and c-IAP2 were determined by Western blot. Results After MUTZ-1 cells were exposed to 0,20,40 and 60 ng/ml triptolide for 12 hours,the apoptotic percentages were 2.66±0.31,9.87±0.99,30.28±4.35 and 51.89±5.38（%）,respectively. The apoptotic rate was positively correlated with drug doses （r= 0. 980, P=0. 020）. Cellular caspase-3, caspase-8 and caspase-9 were activated. The protein expressions of cleaved caspase-3, cleaved caspase-9 and procaspase-8 were significantly correlated with the rate of apoptosis （r1 = 0. 957, P1 = 0. 043;r2 = 0. 994 ,P2 = 0. 006 ; r3= -0. 994 ,P3= 0. 006）, respectively. With the increasing of drug concertration,c-IAP1 and c-IAP2 were down-regulated. The rate of apoptosis was inversely related with the protein levels of c-IAP1 and c-IAP2（r1=-0. 961,P1=0. 039;r2=-0. 963,P2=0. 037）.Conclusion Triptolide induces the apoptosis of MDS MUTZ-1 cells though the activation of caspases, involved with mitochondrial and death receptor pathway. Downregulation of IAPs may be one of the mechanisms of TPL-induced apoptosis.