白花丹醌增强TRAIL诱导Kasumi-1细胞凋亡及其机制

摘要

目的:观察白花丹醌、rsTRAIL单独及其联合体外诱导Kasumi-1细胞凋亡的作用,探讨其作用机制.方法:采用WST-8 (CCK-8)比色法测定rsTRAIL、白花丹醌单独和联合应用对Kasumi-1细胞的生长抑制率;用流式细胞术、TUNEL法观察并且检测凋亡率;实时定量PCR检测白花丹醌作用后DR4和DR5 mRNA水平变化;Western blotting法检测单独应用及联合应用后DR5、caspase-3、caspase-8、caspase-9、Bid、Bax及c-FLIP的变化.结果:(1)白花丹醌和rsTRAIL单独应用均可抑制Kasumi-1细胞的生长,联合应用可增加对Kasumi-1细胞的生长抑制率且呈时间和剂量依赖性(P＜0.05).(2)rsTRAIL(100 μg/L)和白花丹醌(2 μmol/L)单用及其联合使用诱导Kasumi-1细胞Annexin V阳性细胞百分率分别为(27.7±2.9)%、(25.6±3.1)%和(52.1±3.3)%.(3)TUNEL法检测发现联合应用组较单用组凋亡细胞显著增多.(4)实时定量PCR检测发现白花丹醌可以上调DR5的表达.(5)Western blotting发现白花丹醌单独作用及其联合rsTRAIL应用时上调DR5、激活caspase-8和下调c-FLIP表达.结论:白花丹醌具有增强TRAIL诱导Kasumi-1细胞凋亡的作用,其机制与DR5上调、caspase-8激活和c-FLIP下调有关.%AIM : To investigate the effect of plumbagin and tumor necrosis factor - related apoptosis - inducing ligand ( TRAIL ) on the apoptosis of leukemic Kasumi - 1 cells.METHODS: Kasumi - 1 cells were treated with plumbagin alone, recombinant soluble TRAIL( rsTRAIL ) alone or the combination of plumbagin with rsTRAIL to induce apoptosis.The cell proliferation was analyzed by CCK - 8 assay.Apoptosis was determined by flow cytometry with Annexin V/PI double staining and TUNEL staining.The expression of DR4 and DR5 at mRNA level was measured by real - time PCR.The expression of signal transduction proteins, such as DR5 , caspase - 3, caspase - 8, caspasep -9, Bid, Bax and c - FLIP was detected by Western blotting.RESULTS : Both rsTRAIL and plumbagin induced the apoptosis in Kasumi cells, and combination of plumbagin with rsTRAIL enhanced the apoptosis.The ratios of Annexin V - positive Kasumi 1 cells were ( 27.7 ±2.9 )％ , ( 25.6 ±3.1 )％ and ( 52.1 ±3.3 )％ in 100 μg/L rsTRAIL group, 2 μ mol/L plumbagin group and the combination group, respectively, and the positive rate in comhination group was significantly higher than those in other 2 groups.TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone.Plumbagin up - regulated the expression of DR5 at mRNA level in Kasumi - 1 cells, and up - regulation of DR5 , activation of caspase - 8 and down - regulation of c - FLIP at protein level were detected in the cells treated with plumbagin alone and the comhination of plumhagin with rsTRAIL.CONCLUSION : Plumbagin enhances TRAIL - induced apoptosis in Kasumi - 1 cells by up - regulating DR5 , activating caspase - 8and down - regulating c - FLIP.