As_4S_4对人白血病细胞HL-60细胞COX-2表达和PGE_2水平的影响

Experimental studies on As_4S_4-induced human-leukemia cell HL-60 cell apoptosis and molecular mechanism

目的体外研究中药雄黄主要成分(As4S4)对白血病细胞HL-60细胞的增殖和诱导凋亡的作用及其可能的机制。方法以不同浓度的As4S4(7.5、15、30、60mmol/L),分别处理24h,48h,72hHL-60细胞,用四甲基偶氮唑蓝(MTT)法测定细胞增殖抑制率;流式细胞仪(FCM)检测细胞凋亡率;Western blot检测环氧合酶-2(COX-2)蛋白的表达,用放射免疫法检测细胞PGE2释放水平。结果不同浓度(7.5、15、30、60mmol/L)的As4S4处理的HL-60细胞,细胞增殖受到抑制,作用呈明显的时效和量效关系。24h时IC50为30mmo/L,与对照组相比,差异有显著性(P<0.01)。As4S4诱导HL-60细胞的凋亡率分别为(7.28±1.03)%、(26.98±2.21%)、(47.15±2.66)%、(61.73±3.12)%,与对照组比较(3.84±1.88)%,差异有极显著性(P<0.01),并呈浓度依赖性,As4S4明显抑制COX-2的活性和表达,随着浓度的增加,其COX-2蛋白表达逐渐降低,与对照组相比,差异有显著性(P<0.05);不同浓度As4S4作用24h,可明显抑制Hela细胞PGE2释放水平,其值分别为(70.56±2.03)、(48.58±2.28)、(29.25±1.57)和(18.02±1.04),与对照组比较(3.15±1.01)差异有显著性(P<0.01)。结论As4S4体外对HL-60细胞具有增殖抑制作用,并促进其凋亡,其机制可能与抑制COX-2活性表达和抑制细胞释放PGE2水平有关。

Objective To investigate the Human-Leukemia cell HL-60 cells growth inhibition and apoptosis induced by As4S4 and its molecular mechanisms. Methods HL-60 cells were treated with various concentrations（7.5,15,30, 60mmol/ L ） of As4S4.Cell growth was measured by MTT, apoptosis was detected by double staining flow cytometry （FCM）. Levels of PGE2 was measured by radioimmunoassay. The expression of COX-2 protein was also examined by Western blot analysis. Results After treated with different concentrations of As4S4, the cell growth of HL-60 was suppressed significantly with showed dose-and time-dependent manner increase in proliferative inhibition rate. The IC50 of 24 hours was 30mmol/1. （P〈0.01）. The As4S4 can induce apoptosis and the apoptosis rate was 7.28%-60.73% by flow cytometery （FCM）, and the in dose-dependent. As4S4 the release of PGE2 was reduced in HL-60 cells with the values being （70.56 ±2.03）, （48.58 ±2.28）, （29.25 ±1.57）and （18.02±1.04） respectively, as compared with control group （3.15±0.01） （P〈0,01）. The As4S4 could inhibit the activity and expression of COX-2, with the rise of concentration; can down-regnlation the expression of COX-2 protein greatly. Conclusion As4S4 could inhibit the proliferation and increase apoptosis in human leukemia HL-60 cells. These effects maybe depend on the inhibition of the expression of COX-2 and depend on the inhibition of level of PGE2 by As4S4.