蟾蜍灵在诱导HL-60细胞凋亡过程中对Bcl-2和PKC的影响  

Effect of Bufalin-Inducing Apoptosis on Bcl-2 and PKC in HL-60 Cells

作　　者：田昕[1] 王萍萍[1] 刘云鹏[1] 侯科佐[1] 金波[1] 罗颖[1] 曲秀娟[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤内科,沈阳110001

出　　处：《中国实验血液学杂志》2007年第1期67-71,共5页Journal of Experimental Hematology

基　　金：辽宁省科学技术厅基金资助项目;编号2004225004-11

摘　　要：为了研究蟾蜍灵在诱导HL-60细胞凋亡过程中对Bcl-2表达与裂解和磷酸化的作用,及对蛋白激酶C(PKC)活性与PKCs亚细胞定位的影响,分别采用台盼蓝拒染法检测细胞生存率,细胞染色法观察凋亡形态,流式细胞术分析细胞周期,琼脂糖凝胶电泳检测凋亡DNA片段化,[γ-32P]同位素掺入法测定PKC活性,Westernblot分析Bcl-2及PKC蛋白表达。结果表明:①蟾蜍灵抑制HL-60细胞增殖,24、48及72小时的IC50分别为(25.8±2·1)、(8.0±1.2)及(2.3±0.3)nmol/L;②50nmol/L蟾蜍灵作用24小时可诱导HL-60细胞凋亡;③50nmol/L蟾蜍灵处理HL-60细胞6-24小时,Bcl-2蛋白表达水平明显下调,并出现23kD的裂解片段,磷酸化水平逐渐降低;④1-100nmol/L蟾蜍灵分别作用30分钟,对PKC总活性无影响,但可促使PKCβⅡ膜转位。结论:蟾蜍灵诱导HL-60细胞凋亡可能与Bcl-2表达降低、裂解及脱磷酸化有关。Previous study revealed that bufalin can inhibit proliferation, and induce apoptosis in some human cancer cell lines. However, the mechanism of its anticancer effect has not been fully understood. The present study was designed to investigate the effects of bufalin-induced apoptosis on Bcl-2 and PKC in human leukemic HL-60 cells. The cell viability was determined by trypan blue dye exclusion. The apoptosis was detected by morphology, flow cytometry and DNA agarose gel electrophoresis. The expressions of Bcl-2 and PKC were analyzed by Western blot, and activity of PKC was assayed by [ γ-^32P] isotope incorporation method. The results showed as follows： （ 1 ） proliferation of HL-60 cells was inhibited by bufalin and the IC50 at 24, 48, 72 hours were （ 25.8 ± 2.1 ）, （ 8.0 ± 1.2 ） and （ 2.3 ± 0.3 ） nmol/ L, respectively. （2） apoptosis of HL-60 cells was induced when the cells were treated with bufalin at concentration of 50 nmol/L for 24 hours. （3） compared with control, treatment with bufalin at concentration of 50 nmol/L for 6 - 24 hours resulted in downregulation of protein expression, decrease of pbospborylation, and cleavage of Bcl-2, simultaneously. （4） the activity of total PKC was unchanged when HL-60 cells were exposed to 1 - 100 nmol/L bufalin for 30 minutes, but PKCβⅡ underwent translocation from cytosol to membrane. It is concluded that apoptosis induced by bufalin is associated with downregulation of protein expression, dephosphorylation, and cleavage of Bcl-2 in HL-60 cells.

关 键 词：白血病 蟾蜍灵 凋亡 BCL-2 蛋白激酶C 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]