Liu Jia-jun, Liu Wen-da, Liu Xiao-dan, Raj-Shrestha Prem, Liu Pei-qing, Huang He-qing, Wu Chuan-bin, Wang Chun-zhi, Xu Yan, Xiao Ruo-zhi, Lin Dong-jun, Huang Ren-wei
Hematological Department & Institute, Third Hospital of Sun Yat-Sen University, Guangzhou 510630, China.
Zhonghua Yi Xue Za Zhi. 2010 Aug 24;90(32):2270-4.
To investigate the apoptosis-inducing effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone (CGZ) on leukemic HL-60 cells and its mechanisms of action.
HL-60 cells in vitro culture medium were subject to different concentrations of CGZ (10-50 µmol/L) for 24, 48 and 72 h. MTT assay was used to detect the cell inhibitory rate and agarose gel electrophoresis to observe DNA fragmentation. Flow cytometry (FCM) and Annexin V/PI staining were used to detect CGZ and/or GW9662 (PPARγ antagonist)-induced cell apoptosis. The expression of PPARγ was examined by RT-PCR and Western blotting. The caspase-3 and protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) were also detected.
CGZ (over 30 µmol/L) could inhibit the growth of HL-60 cells in both time- and dose-dependent manner. After treatment for 72 h, the cell growth inhibitory rate in 50 µmol/L CGZ (84% ± 11%) treated cells was found more higher than that in both 40 µmol/L and 30 µmol/L CGZ treated cells (72% ± 13%, 59% ± 13%, P < 0.01) and a typical DNA ladder was also observed by agarose gel electrophoresis. The expression of PPARγ was gradually up-regulated by CGZ treatment and could be down-regulated partially by PPARγ antagonist GW9662. The results also revealed that CGZ-induced cell apoptosis (49.7%, 72 h) could not be inhibited thoroughly by GW9662 (36.2%, control:3.2%). It indicated that the CGZ-induced cell apoptosis was partially PPARγ-independent. Western blotting showed a cleavage of caspase-3 zymogen protein and up-regulation of p-P38 protein. Thus it indicated that the activations of caspase-3 and P38 MAPK were involved in CGZ-induced cell apoptosis.
CGZ inhibits cell growth by induction of cell apoptosis in HL-60 cells via PPARγ dependent and independent signaling pathways. The activations of caspase-3 and P38 MAPK may be one of the important mechanisms in CGZ in treated HL-60 cells.
探讨过氧化物酶体增殖物激活受体γ（PPARγ）激动剂吡格列酮（CGZ）对白血病HL-60细胞的凋亡诱导作用及其作用机制。
将体外培养基中的HL-60细胞用不同浓度的CGZ（10 - 50 μmol/L）处理24、48和72小时。采用MTT法检测细胞抑制率，琼脂糖凝胶电泳观察DNA片段化。流式细胞术（FCM）和Annexin V/PI染色用于检测CGZ和/或GW9662（PPARγ拮抗剂）诱导的细胞凋亡。通过RT-PCR和蛋白质印迹法检测PPARγ的表达。还检测了丝裂原活化蛋白激酶信号通路（MAPKs、p-P38、p-ERK和p-JNK）中的半胱天冬酶-3和蛋白水平。
CGZ（超过30 μmol/L）能以时间和剂量依赖性方式抑制HL-60细胞的生长。处理72小时后，发现50 μmol/L CGZ处理的细胞（84% ± 11%）的细胞生长抑制率高于40 μmol/L和30 μmol/L CGZ处理的细胞（72% ± 13%，59% ± 13%，P < 0.01），并且琼脂糖凝胶电泳也观察到典型的DNA梯带。CGZ处理可使PPARγ的表达逐渐上调，PPARγ拮抗剂GW9662可部分下调其表达。结果还显示，GW9662（36.2%，对照组：3.2%）不能完全抑制CGZ诱导的细胞凋亡（49.7%，72小时）。这表明CGZ诱导的细胞凋亡部分不依赖于PPARγ。蛋白质印迹显示半胱天冬酶-3酶原蛋白的裂解和p-P38蛋白的上调。因此表明半胱天冬酶-3和P38 MAPK的激活参与了CGZ诱导的细胞凋亡。
CGZ通过PPARγ依赖性和非依赖性信号通路诱导HL-60细胞凋亡来抑制细胞生长。半胱天冬酶-3和P38 MAPK的激活可能是CGZ处理HL-60细胞的重要机制之一。
