摘要
目的 探讨过氧化物酶体增殖物激活受体γ（PPARγ）激动剂环格列酮（CGZ）对白血病HL-60细胞的增殖抑制作用及其作用机制.方法 以不同浓度的CGZ（10～50μmol/L）作用于体外培养的HL-60细胞24、48、72 h,应用四甲基偶氮唑盐（MTT）法检测细胞生长抑制率,琼脂糖凝胶电泳检测细胞凋亡时的DNA梯状条带,流式细胞术（FCM）及膜联蛋白V（Annexin-V）/碘化丙啶（PI）双染色法检测CGZ单独及联合PPARγ拮抗剂GW9662作用后细胞凋亡率的变化,反转录-聚合酶链反应（RT-PCR）及Western印迹检测药物作用后PPARγ表达水平的变化,并对半胱氨酸蛋白酶3（caspase-3）及丝裂原活化蛋白激酶（MAPK）信号转导通路相关蛋白的表达水平进行检测.结果 30 μmol/L以上的CGZ可显著抑制细胞的生长,并呈现出明显的量-效与时-效关系.药物作用后72 h50 μmol/L CGZ对细胞的生长抑制率为84%±11%,明显高于40、30μmol/L CGZ对细胞的生长抑制率（72%±13%、59%±13%,P＜0.01）,而且药物作用72 h后在琼脂糖凝胶电泳上可见典型的DNA梯状条带.RT-PCR及Western印迹结果表明,作用72 h后随着药物浓度的升高,PPARγ mRNA及蛋白的表达水平均逐渐升高,5μmol/L GW9662可以部分阻滞PPARγ的表达.FCM检测结果显示,CGZ（50 μmol/L）诱导的细胞凋亡只能部分被GW9662所阻滞（药物作用后72 h,CGZ诱导的细胞凋亡率为49.7%,CGZ＋GW9662的细胞凋亡率为36.2%,未加药对照组为3.2%）.Western印迹检测结果显示,MAPK信号途径中磷酸化P38（p-P38）的表达水平随药物浓度逐渐升高,同时caspase-3被活化出现相对分子质量为20 000的亚单位.结论 CGZ可以通过PPARγ依赖性和非依赖性途径诱导HL-60细胞凋亡,通过caspase-3活化以及激活P38 MAPK信号途径是CGZ诱导白血病HL-60细胞发生凋亡的重要作用机制之一.
Objective 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. Methods 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 bloting. The caspase-3 and protein levels in mitogen-activated protein kinase signaling pathways （MAPKs, p-P38, p-ERK and p-JNK） were also detected. Results 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 bloting showed a cleavage of caspase-3 zymogen protein and upregulation of p-P38 protein. Thus it indicated that the activations of caspase-3 and P38 MAPK were involved in CGZ-induced cell apoptosis. Conclusion 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 P38MAPK may be one of the important mechanisms in CGZ in treated HL-60 cells.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第32期2270-2274,共5页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30570786）
教育部新世纪优秀人才支持计划资助项目（NCET-06-0721）
广东省自然科学基金（8151008901000128）
