过氧化物酶体增殖物激活受体γ对人肝癌细胞生长和凋亡的影响及其机制

目的 检测过氧化物酶体增殖物激活受体γ(PPARγ)在人肝癌、癌旁和正常肝组织的表达,探讨激活或抑制PPARγ在肝癌细胞株生长和凋亡中的作用及其机制.方法 应用免疫组织化学方法检测20例肝癌、癌旁和正常肝组织中PPARγ的表达,Western blot检测2种肝癌和1种正常肝细胞株中PPARγ表达;PPARγ激动剂15dPGJ2和拮抗剂T0070907分别干预培养的2种肝癌细胞株,噻唑蓝(MTT)检测细胞生存率,流式细胞术(FCM)检测细胞周期及凋亡,试剂盒检测Caspase-3活性变化.结果 肝癌组织中PPARγ表达高于癌旁和正常肝组织(P＜0.05),PPARγ主要在肝癌细胞质中表达;肝癌细胞株PPARγ表达比正常肝细胞株高(P＜0.05);15dPG-J2抑制肝细胞增殖而T0070907则促进肝癌细胞增殖(P＜0.05).15dPG-J2可使肝癌HepG2和SMMC7721细胞G0/G1期细胞比例由(42.5±0.9)%和(49.0±3.8)%增高至(61.0±2.0)%和(67.5±2.2)%,(P＜0.05),S期由(30.5±0.3)%和(43.0±2.5)%降低至(6.6±1.1)%和(25.1±2.0)%(P＜0.05).而且,15dPG-J2可明显促进两种肝癌细胞凋亡,凋亡率分别增加约24.4%和22.4%,并可增强细胞Caspase-3活性(P＜0.05);Caspase抑制剂Z-VAD-FMK则可逆转15dPG-J2对肝癌细胞的促凋亡作用.结论 PPARγ在肝癌细胞中表达升高,其表达主要位于细胞质中,为无活性状态.PPARγ激动剂可活化PPARγ和Caspase通路,抑制肝癌细胞生长,促进细胞凋亡.

Abstract：

Objective To investigate the expression of peroxisome proliferator-activated receptor γ (PPARγ) in human liver cancer tissue and cells, and to explore the effects and mechanisms of PPARγ on growth and apoptosis in human hepatocellular carcinoma cells. Methods The expression of PPARγ in 20 cases of liver cancer, tumor adjacent tissue, normal liver, one liver cell line and two liver cancer cell lines were detected by immunohistochemistry or Western blotting. Two incubated malignant cell lines were exposed to PPARγ agonist 15dPGJ2 or antagonist T0070907, cell viability was determined by methyl thiazol tetrazolium (MTT) assay and cell cycle distribution and apoptosis were examined by flow cytometry (FCM). Consequently, the activity of caspase-3 following treatments of 15dPGJ2 or T0070907 was observed by the commercial Caspase-3 Activity Kit. Results The expression of PPARγin liver cancer was higher than that in adjacent and normal liver tissue ( P ＜ 0. 05) and PPART was predominantly expressed in the cytoplasm. Further,the expression of PPARγwas higher in hepatoma cell line than that in normal liver cell line (P＜ 0.05 ). PPARγ agonist 15dPG-J2 inhibited the proliferation while the antagonist T0070907 induced the growth of hepatoma cells (P ＜ 0. 05). G0/G1 phase of HepG2 and SMMC772 cells was blocked by 15dPG-J2, and the ratio were raised from (42.5 ±0.9)% and (49.0 ±3.8)% to (61.0 ±2. 0)% and (67.5 ±2. 2)% ,respectively. Meantime,S phase ratio were decreased from (30. 5 ± 0. 3 ) % and (43.0 ± 2. 5 ) % to ( 6. 6 ± 1. 1 ) % and ( 25. 1 ± 2. 0 ) %. Further, 15 dPG-J2 facilitated significant apoptosis in those cell lines and the increased apoptosis ratio were 24. 4% and 22. 4% respectively.Accordingly,the activity of Caspase-3 induced by 15dPGJ2 were 21-and 23-fold higher than those in control groups (P ＜ 0. 05 ). The Caspase inhibitor Z-VAD-fmk expectedly reversed the apoptosis induced by 15dPGJ2 in HepG2 and SMMC772 cells. Conclusion The expression of PPARγ increases in human hepatoma predominantly in cytoplasm with its inactivated form. 15dPG-J2 ,an agonist of PPART inhibits growth and induces apoptotic in hepatoma cells through activation of PPARγ pathway and the consequent Caspase3 signal.