环氧合酶-2对人肝癌细胞增殖和凋亡的调节作用

Modulatory roles of cyclooxygenase-2 in proliferation and apoptosis of hepatocarcinoma cells

目的:观察环氧合酶-2(cyclooxygenase-2, COX-2)在肝癌细胞中表达,探讨COX-2抑制剂celecoxib对肝癌细胞增殖和凋亡的作用．方法:免疫细胞化学、逆转录聚合酶链反应(RT-PCR)方法研究COX-2在肝癌细胞株中表达;MTT法观察COX-2抑制剂对肝癌细胞增殖的影响;透射电镜及流式细胞仪观察 celecoxib诱导肝癌细胞凋亡的作用、对细胞周期的影响及MDR1／P-gp表达的变化;用RTPCR 方法检测Survivin mRNA药物处理后表达的变化．结果:celecoxib对肝癌细胞抑制增殖、诱导凋亡呈时间和剂量依赖性．celecoxib作用HepG2 48 h抑制率为70．98％±0．67％(200 μmol／L)、 47．93％±1．08％(100 μmol／L);Bel-7402为 57．29％±0．67％(200 μmol／L)、43．84％± 0．86％(100 μmol／L);同样浓度但作用20 h, HepG2为45．51％±1．35％(200 μmol／L), 14.35％±1．55％(100 μmol／L);Bel-7402则为34．35％±0．63％(200 μmol／L),15．35％± 0．88％(100 μmol／L),不同浓度以及不同作用时间相比均有显著差异(P<0．01);100 μmol／L celecoxib作用24,48,72,96 h的肝癌细胞凋亡率分别为12．2％±2．44％,4．0％±1．67％,20．4％±4．38％,57．9％±5．74％(HepG2)和3．0％± 1．05％,18．5％±3．51％,29.3％±3．25％,48．4％±4．77％(Bel-7402),与对照组相比有显著差异(P<0．01);细胞周期分布改变,G0／G1期细胞比例增加,24,48,72 h分别为:44．17％±1．01％,59．60％±0．61％,62．7％±1．22％ (HepG2)和47．80％±0．41％,58．60％±0．46％, 78．40％±1．95％(Bel-7402),与对照组比较有显著差异(P<0．01);对照组PCNA蛋白表达呈强阳性(+++),经药物处理后表达减弱,并随时间延长而显著;HepG2中COX-2蛋白表达明显弱于Bel-7402,药物处理后表达也不同．Survivin在肝癌细胞株中呈高表达状态, celecoxib作用48 h mRNA表达降至零,而72 h 后表达水平又有上升;两株肝癌细胞中经 celecoxib处理后,MDR1／P-gp表达有降低的趋势(Bel-7402),或是基本上不受影响(HepG2)．结论:COX-2抑制剂celecoxib体外对肝癌细胞有较强的细胞毒作用且以剂量、时间依赖方式抑制细胞增殖,并诱导凋亡,使细胞周期阻滞于G1／S期．COX-2与P-gp,Survivin表达密切相关．

AIM： To observe the expression of cyclooxygenase-2 （COX-2） and investigate the effects of COX-2 inhibitor, celecoxib, on the proliferation and apoptosis of hepatocarcinoma cells. METHODS： The expression of COX-2 was detected by immunohistochemistry and reverse transcription polymerase chain reaction （RT- PCR）. The effect of celecoxib on the growth of hepatocarcinoma cells （HepG2 and Bel-7402） were evaluated by MTT assay and changes proliferation cell nuclear antigen （PCNA）. The apoptosis of hepatocarcinoma cells induced by celecoxib and P-gp expression were detected by electron microscopy and flow cytometry. The expression of Survivin was analyzed by RT-PCR. RESULTS： Celecoxib inhibited the growth and induced apoptosis of hepatocarcinoma cells in a time- and concentration-dependent manner. The inhibitory rates for HepG2 cells were 70.98% ± 0.67% and 47.93% ± 1.08% after the cells were exposed to 200 and 100 μmol/L celecoxib, respectively, for 48 h, and 45.51% ± 1.35% and 14.35% ± 1.55% for 24 h; while the rates for Bel-7402 cells were 57.29% ± 0.67% and 43.84% ± 0.86% for 48 h, and 34.35% ± 0.63% and 15.35% ± 0.88% for 24 h （all P 〈 0.01）. Substantial apoptosis was revealed by increment of apoptotic body under electron microscopy. After treatment with 100 μmol/L celecoxib for 24, 48, 72 and 96 h, the apoptosis rates of HepG2 cells were 12.2% ± 2.44%, 4.0% ± 1.67%, 20.4% ± 4.38%, and 57.9% ± 5.74%, respectively, while those of Bel-7402 cells were 3.0% ± 1.05%, 18.5% ± 3.51%, 29.3% ± 3.25%, and 48.4% ± 4.77%, respectively, which were significantly different from those in the controls. The percentage of G0/G1 phase cells was markedly increased （HepG2： 44.17% ± 1.01%, 59.60% ± 0.61%, 62.7% ± 1.22%; Bel-7402： 47.80% ± 0.41%, 58.60% ± 0.46%, 78.40% ± 1.95%; at 24, 48, and 72 h, respectively） in comparison with that of the controls （P 〈 0.01）. COX-2 protein expression was obviously lower in HepG2 cells than that in Bel-7402 cells. PCNA expression was greatly lessened after celecoxib treatment. COX-2 and Survivin were down-regulated by celecoxib, but not linearly correlated. MDR1/P-gp expression was reduced or changed little as time went by after celecoxib treatment. CONCLUSION： COX-2 inhibitor, celecoxib, inhibits the proliferation and induces the apoptosis of hepatocarcinoma cells in vitro a timeand concentration-dependent manner by downregulating the expression of COX-2 and Survivin. COX-2 is correlated with MDR/P-gp and Survivin expression.