戊地昔布对直肠癌Colo320细胞增殖与凋亡的影响  

Effects of Valdecoxib on the proliferation and apoptosis of human rectal cancer Colo320 cells

作　　者：冷洁[1] 赵晶[2] 高向阳[2] 宋超[2] 高超[2] 

机构地区：[1]徐州医学院研究生学院,江苏徐州221004 [2]徐州医学院附属医院肿瘤科,江苏徐州221004

出　　处：《中华肿瘤防治杂志》2016年第10期636-641,647,共7页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的戊地昔布为第2代选择性环氧合酶-2（cyclooxygenase-2，COX-2）抑制剂，主要用于抗炎止痛，已被证实具有拮抗多种肿瘤细胞的作用，但其对直肠癌细胞的作用鲜有报道。本研究探讨其对人直肠癌细胞Colo320增殖与凋亡的影响并探讨其可能的机制。方法以药物终浓度为0、5、10、25、50、100、200μmol／L的戊地昔布分别处理Colo320细胞24、48、72及96h后，采用Cell Counting Kit-8（CCK-8）实验检测戊地昔布对细胞增殖能力的影响；采用细胞克隆形成实验检测戊地昔布对Colo320细胞克隆形成率的影响；采用流式细胞术检测戊地昔布对细胞凋亡率及细胞周期分布的影响；采用蛋白质印迹实验检测戊地昔布作用后细胞内COX-2蛋白的表达变化，以及凋亡相关蛋白Caspase-3、cleaved Caspase-3、Bax、Bcl-2、p38MAPK及P—p38MAPK蛋白的表达变化。结果CCK8结果经统计学分析提示，不同浓度（0、5、10、25、50、100、200μmol／L）戊地昔布分别作用于直肠癌Colo320细胞24、48、72及96h后，细胞增殖受到不同程度的抑制，并呈时间（r=0．686～0．972，P〈0．001）及浓度（r=0．829～O．976，P〈0．001）依赖性。24、48、72、96h的IC50值分别为582、153、136和61μmol／L；细胞克隆形成实验显示，戊地昔布处理后细胞克隆形成率由（28．5±1．2）％下降至（3．3±1.0）％，各组比较差异有统计学意义，F=454．227，P〈0．001。流式结果显示，戊地昔布使细胞凋亡率明显增加，由9．3％增加到30．8％，除50umol／L组与对照组之间差异无统计学意义（t=-3．849，P=0．613）外，其余各组之间差异有统计学意义（F=224．694，P=0．001），但对细胞周期阻滞现象不明显。蛋白质印迹实验结果显示，戊地昔布使细胞内COX-2表达量降低（F=36．771，P=0．002），cleavedCaspase-3／Caspase=3（F=161．097，P〈0．001）、P—p38MAPK／p38MAPK（F=104．770，POBJECTIVE Valdeeoxib is the second generation of selective COX-2 inhibitor, which mainly used for anti-inflammatory and analgesic, has been proved to have inhibiting effects on various kinds of tumor cells, but few re- ports of its effect on colorectal cancer cells were reported. This study discussed the effects of valdecoxib on proliferation and apoptosis of human colorectal cancer Colo320 cells and investigated the possible mechanisms involved. METHODS Colo320 cells were cultured in vitro and treated with valdecoxib （0, 5, 10, 25, 50, 100, 200 μmol/L） for 24, 48, 72 and 96 hours. The proliferation inhibitory rates were detected by CCK-8 assay. Colony formation assay was performed to de- tect the effect of valdecoxib on growth of Colo320 cells. The cell cycle and apoptotic rate were detected by propidiumio- dide staining for fluorescence-activated cell sorting （FACS）. The apoptosis-related protein expression levels of COX=2,Bax, Bcl-2, p38MAPK, P-p38MAPK, Caspase-3 and cleaved Caspase-3 were measured and analyzed by western blotting. RESULTS CCK-8 results through statistical analysis suggest that valdecoxib treatment could remarkably inhibit the pro- liferation of Colo320 cells in a dose （r=0. 829-0. 976,P〈0.01） and time （r=0. 686-0. 972,P〈0.01） dependent man- ner compared with the control. After treating Colo320 for 24, 48, 72 and 96 h, the IC50 values were 582, 153, 136, 61 μmol/L respectively. As demonstrated in colony formation assay, the colony formation rate decreased significantly from （28.5±1.2）% to （3.3± 1.0）% （F=454. 227,P〈0. 001）. Annexin V-FITC/PI double staining flow cytometry showed valdecoxib induced apoptosis in Colo320 cells in a concentration- and time- dependent manner. The apoptosis rate of Colo320 cells was significantly increased from 9. 3% to 30. 8% after treated with valdecoxib. In addition to the 50 μmol/L group （t=- 3. 849, P= 0. 613）, the difference between the other groups was statistically significant （F= 224. 694, P= 0. 001）. The phenom

关 键 词：直肠肿瘤 Colo320细胞 戊地昔布 环氧合酶-2抑制剂 增殖 凋亡 

分 类 号：R735.37[医药卫生—肿瘤]