戊地昔布对直肠癌Colo320细胞增殖与凋亡的影响
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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 CCK-8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=0.829~0.976,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50μmol/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),cleaved Caspase-3/Caspase-3(F=161.097,P<0.001)、P-p38MAPK/p38MAPK(F=104.770,P<0.001)和Bax/Bcl-2(F=370.001,P<0.001)比率明显升高。结论戊地昔布能够抑制直肠癌Colo320细胞增殖,促进其凋亡,可能是通过抑制COX-2蛋白的表达,调节凋亡相关蛋白Bax和Bcl-2的表达,及激活MAPK和Caspase信号通路而实现的。
OBJECTIVE Valdecoxib is the second generation of selective COX-2inhibitor,which mainly used for anti-inflammatory and analgesic,has been proved to have inhibiting effects on various kinds of tumor cells,but few reports 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.METHODSColo320 cells were cultured in vitro and treated with valdecoxib(0,5,10,25,50,100,200μmol/L)for 24,48,72and96 hours.The proliferation inhibitory rates were detected by CCK-8assay.Colony formation assay was performed to detect the effect of valdecoxib on growth of Colo320 cells.The cell cycle and apoptotic rate were detected by propidiumiodide staining for fluorescence-activated cell sorting(FACS).The apoptosis-related protein expression levels of COX-2,Bax,Bcl-2,p38 MAPK,P-p38 MAPK,Caspase-3and cleaved Caspase-3were measured and analyzed by western blotting.RESULTS CCK-8results through statistical analysis suggest that valdecoxib treatment could remarkably inhibit the proliferation 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 manner compared with the control.After treating Colo320 for 24,48,72 and 96h,the IC50 values were 582,153,136,61μmol/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 the50μmol/L group(t=-3.849,P=0.613),the difference between the other groups was statistically significant(F=224.694,P=0.001).The phenomenon of cell cycle arresting is not obvious.Western blot assay showed that after valdecoxib treatment,the protein expression level of COX-2was significantly dowregulated(F=36.771,P=0.002),the ratio of cleaved Caspase-3/Caspase-3was dowregulated(F=161.097,P<0.001),the ratio of Bax/Bcl-2(F=370.001,P<0.001)and P-p38MAPK/p38MAPK(F=104.770,P<0.001)were apparently upregulated.CONCLUSIONS Valdecoxib can inhibit proliferation and promoted apoptosis of Colo320 cells.The potential mechanism by which valdecoxib exhibited its antitumor activity is increasing apoptosis rate,downregulating the expression of COX-2,regulating the expression of apoptosis-related proteins Bax and Bcl-2as well as activating caspase or MAPK signaling pathways.
OBJECTIVE Valdecoxib is the second generation of selective COX-2inhibitor,which mainly used for anti-inflammatory and analgesic,has been proved to have inhibiting effects on various kinds of tumor cells,but few reports 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.METHODSColo320 cells were cultured in vitro and treated with valdecoxib(0,5,10,25,50,100,200μmol/L)for 24,48,72and96 hours.The proliferation inhibitory rates were detected by CCK-8assay.Colony formation assay was performed to detect the effect of valdecoxib on growth of Colo320 cells.The cell cycle and apoptotic rate were detected by propidiumiodide staining for fluorescence-activated cell sorting(FACS).The apoptosis-related protein expression levels of COX-2,Bax,Bcl-2,p38 MAPK,P-p38 MAPK,Caspase-3and cleaved Caspase-3were measured and analyzed by western blotting.RESULTS CCK-8results through statistical analysis suggest that valdecoxib treatment could remarkably inhibit the proliferation 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 manner compared with the control.After treating Colo320 for 24,48,72 and 96h,the IC50 values were 582,153,136,61μmol/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 the50μmol/L group(t=-3.849,P=0.613),the difference between the other groups was statistically significant(F=224.694,P=0.001).The phenomenon of cell cycle arresting is not obvious.Western blot assay showed that after valdecoxib treatment,the protein expression level of COX-2was significantly dowregulated(F=36.771,P=0.002),the ratio of cleaved Caspase-3/Caspase-3was dowregulated(F=161.097,P<0.001),the ratio of Bax/Bcl-2(F=370.001,P<0.001)and P-p38MAPK/p38MAPK(F=104.770,P<0.001)were apparently upregulated.CONCLUSIONS Valdecoxib can inhibit proliferation and promoted apoptosis of Colo320 cells.The potential mechanism by which valdecoxib exhibited its antitumor activity is increasing apoptosis rate,downregulating the expression of COX-2,regulating the expression of apoptosis-related proteins Bax and Bcl-2as well as activating caspase or MAPK signaling pathways.
引文
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[11]张曼丽,王慧慈,柳金金,等.戊地昔布通过上调ROS诱导人乳腺癌MCF-7细胞凋亡[J].基础医学与临床,2015,35(4):491-495.
[12]徐清华,侯晓敏,王慧慈,等.戊地昔布联用吡柔比星对人肺癌A549细胞凋亡的影响[J].中国药学杂志,2013,48(12):972-975.
[13]Cheng J,Fan XM.Role of cyclooxygenase-2in gastric cancer development and progression[J].World J Gastroenterol,2013,19(42):7361-7368.
[14]Masunaga R,Kohno H,Dhar DK,et al.Cyclooxygenase-2expression correlates with tumor neovascularization and prognosis in human colorectal carcinoma patients[J].Clin Cancer Res,2000,6(10):4064-4068.
[15]Tomozawa S,Tsuno NH,Sunami E,et al.Cyclooxygenase-2overexpression correlates with tumour recurrence,especially haematogenous metastasis,of colorectal cancer[J].Br J Cancer,2000,83(3):324-328.
[16]韩宏斌,梁宁震,周怡萍,等.NF-κB、COX-2在结直肠癌组织中的表达及其临床意义[J].临床和实验医学杂志,2015,14(16):1342-1344.
[17]李焕焕,苏帆,李晶,等.塞来昔布联合吉非替尼对HCC827细胞凋亡影响机制探讨[J].中华肿瘤防治杂志,2015,22(6):432-435.
[18]Chen C,Zhang X,Zhang S,et al.Quantification of protein copy number in single mitochondria:The Bcl-2family proteins[J].Biosens Bioelectron,2015,(74):476-482.
[19]Frejlich E,Rudno-rudzinska J,Janiszewski K,et al.Caspases and their role in gastric cancer[J].Adv Clin Exp Med,2013,22(4):593-602.
[20]Hakozaki M,Tajino T,Konno S,et al.Overexpression of cyclooxygenase-2in malignant peripheral nerve sheath tumor and selective cyclooxygenase-2inhibitor-induced apoptosis by activating caspases in human malignant peripheral nerve sheath tumor cells[J/CD].PLoS One,2014,9(2):e88035.
[21]Chun J,Joo EJ,Kang M,et al.Platycodin D induces anoikis and caspase-mediated apoptosis via p38 MAPK in AGS human gastric cancer cells[J].J Cell Biochem,2013,114(2):456-470.
[22]Kim EK,Choi EJ.Compromised MAPK signaling in human diseases:an update[J].Arch Toxicol,2015,89(6):867-882.
[2]Vosooghi M,Amini M.The discovery and development of cyclooxygenase-2inhibitors as potential anticancer therapies[J].Expert Opin Drug Discov,2014,9(3):255-267.
[3]辛兵,吉凯强.非甾体类消炎药对卵巢癌抑制作用的研究[J].中华肿瘤防治杂志,2015,22(16):1266-1269.
[4]柯晗昵,曲显俊.乙酰基-11-酮基-β-乳香酸与阿斯匹林对HCT-116作用的比较研究[J].中华肿瘤防治杂志,2015,22(2):103-108.
[5]Souza RF,Shewmake K,Beer DG,et al.Selective inhibition of cyclooxygenase-2suppresses growth and induces apoptosis in human esophageal adenocarcinoma cells[J].Cancer Res,2000,60(20):5767-5772.
[6]张德庆,祝建红,陈卫昌.塞来昔布联合5-FU抑制裸鼠结肠癌生长及其机制的探讨[J].中华肿瘤防治杂志,2013,20(1):15-20.
[7]Shao D,Kan M,Qiao P,et al.Celecoxib induces apoptosis via a mitochondriadependent pathway in the H22mouse hepatoma cell line[J].Mol Med Rep,2014,10(4):2093-2098.
[8]Liu DB,Long GX,Mei Q,et al.Anticancer effects of celecoxib through inhibiton of STAT3phosphorylation and AKT phosphorylation in nasopharyngeal carcinoma cell lines[J].Pharmazie,2014,69(5):358-361.
[9]Dong X,Li R,Xiu P,et al.Meloxicam executes its antitumor effects against hepatocellular carcinoma in COX-2-dependent and-independent pathways[J/CD].PLoS One,2014,9(3):e92864.
[10]韦金英,刘玉,李宏博,等.戊地昔布对人结肠癌HT-29细胞凋亡的影响[J].肿瘤防治研究,2014,41(6):549-551.
[11]张曼丽,王慧慈,柳金金,等.戊地昔布通过上调ROS诱导人乳腺癌MCF-7细胞凋亡[J].基础医学与临床,2015,35(4):491-495.
[12]徐清华,侯晓敏,王慧慈,等.戊地昔布联用吡柔比星对人肺癌A549细胞凋亡的影响[J].中国药学杂志,2013,48(12):972-975.
[13]Cheng J,Fan XM.Role of cyclooxygenase-2in gastric cancer development and progression[J].World J Gastroenterol,2013,19(42):7361-7368.
[14]Masunaga R,Kohno H,Dhar DK,et al.Cyclooxygenase-2expression correlates with tumor neovascularization and prognosis in human colorectal carcinoma patients[J].Clin Cancer Res,2000,6(10):4064-4068.
[15]Tomozawa S,Tsuno NH,Sunami E,et al.Cyclooxygenase-2overexpression correlates with tumour recurrence,especially haematogenous metastasis,of colorectal cancer[J].Br J Cancer,2000,83(3):324-328.
[16]韩宏斌,梁宁震,周怡萍,等.NF-κB、COX-2在结直肠癌组织中的表达及其临床意义[J].临床和实验医学杂志,2015,14(16):1342-1344.
[17]李焕焕,苏帆,李晶,等.塞来昔布联合吉非替尼对HCC827细胞凋亡影响机制探讨[J].中华肿瘤防治杂志,2015,22(6):432-435.
[18]Chen C,Zhang X,Zhang S,et al.Quantification of protein copy number in single mitochondria:The Bcl-2family proteins[J].Biosens Bioelectron,2015,(74):476-482.
[19]Frejlich E,Rudno-rudzinska J,Janiszewski K,et al.Caspases and their role in gastric cancer[J].Adv Clin Exp Med,2013,22(4):593-602.
[20]Hakozaki M,Tajino T,Konno S,et al.Overexpression of cyclooxygenase-2in malignant peripheral nerve sheath tumor and selective cyclooxygenase-2inhibitor-induced apoptosis by activating caspases in human malignant peripheral nerve sheath tumor cells[J/CD].PLoS One,2014,9(2):e88035.
[21]Chun J,Joo EJ,Kang M,et al.Platycodin D induces anoikis and caspase-mediated apoptosis via p38 MAPK in AGS human gastric cancer cells[J].J Cell Biochem,2013,114(2):456-470.
[22]Kim EK,Choi EJ.Compromised MAPK signaling in human diseases:an update[J].Arch Toxicol,2015,89(6):867-882.