吲哚美辛联合奥沙利铂对大肠癌细胞生物学行为的影响及机制研究
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摘要
目的研究抗炎类药物吲哚美辛联合抗癌药物奥沙利铂对人大肠癌细胞SW620生物学行为的影响,并初步探讨其作用机制。方法体外培养人大肠癌细胞株SW620,将细胞随机分为四组进行处理,对照组、吲哚美辛组、奥沙利铂组、吲哚美辛联合奥沙利铂组,采用噻唑蓝(MTT)法检测各组大肠癌细胞增殖抑制情况,采用流式细胞术检测各组大肠癌细胞凋亡情况,采用Transwell实验检测各组大肠癌细胞体外迁移和侵袭能力,采用Western blotting检测各组大肠癌细胞活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(Cleaved Caspase-3)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)的蛋白表达情况。结果与对照组比较,吲哚美辛组和奥沙利铂组大肠癌细胞增殖抑制率显著升高(P<0.05)。与单用药组比较,联合用药组大肠癌细胞增殖抑制率、凋亡率、迁移和侵袭能力均明显升高,细胞中Cleaved Caspase-3蛋白表达量明显升高,MMP-2、MMP-9蛋白表达量明显降低,差异有统计学意义(P<0.05)。结论吲哚美辛联合奥沙利铂可协同增强对大肠癌细胞增殖、侵袭和转移抑制作用,协同诱导细胞凋亡,其作用机制可能与吲哚美辛、奥沙利铂调控细胞中Cleaved Caspase-3、MMP-2、MMP-9蛋白表达有关。
Objective To study the effect of Indomethacin combined with anticancer drug Oxaliplatin on the biological behavior of human colorectal cancer cell line SW620,and to explore its mechanism.Methods Human colon cancer cell line SW620 was cultured in vitro.The cells were randomly divided into four groups:control group,Indomethacin group,Oxaliplatin group,Indomethacin combined with Oxaliplatin group.The proliferation inhibition of colorectal cancer cells was detected by MTT method.The apoptosis of colorectal cancer cells was detected by flow cytometry.Transwell assay was used to detect the migration and invasion ability of colorectal cancer cells in vitro.Western blotting was used to detect the expressions of caspase-containing caspase-3(Cleaved caspase-3),matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) proteins activated by cancer cells.Results Compared with the control group,the inhibition rate of colorectal cancer cells in the Indomethacin group and the Oxaliplatin group was significantly increased(P<0.05).Compared with the single-agent groups,the proliferation inhibition rate,apoptosis rate,migration and invasion ability of colorectal cancer cells in the combination group were significantly increased,and the expression of Cleaved Caspase-3 protein was significantly increased in the cells.The expressions of MMP-2 and MMP-9 were significantly reduced,and the differences were statistically significant(P<0.05).Conclusion Indomethacin combined with Oxaliplatin can synergistically enhance the growth,invasion and metastasis of colorectal cancer cells,and synergistically induce apoptosis.The mechanism may be related to expressions of Cleaved Caspase-3,MMP-2,MMP-9 proteins in Indomethacin and Oxaliplatin-regulated cells.
Objective To study the effect of Indomethacin combined with anticancer drug Oxaliplatin on the biological behavior of human colorectal cancer cell line SW620,and to explore its mechanism.Methods Human colon cancer cell line SW620 was cultured in vitro.The cells were randomly divided into four groups:control group,Indomethacin group,Oxaliplatin group,Indomethacin combined with Oxaliplatin group.The proliferation inhibition of colorectal cancer cells was detected by MTT method.The apoptosis of colorectal cancer cells was detected by flow cytometry.Transwell assay was used to detect the migration and invasion ability of colorectal cancer cells in vitro.Western blotting was used to detect the expressions of caspase-containing caspase-3(Cleaved caspase-3),matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) proteins activated by cancer cells.Results Compared with the control group,the inhibition rate of colorectal cancer cells in the Indomethacin group and the Oxaliplatin group was significantly increased(P<0.05).Compared with the single-agent groups,the proliferation inhibition rate,apoptosis rate,migration and invasion ability of colorectal cancer cells in the combination group were significantly increased,and the expression of Cleaved Caspase-3 protein was significantly increased in the cells.The expressions of MMP-2 and MMP-9 were significantly reduced,and the differences were statistically significant(P<0.05).Conclusion Indomethacin combined with Oxaliplatin can synergistically enhance the growth,invasion and metastasis of colorectal cancer cells,and synergistically induce apoptosis.The mechanism may be related to expressions of Cleaved Caspase-3,MMP-2,MMP-9 proteins in Indomethacin and Oxaliplatin-regulated cells.
引文
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[17]CHOUDHARY G S,ALHARBI S,ALMASAN A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219(414):1-9.DOI:10.1007/978-1-4939-1661-0_1.
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[19]XIAN J,SHAO H,CHEN X,et al.Nucleophosmin mutants promote adhesion,migration and invasion of Human leukemia THP-1 cells through MMPs up-regulation via Ras/ERK MAPK signaling[J].Int JBiol Sci,2016,12(2):144-155.DOI:10.7150/ijbs.13382.
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[21]TONG W,WANG Q,SUN D,et al.Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB,u PA activator and MMP9[J].Oncol Lett,2016,12(5):4139-4146.DOI:10.3892/ol.2016.5148.
[22]许崇文,秦思达,李硕,等.吲哚美辛诱导食管癌EC109细胞凋亡的Smac依赖性机制[J].现代肿瘤医学,2015,23(14):1935-1939.DOI:3969/j.issn.1672-4992.2015.14.01.XU C W,QIN S D,LI S,et al.The study of Smac-dependent opoptosis induced by indomethacin in EC109 esophageal cancer cell line[J].Modern Oncology,2015,23(14):1935-1939.DOI:3969/j.issn.1672-4992.2015.14.01.
[23]孙维忆.吲哚美辛联合奥沙利铂对人肺癌裸鼠移植瘤的治疗作用及其作用机制的研究[D].石家庄:河北医科大学,2013.
[2]KWASIGROCH B,ESCRIBANO E,MORN M D C,et al.Oil-inwater nanoemulsions are suitable for carrying hydrophobic compounds:Indomethacin as a model of anti-inflammatory drug[J].Int J Pharm,2016,515(1-2):749-756.DOI:10.1016/j.ijpharm.2016.11.016.
[3]GENARI B,FERREIRA M,MEDEIROS L F,et al.Anti-inflammatory effect of an adhesive resin containing indomethacin-loaded nanocapsules[J].Arch Oral Biol,2017,84(9):106-111.DOI:10.1016/j.archoralbio.2017.09.016.
[4]SILVA A A,BEZERRA M M,CHAVES H V,et al.Protective effect of Chresta martii extract against indomethacin-induced gastric lesions in mice[J].J Nat Med,2013,67(1):143-151.DOI:10.1007/s11418-012-0663-x.
[5]彭晓斌,王小云,吴高珏,等.吲哚美辛栓对ERCP术后血清淀粉酶、炎症因子和免疫功能的影响[J].海南医学院学报,2016,22(19):2290-2293.
[6]WANG S,TAN X,LI S,et al.Indomethacin-based stimuli-responsive micelles combined with paclitaxel to overcome multidrug resistance[J].Oncotarget,2017,8(67):111281-111294.DOI:10.18632/oncotarget.22781.
[7]MODI S,KIR D,GIRI B,et al.Minnelide overcomes oxaliplatin resistance by downregulating DNA repair pathway in pancreatic cancer[J].J Gastrointest Surg,2015,20(1):13-24.DOI:10.1007/s11605-015-3000-3.
[8]韩江琼,陈云兰,秦锴,等.5-氟尿嘧啶联合亚叶酸钙与奥沙利铂化疗方案治疗晚期结直肠癌的临床研究[J].中国临床药理学杂志,2014,30(8):674-675.HAN J Q,CHEN Y L,QIN K,et al.Clinical study of 5-FU/LV and L-OHP chemotherapy regimen for adcanced colorectal cancer[J].Chin J Clin Pharmacol,2014,30(8):674-675.
[9]LIU M Y,HUANG X E.Effects of analgecine on oxaliplatin-induced neurotoxicity in patients with gastrointestinal cancer[J].Asian Pac JCancer Prev,2015,16(10):4465-4468.
[10]刘晓雪,宇传华,周薇,等.中国近30年间结直肠癌死亡趋势分析[J].中国癌症杂志,2018,28(3):177-183.DOI:10.19401/j.cnki.1007-3639.2018.03.002.LIU X X,YU C H,ZHOU W,et al.Trends in colorectal cancer mortality for the last 30 years in China[J].China Oncol,2018,28(3):177-183.DOI:10.19401/j.cnki.1007-3639.2018.03.002.
[11]BIAGI J J,RAPHAEL M J,MACKILLOP W J,et al.Association between time to initiation of adjuvant chemotherapy and survival in colorectal cancer:a systematic review and Meta-analysis[J].JAMA,2016,160(1):1-12.DOI:10.1001/jama.2011.749.
[12]WANG J,ZHANG G Y,LI X H.Effect of indomethacin on Bfl-1,WISP-1 and proliferating cell nuclear antigen in colon cancer cell line HCT116 cells[J].Chin J Dig Dis,2010,7(4):219-224.DOI:10.1111/j.1443-9573.2006.00272.x.
[13]徐晖,俞佳,吕良忠.吲哚美辛对宫颈癌Hela细胞增殖和凋亡作用的影响[J].中国药房,2015,26(31):4375-4377.DOI:10.6039/j.issn.1001-0408.2015.31.18.XU H,YU J,LYU L Z.Effects of indometacin on apoptosis and proliferation of cervical cancer Hela cell[J].China Pharmacy,2015,26(31):4375-4377.DOI:10.6039/j.issn.1001-0408.2015.31.18.
[14]ZHOU Y,WANG S,YING X,et al.Doxorubicin-loaded redox-responsive micelles based on dextran and indomethacin for resistant breast cancer[J].Int J Nanomedicine,2017,12(22):6153-6168.DOI:10.2147/IJN.S141229.
[15]XING D,CHEN Y Q,WANG D C,et al.Combined effects off indomethacin and oxaliplatin on lymph node metastasis related factors in human lung cancerxenografts in nude mice[J].Pak J Pharm Sci,2016,29(6):2083-2088.
[16]YANG C,FU Z X.Liposomal delivery and polyethylene glycol-liposomal oxaliplatin for the treatment of colorectal cancer(Review)[J].Biomed Rep,2014,2(3):335-339.DOI:10.3892/br.2014.249.
[17]CHOUDHARY G S,ALHARBI S,ALMASAN A.Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219(414):1-9.DOI:10.1007/978-1-4939-1661-0_1.
[18]LIN H L,LIU T Y,WU C W,et al.2-Methoxyestradiol-induced caspase-3 activation and apoptosis occurs through G(2)/M arrest dependent and independent pathways in gastric carcinoma cells[J].Cancer,2015,92(3):500-509.
[19]XIAN J,SHAO H,CHEN X,et al.Nucleophosmin mutants promote adhesion,migration and invasion of Human leukemia THP-1 cells through MMPs up-regulation via Ras/ERK MAPK signaling[J].Int JBiol Sci,2016,12(2):144-155.DOI:10.7150/ijbs.13382.
[20]ITOH Y,TAKAMURA A,ITO N,et al.Homophilic complex formation of MT1-MMP facilitates pro MMP-2 activation on the cell surface and promotes tumor cell invasion[J].EMBO J,2014,20(17):4782-4793.DOI:10.1093/emboj/20.17.4782.
[21]TONG W,WANG Q,SUN D,et al.Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB,u PA activator and MMP9[J].Oncol Lett,2016,12(5):4139-4146.DOI:10.3892/ol.2016.5148.
[22]许崇文,秦思达,李硕,等.吲哚美辛诱导食管癌EC109细胞凋亡的Smac依赖性机制[J].现代肿瘤医学,2015,23(14):1935-1939.DOI:3969/j.issn.1672-4992.2015.14.01.XU C W,QIN S D,LI S,et al.The study of Smac-dependent opoptosis induced by indomethacin in EC109 esophageal cancer cell line[J].Modern Oncology,2015,23(14):1935-1939.DOI:3969/j.issn.1672-4992.2015.14.01.
[23]孙维忆.吲哚美辛联合奥沙利铂对人肺癌裸鼠移植瘤的治疗作用及其作用机制的研究[D].石家庄:河北医科大学,2013.