金雀异黄素联合5-氟尿嘧啶抑制人结肠癌细胞株 SW480增殖的分子机制
금작이황소연합5-불뇨밀정억제인결장암세포주 SW480증식적분자궤제
Investigation of the molecular mechanism how genistein combined with 5-fluorouracil inhibits proliferation of human colon cancer cell line SW480
  • 万方数据
    中华消化杂志 중화소화잡지
    Chinese Journal of Digestion
    2015年 6期 390-394 ,共5页

    凌航%陈裕庆%高美钦%张文敏

    릉항%진유경%고미흠%장문민

    金雀异黄素%氟尿嘧啶%结肠肿瘤%凋亡%机制 金雀異黃素%氟尿嘧啶%結腸腫瘤%凋亡%機製
    금작이황소%불뇨밀정%결장종류%조망%궤제
    Genistein%Fluorouracil%Colonic neoplasms%Apoptosis%Mechanism
    目的：初步探讨金雀异黄素提高结肠癌细胞对5-氟尿嘧啶（5-FU）的敏感性，促进结肠癌细胞凋亡的分子机制。方法选择 SW480结肠癌细胞株为实验对象，将金雀异黄素80μmol／L 和5-FU 30μg／mL 单用或联用48 h 作为药物实验组，另设不加药组为对照组。分别采用实时定量 PCR 和Western 印迹法检测各组细胞的生存素、Bcl-2、p21、caspase3和 caspase 9基因和蛋白表达情况。采用比较阈值法（2－ΔΔCT 法）来确定基因的相对表达量。以β-actin 作为内参照，进行半定量分析计算蛋白相对表达量。电泳迁移率改变分析（EMSA）检测各组细胞 NF-κB 的 DNA 结合活性。多组间样本均数比较采用单因素方差分析，组间两样本均数比较采用 LSD 检验。结果联合用药组的 caspase3、caspase9和p21基因表达（1．903±0．122、2．726±0．050、2．541±0．393）与蛋白表达（0．534±0．077、1．161±0．172、0．463±0．016）高于对照组（1．001±0．052、1．000±0．014、1．001±0．037和0．080±0．043、0．248±0．059、0．139±0．021）、金雀异黄素组（1．559±0．038、2．394±0．095、1．686±0．061和0．335±0．052、0．478±0．059、0．304±0．018）和5-FU 组（1．198±0．063、1．051±0．043、1．399±0．055和0．194±0．015、0．337±0．036、0．231±0．011），联合用药组的生存素基因与蛋白表达（0．165±0．018和0．216±0．014）低于对照组、金雀异黄素组和5-氟尿嘧啶单组（1．001±0．033、0．775±0．044、0．395±0．030和0．594±0．079、0．375±0．014、0．295±0．014），差异均有统计学意义（基因表达，F ＝802．865、52．760、39．992、187．288、37．435；蛋白表达，F ＝10．466、44．483、19．490、200．011、45．238；P 均＜0．01）， caspase3、p21在两单药组的表达与对照组相比，差异也有统计学意义（LSD 检验，P ＜0．05）。EMSA 检测显示，与对照组（1067．97±36．01）和5-FU 组（718．83±23．18）相比，金雀异黄素组（461．64±15．41）和联合用药组（585．28±7．82）的 NF-κB 蛋白 DNA 结合活性均有明显降低（LSD 检验，P ＜0．05）。结论金雀异黄素联合5-FU 协同促进结肠癌细胞凋亡，可能通过金雀异黄素抑制 NF-κB 的 DNA 结合活性，进一步上调 caspase3、caspase9、p21以及下调生存素起作用，提示金雀异黄素可能为结肠癌化学治疗提供辅助。
    목적：초보탐토금작이황소제고결장암세포대5-불뇨밀정（5-FU）적민감성，촉진결장암세포조망적분자궤제。방법선택 SW480결장암세포주위실험대상，장금작이황소80μmol／L 화5-FU 30μg／mL 단용혹련용48 h 작위약물실험조，령설불가약조위대조조。분별채용실시정량 PCR 화Western 인적법검측각조세포적생존소、Bcl-2、p21、caspase3화 caspase 9기인화단백표체정황。채용비교역치법（2－ΔΔCT 법）래학정기인적상대표체량。이β-actin 작위내삼조，진행반정량분석계산단백상대표체량。전영천이솔개변분석（EMSA）검측각조세포 NF-κB 적 DNA 결합활성。다조간양본균수비교채용단인소방차분석，조간량양본균수비교채용 LSD 검험。결과연합용약조적 caspase3、caspase9화p21기인표체（1．903±0．122、2．726±0．050、2．541±0．393）여단백표체（0．534±0．077、1．161±0．172、0．463±0．016）고우대조조（1．001±0．052、1．000±0．014、1．001±0．037화0．080±0．043、0．248±0．059、0．139±0．021）、금작이황소조（1．559±0．038、2．394±0．095、1．686±0．061화0．335±0．052、0．478±0．059、0．304±0．018）화5-FU 조（1．198±0．063、1．051±0．043、1．399±0．055화0．194±0．015、0．337±0．036、0．231±0．011），연합용약조적생존소기인여단백표체（0．165±0．018화0．216±0．014）저우대조조、금작이황소조화5-불뇨밀정단조（1．001±0．033、0．775±0．044、0．395±0．030화0．594±0．079、0．375±0．014、0．295±0．014），차이균유통계학의의（기인표체，F ＝802．865、52．760、39．992、187．288、37．435；단백표체，F ＝10．466、44．483、19．490、200．011、45．238；P 균＜0．01）， caspase3、p21재량단약조적표체여대조조상비，차이야유통계학의의（LSD 검험，P ＜0．05）。EMSA 검측현시，여대조조（1067．97±36．01）화5-FU 조（718．83±23．18）상비，금작이황소조（461．64±15．41）화연합용약조（585．28±7．82）적 NF-κB 단백 DNA 결합활성균유명현강저（LSD 검험，P ＜0．05）。결론금작이황소연합5-FU 협동촉진결장암세포조망，가능통과금작이황소억제 NF-κB 적 DNA 결합활성，진일보상조 caspase3、caspase9、p21이급하조생존소기작용，제시금작이황소가능위결장암화학치료제공보조。
    [Abstract ] Objective To investigate the molecular mechanism how genistein increased the sensitivity of colon cancer cell to 5-fluorouracil(FU)and promoted colon cancer cell apoptosis.Methods SW480 colon cancer cell line was chosen as experimental object.Genistein 80 μmol/L and 5-FU 30 μg/mL used separated or combined for 48 hours were set as drug experiment group.There were four experiment groups in this study:control group (without any drug),5-FU group,genistein group,5-FU and genistein combined group.The expression of survivin,Bcl-2,p21 ,caspase3 and caspase 9 in the tumor cells of each group at mRNA and protein level was deteced by real-time quantitative polymerase chain reaction (PCR) and Western blotting.The relative expression quantity of genes was determined by comparative threshold method (2 -ΔΔCT )andβ-actin was taken as an internal reference.Semi quantitative analysis was performed for protein relative expression quantity.The DNA combination activity of nuclear factor(NF)-κB of each&nbsp;group was measured by electrophoretic mobility shift assay (EMSA).Single factor analysis of variance was used for mean comparison among multiple groups and LSD test was for mean comparison between two groups.Results The expression of caspase3,caspase 9 and p21 of 5-FU and genistein combination group at mRNA (1 .903±0.122,2.726±0.050 and 2.541 ±0.393)and protein level (0.534±0.077,1 .161 ± 0.172 and 0.463±0.016)were all higher than those of control group (1 .001 ±0.052,1 .000±0.014 and 1 .001 ±0.037;0.080 ±0.043,0.248±0.059 and 0.139 ±0.021 ),genistein group (1 .559 ±0.038, 2.394±0.095 and 1 .686 ±0.061 ;0.335 ±0.052,0.478 ±0.059 and 0.304 ±0.018)and 5-FU group (1 .198±0.063,1 .051 ±0.043 and 1 .399±0.055 ;0.194±0.015 ,0.337 ±0.036 and 0.231 ±0.011 );the expression of survivin of 5-FU and genistein combined group at mRNA and protein level (0.165 ± 0.018 and 0.216±0.014)were all lower than those of control group,genistein group and 5-FU group (1 .001 ±0.033,0.775 ±0.044 and 0.395 ±0.030;0.594±0.079,0.375 ±0.014 and 0.295 ±0.014), and all the differences were statistically significant (gene,F = 802.865 ,52.760,39.992,187.288, 37.435 ;protein,F =10.466,44.483,19.490,200.011 ,45 .238;all P <0.01);the difference was also statistically significant in the expression of caspase3 and p21 of 5-FU group and genistein group compared with those of control group (LSD test,P <0.05 ).The results of EMSA assay showed that the DNA binding activity of NF-κB protein of genistein group (461.64 ±15.41 )and combined group (585.28 ±7.82) significantly decreased compared with that of control group (1 067.97 ±36.01)and 5-FU group (718.83± 23.18,LSD-test,P < 0.05).Conclusions Genistein combined with 5-FU seemed to have synergistic effects on apoptosis of colon cancer cell.The mechanism was that genistein inhibited the DNA binding activity of NF-κB mediated by genistein,further up-regulated caspase 3,caspase 9,p21 and down regulated survivin.Therefore,genistein may provide assistance in colon cancer chemotherapy.
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