金雀异黄素联合5-氟尿嘧啶抑制人结肠癌细胞株SW48O增殖的分子机制

Investigation of the molecular mechanism how genistein combined with 5-fluorouracil inhibits proliferation of human colon cancer cell line SW480

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-Fu30μg／mL单用或联用48h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、Bcl2、p21、caspase3和caspase9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土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士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。

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） and β-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 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±0.39a） 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. 586±0,051; 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. 215±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 （I 067.97±35.01） and 5-FU group （718.83±23.18, LSD-test, P〈0.05）. Coneluslons 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.