Investigation of the molecular mechanism how genistein combined with 5-fluorouracil inhibits proliferation of human colon cancer cell line SW480 / 中华消化杂志

ABSTRACT

[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 studycontrol 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 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.