Promotion Apoptosis Effect And Its Mechanism Of Genistein Combined With 5-fu On Human Colon Carcinoma Cell

Objective To investigate the coordinate repression of genistein combined with 5-fluorouracil(5-Fu) on the growth of human colon carcinoma cell SW480 in vitro and the mechanism of apoptosis on colon carcinoma cell, so as to provide important theory accordance for improving chemotherapy effect of colon carcinoma in clinical.Methods The experiment were divided into four groups: control group(no drug group); 5-Fu group; genistein group; combination group(5-Fu+genistein).The growth inhibitory effect of different concentrations genistein and 5-Fu single or combination using on colon carcinoma cell SW480 was studied by MTT growth assay. TUNEL (terminal deoxynucleotidyl transferase mediated dutp-biotinnick end-labeling assay) technology investigated the apoptosis of colon carcinoma cell and electron microscopy was used to observe the ultra-microstructure changes of tumor cells. Cell cycle distribution was detected by flow cytometry (FCM) after using drugs. The gene expression of survivin,Bcl-2,p21,caspase3 and caspase 9 in tumor cells was deteced by real-time quantitative PCR ; survivin,Bcl-2,p21,caspase3 and caspase 9 protein was deteced by Western blot; EMSA(electrophoretic mobility shift assay) investigated the DNA combination activity of NF-κB.Result 1. The MTT results showed that both genistein and 5-Fu exhibited a dose- and time- dependent growth inhibitory effect on SW480 cells after 24h,48h,72h,96h.2. The combination of genistein and 5-Fu through Latin square permutation and combination, we calculated the q value. Among, when 5-Fu in the concentration of 30μg/ml and genistein in the concentration of 80μmol/l interaced for 48h, the q value is the highest, q=1.618.3. In TUNEL, the AI of genistein group was (10.32±1.26)%, the 5-Fu group was (16.45±2.56)%, the combination group was (29.32±5.65)%, the control group only was (1.28±0.55)%. Among the four groups, the positive cell compared with each other had statistical significance, F=49.780, P<0.001. The combination group compared with control grop,5-Fu group and genistein group both had statistical significance(P<0.001).4. We found nucleus electron density increased, nuclear fragmention of apoptotic cell and apoptotic body by transmission electron microscopy, all of which was most significant in the of two drugs.5. FCM showed the cell ratio of G1 perior in control group,5-Fu group,genistein group and combination group is 46.0%,43.2%,21.0% and 15.4%, the cell ratio of S perior decreased from40.0% to 19.0%,24.5% and 36.4%, the cell ratio of G2 perior increased from 14.1% to 27.8%,54.5% and 48.2%. All of this showed a G2 arrest on SW480cell after treatment of genistein.6. The real-time quantitative PCR showed that compared with control group, 5-Fu group and genistein group, the 2-ΔΔCT value of caspase3,caspase9 and p21 in combination group was higher, the difference had statistical significance(P<0.05); comparion with control group, 5-Fu group and genistein group, the 2-ΔΔCT value of survivin in combination group was lower, the difference had statistical significance(P<0.05). Comparion with control group and genistein group, the 2-ΔΔCT value of Bcl-2 in combination group was lower, the difference had statistical significance(P<0.05). But, the combination group had no statistical significanc, compared with 5-Fu group (P>0.05).7. In Western blot, compared with control group, 5-Fu group and genistein group, the gray level ratio of caspase3,caspase9 and p21 in combination group was higher, the difference had statistical significance(P<0.05); comparion with control group, 5-Fu group and genistein group, the gray level ratio of survivin in combination group was lower, the difference had statistical significance(P<0.05). Comparion with control group and genistein group, the gray level ratio of Bcl-2 in combination group was lower, the difference had statistical significance(P<0.05). But, the combination group had no statistical significanc, compared with 5-Fu group (P>0.05).8. EMSA showed the expression of NF-κB protein in genistein group and combination group was lower than the 5-Fu group and control group(P<0.05). The result showed that genistein could degrade the expression of NF-κB, and 5-Fu had little influence on NF-κB. When the two drugs combined, the expression of NF-κB was inhibited, them induced more apoptosis. Genistein upgraded the sensitivity of chemotherapertic agents through inhibiting the signal passsway of NF-κB. Conclution 5-Fu combined with genistein could significantly inhibit the growth of colon carcinoma cell. They had synergistic antitumor effect. The mechanism could be concerned with below: through inhibiting the activity of NF-κB, genistein reinforce the chemotherapy sensitivity of colon carcinoma cell; genistein reinforce the effect of inducing apoptosis by 5-Fu in colon carcinoma cell through inactiving caspase-3,caspase9 and p21, decreasing survivin.