| Backgrounds and Purpose:Environmental endocrine disruptors(EEDs) are the considerable elements of the environmental pollution.Such contaminants are derived from pesticides,plastics,combustion by-products,plants and agricultural products. They enter the body and disrupt normal endocrine functions,having adverse effects on wildlife and human health.In recent years,widespread attention has been paid to EEDs.There has been increasing interest in the effects of EEDs on estrogen-sensitive malignancies.Neuroblastoma(NB) is a common and embryonal pediatric malignant solid tumor.The cause of NB remains unknown.The promoting effect of EEDs on NB has been proved in vivo and vitro,and it is necessary and urgent to explore inhibitions to prevent this growth-promoting effects.Phosphatidylinositol-3-Kinase/Akt(PI3K/Akt) signaling pathway is one of the common pathways for cell proliferation and differentiation,and can be activated by the G-protein-coupled receptor(GPCR),receptor of the protein tyrosine kinase(PTK) or the Ras protein.It has been proved that PI3K/Akt pathway is one of the most general ways for many growth factors.It is confirmed that PI3K/Akt signaling pathway plays an important role in the initiation and promotion of some human malignant tumors.Genistein(4',5,7-trihydroxyisoflavone,GEN) as an inhibitor of PTK,is a general flavone derived from soybean,and a popular isoflavone for its anticancer biological activity.As a phytoestrogen,it can bind with estrogen receptor(ER),especially ER-βto educe biological activities as an agonistic and antagonistic properties of estrogen with respect to cell growth and differentiation.However,few researches have been made to observe the effect of GEN on the biologic activity of EEDs.In order to find the effects of GEN on the growth promotion of EEDs,we first devised several EEDs as intervention substances to observe their effects on proliferation of SK-N-SH human neuroblastoma cells.Then we use the mixture of GEN and selected EEDs to observe the depression effects of GEN.Furthermore,the cell cycle,cell apoptosis,the protein caspase-3,Akt and its phosphorylation were investigated to observe the underlying mechanisms.Materials and Methods:1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI 1640 without phenol red and divided into twenty-four groups based on different concentration of GEN treatments:group 1,no treatment(control);group 2,treated with GEN1(CO:2μmol/L);group 3,treated with GEN2(CO:5μmol/L);group 4, treated with GEN3(CO:12.5μmol/L);group 5,treated with GEN4(CO:25μmol/L); group 6,treated with GEN5(CO:50μmol/L );group 7,treated with 17β-estradiol(E2); group 8,treated with bisphenol A(BPA);group 9,treated with Di-2-Ethylhexl Phthalate(DEHP);group 10,treated with both E2 and GEN1;group 11,treated with both E2 and GEN2;group 12,treated with both E2 and GEN3;group 13,treated with both E2 and GEN4;group 14,treated with both E2 and GENS;group 15,treated with both BPA and GEN1;group 16,treated with both BPAand GEN2;group 17,treated with both BPA and GEN3;group 18,treated with both BPA and GEN4;group 19, treated with both BPA and GENS;group20,treated with both DEHP and GEN1; group21,treated with both DEHP and GEN2;group22,treated with both DEHP and GEN3;group23,treated with both DEHP and GEN4;group24,treated with both DEHP and GEN5.Absorbance value(AV) was determined at time 72 hours.2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment (control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2; group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN.AV was determined at time 24,48 and 72 hours.Flow cytometer was employed to monitor cell cycles,terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling(TUNEL) were employed to monitor cell apoptosis.Western blot analysis was employed to monitor the expression of caspase-3.3.Interruption of GEN to PI3K/Akt signaling pathway when combined with EEDs: NB cells were cultured in estrogen-free RPMI1640 without phenol red and divided into eight groups based on different treatments:group 1,no treatment(control);group 2,treated with GEN(CO:12.5μmol/L);group 3,treated with E2;group 4,treated with both E2 and GEN;group 5,treated with BPA;group 6,treated with both BPA and GEN;group 7,treated with DEHP;group 8,treated with both DEHP and GEN. Western blot analysis was employed to monitor the expression of Akt and its phosphorylation,p-Akt.Results:1.Experiment system establishment of the effect of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:At 72 hours,AV was 0.97±0.05 for group 2,0.94±0.08 for group 3;both of them were increased when compared with that of control group(p＜0.01).There was no difference between group 4(AV:0.77±0.07) and the control group.Compared with that of the control group,the AV of group5(AV:0.63±0.02) and group6(AV: 0.43±0.02) were definitely decreased with obvious difference.When treated with E2 and GEN,the AV of group12,13,14 and 15 were 93±0.03,0.89±0.03,0.82±0.06 and 0.72±0.05;compared with that of the group treated with E2 only,the AV was decreased and had statistical difference(p＜0.01).When treated with BPA and GEN, the AV each group was also decreased,compared with that of the group treated with BPA only,statistical difference existed(p＜0.001).When treated with both DEHP and GEN,the AV of group22,23 and 24 were decreased,compared with that of the group treated with DEHP only,the results had statistical significance(p＜0.01),however there were no statistical significances between the group20,21 and group9.2.Inhibitory action of GEN on the growth promotion of SK-N-SH cell line induced by EEDs:At 24 hours,there's no difference of the AV between the group treated with GEN and without GEN.However,at 48 hours,the differences were found.The AV of group 4 was decreased by 25%,compared to the group 3 treated with E2 only(p＜0.001). The same phenomenon could be found in the left group.Comparing with group 5,the AV of group 6 was decreased to 77%;the differences between the two groups had statistical significance(p＜0.001).When treated with both DEHP and GEN,the results also had statistical significance(p＜0.001).The same phenomenon could be found at 72 hours,and the differences between the groups treated with GEN and with EEDs only also had statistical significance.The percentage of cells in G2/M at 72 hours treated with GEN was also different from the ones treated with no GEN,and the results had statistical significance(p＜0.05).However,the percentage of cells in sub-G0 of each group had no difference.The result of TUNEL was similar to flow cytometer.We also monitored the expression of Caspase-3 by Western blot analysis, but there was no difference of expression between the groups(p＞0.05).3.Interruption of GEN to PI3K/Akt signaling pathway combined with EEDs:At 72 hours,the expression of Akt and p-Akt was abundant in E2,BPA,and DEHP groups,when treated with the mixture of GEN and EEDs,the expression of the two proteins was decreased.The differences between the group with the mixture and the EEDs only had statistical significance(p＜0.05).Conclusions:1.GEN can inhibit the growth of SK-N-SH cells in a dose-dependent manner.2.GEN can inhibit the growth-promoting effect of the EEDs on SK-N-SH cells.3.The biological effects of GEN may be activated by blocking the G2/M of cell cycle and by disrupting the PI3K/Akt signaling pathway.And the activation exists even though in the environment with high level of estrogen made by EEDs.4.No obvious apoptosis can be found in this experiment. |
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