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Background: environmental endocrine disruptors (Environmental Endocrine Disruptors, EED) is an important component of environmental pollutants, hormone-like effects in the body; comes mainly from pesticides, plastics, detergents, combustion products and industrial and agricultural products . In recent years, EED pollution has become the important issues of global concern, EED has become a hot topic of sex hormone-sensitive malignancies. Neuroblastoma is the most common pediatric malignant solid tumors, one of its etiology and mechanism is unclear. Environmental endocrine disruptors have been found to promote tumor neuroblastoma factors, choose a can effectively inhibit the environmental endocrine disruptors promote tumor effect of substances of great significance for the prevention and treatment of tumor. PI3K/Akt signal transduction pathway is widely present in cells, is involved in cell growth, proliferation, differentiation regulation of signal transduction pathways may be G protein-coupled receptor and (or) the protein tyrosine kinase receptor activation can also be Ras protein is activated. Variety of growth factors through the PI3K/Akt signaling pathway play a role. It has been confirmed that the PI3K/Akt pathway plays an important role in the occurrence of certain human malignancies, development. 5,7,4 '- genistein (Genistein, GEN) is a protein tyrosine kinase (PTK) inhibitors, a common flavonoids, is an important component of soybean; current research soy isoflavones the main representative of the biological effects thereof. GEN as a phytoestrogen, 17 beta-estradiol similar structure, combined with competition in the 17 beta-estradiol and estrogen-beta receptors play a weak estrogen or anti-estrogen effect in tumorigenesis, stage of development is the existence of multiple inhibitory effect. GEN whether the inhibition of tumor cell proliferation effect caused by environmental endocrine disruptors, at home and abroad has not been reported. Objective: This study was GEN environmental endocrine disruptors bisphenol A (BPA) and phthalate (2 - ethylhexyl) hexyl phthalate (DEHP)-induced neuroblastoma cell proliferation, and to explore its mechanism of action and PI3K/Akt signaling pathway in which changes, open new avenues for children exposed to environmental pollution prevention neuroblastoma. Materials and Methods: First, GEN inhibition of BPA, DEHP induced neuroblastoma tumor SK-N-SH cell proliferation effect experimental system establishment: SK-N-SH cells amplified by conventional culture were divided into 24 groups: Group 1 RPMI1640 phenol red-free culture solution, add ethanol (control group); group 2, plus Gen1 the (concentration 2μmol / L): Group 3 plus GEN2 (concentration of 5 micromol / L); Group 4, plus GNE3 (concentration of 12.5μmol / L); The group 5 add GEN4 (concentration 25μmol / L); group 6, plus GEN5 (concentration of 50 micromol / L); group 7, plus E2; group 8, plus BPA; Group 9, plus DEHP; group of 10, while adding E2 Gen1; group 11, while adding E2 Gen2; group 12, while adding E2 Gen3; group 13, while adding E2 GEN4; group 14, while adding E2 GEN5; group 15, while adding BPA Gen1; Group 16 , while adding BPA GEN2; Group 17, while adding BPA GEN3; Group 18, while adding BPA GEN4; group of 19 and while adding BPA GEN5; Group 20, while adding DEHP Gen1; Group 21, Gen2 group joined DEHP; 22, The added to DEHP Gen3; Group 23, while adding DEHP GEN4; Group 24, while adding DEHP GEN5. Observe the proliferation of cells cultured 72 h cells was detected light absorption value (AV). Second, GEN BPA, DEHP induced SK-N-SH cells proliferation effect in vitro proliferation assay: SK-N-SH cells were routinely cultured amplified divided into eight groups: group 1, RPMI 1640 without phenol red culture solution plus over anhydrous ethanol (control group); group 2, add GEN (the Gen group); group 3, plus E2 (E2 group); group 4 plus E2 and GEN (E2 GEN group); group 5 plus BPA (BPA group ); Group 6, while adding BPA and GEN (BPA GEN group); group 7 plus DEHP (DEHP group); Group 8, plus DEHP and GEN (DEHP GEN group). Respectively 24, 48 and 72 h, cells was detected light absorption value (AV). At the same time 72 h using flow cytometry cell cycle and apoptosis index (AI). Deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) to observe the apoptosis and 72 h, Western blot analysis to detect the expression of Caspase-3 protein. PI3K/Akt signal transduction pathway inhibition in GEN environmental endocrine disruptors promote Experimental study on changes in the proliferation of neuroblastoma effect: SK-N-SH cells were routinely cultured amplified divided into eight groups: group 1, RPMI 1640 phenol red-free culture solution plus ethanol (control group); Group 2, plus GEN (the GEN Group); Group 3, plus E2 (E2 group); Group 4, plus E2 and GEN (E2 GEN group); Group 5 , plus BPA (the BPA Group); Group 6, plus BPA and GEN (BPA GEN group); Group 7, plus DEHP (DEHP group); Group 8, plus DEHP and GEN (DEHP GEN group). 72 h cells was detected light absorption value (AV). By Western blot analysis to detect the expression of Akt protein and phosphorylated protein (p-Akt). Results: First, GEN inhibition of BPA DEHP induced neuroblastoma tumor SK-N-SH cell proliferation effect experimental system to establish: SK-N-SH cells were treated with different concentrations of GEN role 72 h the GEN different concentrations on cell growth. 12.5μmol / L concentration group AV and the control group was 0.79 ± 0.14) (AV Comparative 2μmol / AV L and 5μmol / L concentration group were 0.97 ± 0.05 and 0.94 ± 0.08, having pro-proliferative effect (p <0.01); 0.77 ± 0.07, compared with the control group, inhibit the growth trend, but no significant difference; AV 25μmol / L, 50μmol / L concentration group were 0.63 ± 0.02 and 0.43 ± 0.02, has a significant inhibitory effect (p < 0.05). Add to E2 and BPA, DEHP, GEN have different effects on the growth of cells in each group. E2-induced cell proliferation, while adding the cells of each group AV GEN and E2 were 0.93 ± 0.03,0.89 ± 0.03,0.82 ± 0.06 and 0.72 ± 0.05, compared with separate E2 group decreased, with a statistically significant difference (p <0.01). BPA-induced cell proliferation, while adding GEN and BPA cells in each group AV were 0.97 ± 0.07,0.92 ± 0.02,0.85 ± 0.07,0.84 ± 0.07,0.83 ± 0.03, compared with a separate BPA group were decreased with significant statistical learning differences (p <0.001). And DEHP-induced cell proliferation, the GEN groups 22, 23, 24 AV 0.88 ± 0.04,0.87 ± 0.01 and 0.72 ± 0.11, lower than the separate DEHP group, a significant difference (p <0.01), and 20, 21 with the not GEN added group no significant difference between groups. , GEN on BPA, DEHP induced SK-N-SH cell proliferation effect in vitro: SK-N-SH cells by GEN E2, BPA, after DEHP role, at each time point AV different, 24h adding GEN group with not GEN added group comparison, cell AV slightly lower, but the difference was not statistically significant. Cells at 48h, all the EEDs group co-GEN role AV than separate EEDs group have reduced. 4 (E2 GEN group) AV group 3 (E2 group), 75%, 25%, in comparison with a statistically significant difference (p <0.001). Of BPA Add GEN and BPA cells AV reduced to 0.58 ± 0.09, 77% of the separate BPA action group (0.75 ± 0.02), both compared with a statistically significant difference (p <0.001); Similarly, Group (DEHP the GEN group of) AV group 7 (DEHP group) also decreased, the difference between the difference was statistically significant (p <0.001). 72h, results similar to 48h; the EEDs cells of the role of the United GEN AV more separate EEDs group decreased, in comparison with statistically significant difference (p <0.001). 72h, cell cycle analysis, for each the EEDs joint GEN group G2 / M phase cells significantly increased proportion EEDs alone group was statistically significant (p <0.01), while the sub-G0 period (apoptosis hypodiploid peak), apoptotic index and Caspase-3 protein expression was no significant difference (p> 0.05) between the groups. PI3K/Akt signal transduction pathway inhibition in GEN environmental endocrine disruptors promote Experimental study on changes in the proliferation of neuroblastoma effect: 72h when Western blot analysis found that the the EEDs United GEN group than alone EEDs group Akt and p- Akt protein expression decreased, the difference between the two was statistically significant (p <0.05). Conclusion: 1.GEN neuroblastoma cell growth was a two-way role in a concentration-dependent manner. 2.GEN promoting proliferation inhibition of environmental endocrine disruptors on human neuroblastoma cells through the cell cycle G2 / M phase arrest. 3.GEN may interfere with PI3K/Akt signaling transduction pathway in cellular signal transduction, thereby inhibiting the environmental endocrine disruptors promote the proliferation of human neuroblastoma cells. 4.GEN environmental endocrine disruptors promote neuroblastoma tumor cell growth inhibition, apoptosis may not be the main way.
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