| Neuroblastoma (NB) derives from neural crest cells which can not be differentiated normally during embryo development. It is the most common extracranial tumor in children. Clinically NB is characterized by its rapid growth, susceptibility to multidrug resistance and metastasis. Both genetic and environmental factors may contribute to neuroblastoma oncogenesis. However, little is known about risk factors and their mechanisms that induce NB. Exploring risk factors and their mechanisms related to NB will improve NB early diagnosis and clinical therapies as well as the prognosis. Environmental endocrine disruptors (EED) are a large number of either natural or man-made exogenous compounds. Most of them mainly mimick the action of natural hormone estrogens such as 17β-estradiol (E2) in the body. With aggravation of global environmental problems, there have been growing concerns on relationships between daily exposure to EED and human health. Accumulating evidence suggested that EED are causative factors for multiple human diseases including birth defects, neurological disorders and metabolism disorders as well as adult malignant tumors such as breast cancer, prostate cancer, and etc. However, no studies is focused on the relationship between EED and children solid malignant tumors now. It is necessary to investigate the role of EED on pediatric diseases including cancer since they are more susceptible to adverse effects of EED exposure. Copy number variant (CNV) is a novel structural variation in human chromosomes. CNV is highly associated with various human diseases although it widely exists in the normal population. It is reported that CNV also contributes to the NB's initiation. However, the relationship between NB and CNV in Chinese patients is still unclear. Therefore, it is necessary to explore genetic mechanisms of NB in Chinese population, which will be useful for NB's early diagnosis, therapies and prognosis evaluation.In the current study, we investigate the effects of biphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP) as well as E2 on human SK-N-SH neuroblastoma cell line in vitro. The number of variable cells was detected by using cell proliferation assay. The percentage of cells in both S and G2-M phases was tested with cell flow cytometry analysis. Also, caspase-3 activation was determined by western blotting. Moreover, cell survival rate and its sensitivity to the chemotherapy were detected by using cell cytotoxicity assay. MDR1, MRP1, MVP and p53 mRNA and protein expression were investigated by using both real-time PCR and western blotting. Furthermore, the capacity of cell migration and invasion were detected by cell migration and invasion assay. MMP-2, MMP-9 and TIMP-2 RNA and protein expression were determined by using both real-time PCR and western blotting respectively. Then cells were pretreated with estrogen receptor (ER) antagonist ICI 182,780 or phosphoinositide 3-kinase (PI3K) specific inhibitor LY294002 to see whether these agents can block EED-induced effects on NB cells with the same methods described above. In addition,11 NB samples from Chinese Han patients were collected and the whole genome CNV was detected in these samples by using Agilent 244k array comparative genomic hybridization (aCGH). Data were analysized by DNA analytics, Nexus Copy Number and Ingeunity pathway analysis software to explore NB related CNVs, recurrent CNVs and phenotype dependent CNVs as well as their molecular mehchanisms.After 24 h 0.1μM BPA,50μM DEHP or 10μM E2 treatments, the number of variable cells was significantly increased and the cell activity was elevated (P< 0.001 vs. Control). This trend maintained to 120 h. The percentage of cells in S and G2-M phase obviously increased in group treated with BPA, DEHP or E2 (P< 0.001 vs. Control). However, no significant change was observed in caspase-3 expression (P> 0.05). In the cell cytotoxicity assay, BPA, DEHP or E2-treated SK-N-SH cells had a higher cell survival rate than the control (P< 0.01). Significantly elevated mRNA and protein expression of MDR1, MRP and MVP gene were detected in groups treated with BPA, DEHP or E2 (P< 0.01 vs. Control) but decreased mRNA and protein expression of p53 were detected in the BPA, DEHP or E2 group (P< 0.05 vs. Control). Moreover, the cell migration and invasion assay showed enhanced capacity of cell migration and invasion in the BPA, DEHP or E2 group (P< 0.001 vs. Control). Significantly elevated mRNA and protein expression of MMP-2 and MMP-9 gene were detected in groups treated with BPA, DEHP or E2 (P< 0.001 vs. Control) but decreased mRNA and protein expression of TIMP-2 were also detected in the BPA, DEHP or E2 group (P< 0.01 or P< 0.05 vs. Control). Furthermore, cells pretreated with ICI182,780 or LY294002 showed lower cell growth rate, lower percentage of cell in S and G2-M phase but higher sensitivity to chemotherapy compared with BPA, DEHP or E2-only treated groups (P< 0.001 or P< 0.01). Lower mRNA and protein expression of MDR1, MRP1,and MVP as well as higher mRNA and protein expression of p53 were detected in the groups treated both ICI182,780 or LY294002 and EED compared with BPA, DEHP or E2-only treated groups (P< 0.01). The capacity of cell migration and invasion can not be enhanced by BPA, DEHP or E2 after pretreatment with ICI182,780 or LY294002. Lower mRNA and protein expression of MMP-2 and MMP-9 as well as higher mRNA and protein expression of TIMP-2 were observed in the groups treated both ICI 182,780 or LY294002 and EED compared with BPA, DEHP or E2-only treated groups (P< 0.001 or P< 0.05). Akt (Ser473) phosphorylation level was enhanced by BPA, DEHP or E2 treatement compared with the control group but not in ICI182,780 or LY294002 pretreated groups (P<0.01).Furthermore, various CNVs were observed in 10/11 samples and pathogenic CNVs also existed in 9/10 samples. CNV amounts could be dependent on NB's phathological grades. CNV- induced loss was significantly more than CNV-induced gain.14q, 1p,9q,16p and Xq could be recurrent region related to NB. Genes in CNV regions contributed to cell to cell transaction, tissue development, post-transcription modification and protein folds. These genes were involved in Tight Junction, and ILK pathway. The action among these genes contributed to neurological disease, organ injury and cancer.In summary, BPA, DEHP and E2 can promote SK-N-SH cell proliferation, multidrug resistance, migration and invasion in vitro. ER dependent and PI3K/Akt pathway may be involved. CNV exists widely in Chinese Han NB patients,14q, 1p, 9q,16p and Xq may be key regions to NB pathogenesis. Multiple mechanisms may be involved in NB oncogenesis.
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