| Backgrounds and Purpose: Recently, global attention has been paid to the environmental endocrine disruptors (EED). 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. There has been increasing interest in the effects of EED on estrogen-sensitive malignancies. However, little attention has been focused on the potential deleterious effects of exposure to EED on tumors in children. Neuroblastoma (NB) is a common and embryonal pediatric malignant solid tumor. The cause of NB remains unknown. Because of the early age of onset of most cases of NB, research has focused on maternal and paternal exposures occurring prior to conception of during gestation, and early childhood exposures. The development of solid tumor is largely dependent on angiogenesis. Additionaly, NB had a strong ability of angiogenesis and was dependent on it. People now have paid more attention to the role of angiogenesis on NB. In order to find the effects of EED on the development of NB, we first devised several EED as intervention substances to observe their effects on proliferation of SK-N-SH human neuroblastoma cells and human umbilical vein endothelial cells. Then we made one kind of EED as the intervention condition to observe its effect on the development of NB in vivo induced by injection of SK-N-SH cells subcutaneously. Furthermore, we investigate the cell cycle, cell apoptosis, and angiogenesis to observe the underlying mechanisms.Materials and Methods:1. Proliferation of SK-N-SH human NB cells in vitro: NB cells were cultured in estrogen-free improved minimum essential medium and divided into seven groups based on different treatments: group 1, no treatment (control); group 2, treated with 17p-estradiol (E2); group 3, treated with bisphenol A (BPA); group 4, treated with zearalenone (ZEA); group 5, treated with both E2 and ICI 182,780; group 6, treated with both BPA and ICI 182,780; group 7, treated with both ZEA and ICI 182,780. Absorbance value (A V) was determined at time zero, 24, 48 and 72 hours, and DNA proliferation index (PI) at 72 h. Flow cytometer, terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) were employed to monitor cell apoptosis. Western Blot analysis and immunocytochemistry were employed tomonitor the expression of Cyclin D1, CDK4, p21, and PCNA.2. Proliferation of Human umbilical vein endothelial cells (HUVEC) in vitro: HUVEC cells were cultured in estrogen-free improved minimum essential medium and divided into eleven groups based on different treatments: group 1, no treatment (control); group 2, treated with 17p-estradiol (E2); group 3, treated with bisphenol A (BPA); group 4, treated with zearalenone (ZEA); group 5, treated with nonylphenol (NP); group 6, treated with di-2-ethylhexl phthalate (DEHP); group 7, treated with both E2 and ICI 182,780; group 8, treated with both BPA and ICI 182,780; group 9, treated with both ZEA and ICI 182,780; group 10, treated with both NP and ICI 182,780; group 11, treated with both DEHP and ICI 182,780. Absorbance value (AV) was determined at time zero, 24, 48 and 72 hours, and DNA proliferation index (PI) at 72 h. Flow cytometer, TUNEL were employed to monitor cell apoptosis. Western Blot analysis and immunocytochemistry were employed to monitor the expression of Cyclin D1, CDK4, p21, and PCNA.3.Development of NB in vivo: NB cells were injected subcutaneously into ovariectomized or sham operation nude mice. When the tumors were palpable, the mice were divided into four groups based on different treatments: group 1, ovariectomy (control group); group 2, sham operation (SHAM); group 3, treated both OVX and E2 (E2); group 4, treated both OVX and BPA (BPA). The tumor volume was measured twice a week. On the 18th day, all nude mice were executed, tumor weight and body weight were measured. PI, microvessel density, and VEGF expression in tumor tissue were determined by flow cytometer, immunohistochemistry, and Western Blot, respectively.Results:1. Proliferation of SK-N-SH human NB cells in vitro:At 72 hours, AV was 0.86± 0.25 for the control group, 1.02 ± 0.11 for the BPA group, and 1.05 + 0.04 for the ZEA group. AV was increased by 19.8% for the BPA group and by 22.1% for the ZEA group when compared with that of control group (p <0.05). At 72 hours, PI was 17.96±1.31 % for the control group, 25.57±1.89% for the BPA group, and 25.70± 0.60% in the ZEA group. PI was enhanced by 42.4% in the BPA group and by 43.1% in the ZEA group when compared with that of control group (p <0.01). AV and PI in the E2 group were similar to those of the BPA and ZEA groups (E2 group versus BPA or ZEA group, p >0.05). When ICI 182,780 was added to the treatments with E2, BPA, and ZEA, cell proliferation was inhibited completely.In the E2+ICI 182,780, BPA+ICI 182,780, and ZEA+ICI 182,780 groups, AV and PI at 72 hours were decreased significantly, reaching the zero time level (p <0.01, when compared with the E2, BPA, and ZEA groups, respectively). At 72 hours, apoptosis cells were abundant in controls and ICI 182,780 groups, but little in E2, BPA, and ZEA groups. AI was 35.75 ±15.90% for the control group, 7.41 ±2.34% for the BPA group, and 8.08 ± 1.23% for the ZEA group. AI was decreased significantly compared with control group (p <0.05). The results of TUNEL was similar to flow cytometer. At 72 hours, the expression of CyclinD1, CDK4, and PCNA was abundant in E2, BPA, and ZEA groups, but little in control and ICI 182,780 groups (p <0.05). There was no difference of expression of p21 between the groups (p >0.05).2. Proliferation of HUVEC in vitro:At 72 hours, AV was 0.56 ± 0.12 for the control group, 0.76± 0.04 for the BPA group, 0.74 + 0.07 for the ZEA group, 0.90 ±0.15 for the NP group,and 1.00±0.04 for the DEHP group. AV in BPA, ZEA, NP, and DEHP from 1.3 to 1.7 times that of control group (p <0.05). At 72 hours, PI was 30.86±7.08% for the control group, 44.81 ±4.29% for the BPA group, 44.21 ±2.60% for the ZEA group, 44.13±3.06% for the NP group, and 44.67±3.41% for the DEHP group. PI in BPA, ZEA, and DEHP group was enhanced by 50% when compared with that of control group (p <0.05). AV and PI in the E2 group were similar to those of the BPA and ZEA groups (E2 group versus BPA or ZEA group, p >0.05). When ICI 182,780 was added, cell proliferation was inhibited. At 72 hours, apoptosis cells were abundant in controls and ICI 182,780 groups, but little in E2, BPA, ZEA, NP, and DEHP groups. ,AI was 45.16 ±11.50% for the control group, 8.25±3.32% for the E2 group, and 10.59±4.92% for the BPA group, 9.81 ±4.60% for the ZEA group, 10.65±3.63% for the NP group, 9.83 ±5.29% for the DEHP group, AI was decreased significantly compared with control group (p <0.05). The results of TUNEL was similar to that of flow cytometer. At 72 hours, the expression of CyclinD1, CDK4, and PCNA was abundant in E2, BPA, and ZEA, groups, but little in control and ICI 182,780 groups (p <0.05). There was no difference of expression of p21 between the groups (p >0.05).3. Development of NB in vivo:In BPA group in vivo, there was 59% increase in tumor volume (p <0.05), 82% increase in tumor weight (p <0.05), 19% increase in PI (p <0.05), 83% increase in microvessel density (p <0.01), and 54% increase in VEGF expression (p <0.05) in comparison with controls. The results of E2 groups both in vitro and in vivo weresimilar to those in BPA groups. The expression of CyclinDland PCNA was abundant in E2, BPA, and SHAM groups, but little in control (p <0.05). There was no difference of expression of CDK4, p21 between the groups (p >0.05).Conclusions:1. Two environmental endocrine disruptors, Bispaenol A and Zearalenone, could promote the proliferation of SK-N-SH cells. Estrogen receptor pathway, depressing apoptosis, or disrupting cell cycle may be involved in the process of growth of SK-N-SH cells, which is directed by Bispaenol A and Zearalenone.2. Bispaenol A and Zearalenone could promote the proliferation of Human Umbilical Vein Endothelial Cells. Estrogen receptor pathway, depressing apoptosis, or disrupting cell cycle may be involved in the process of growth of Human Umbilical Vein Endothelial Cells, which is directed by Bispaenol A and Zearalenone.3. The growth-promoting effect of Nonylphenol and Di-2-ethylhexl phthalate on Human Umbilical Vein Endothelial Cells, and the mechanism was not clear, needing to be further study.4. Bispaenol A could promote the growth of tumor transplanted with human neuroblastoma SK-N-SH cell line in nude mice.5. Cell cycle, and tumor angiogenesis may be involved in the development of tumor transplanted with SK-N-SH human neuroblastoma cell line in nude mice. |
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