Effect And Mechanism Of DP On The Stem Cells Properties In Human Ovarian Cancer Cell Line OVCAR-3

Dechlorane Plus（DP）is a widely used new chlorinated flame retardant,which can persist in environment and accumulate in organisms.Our previous studies have shown that DP has an estrogen-like effect and can cause the proliferation in human breast cancer cell MCF-7.Ovarian cancer is an estrogen-dependent high-mortality female cancer.Some environmental estrogens can lead to its formation and deterioration.It has been proven that cancer stem cells（CSCs）are closely related to tumor growth,invasion and metastasis.In this study,the effects of DP on cell viability and stem cells properties in human ovarian cancer cells were investigated.Human ovarian epithelial cancer OVCAR-3 cells were exposed to environmental relevant concentrations of DP(10^-5-10^-10M),using estradiol（E2）as a positive control.After exposure for 24 and 48 hours,cell proliferation was detected by MTT assay and cell cycle was detected by flow cytometry.The effects of different concentrations of DP on the formation of OVCAR-3 stem cell spheres were investigated by cell spheroidization experiment.DP concentrations with significant effects on cell spheroidization were selected for mechanism study.RT-q PCR and Western Blot were used for quantitative analysis of CSCs biomarkers andβ-catenin in Wnt/β-catenin signaling pathway.The biomarkers of CSCs were ALDH1 and CD133 related to the occurrence,metastasis and chemotherapy resistance risks of ovarian cancer CSCs,as well as NANOG,OCT4 and SOX2 related to the maintenance of ovarian cancer stem cell characteristics.Results of cell viability and cycle assay demonstrated the non-monotonic dose-dependent effects of DP.High dose(10^-7M)of DP exposure could significantly upregulate the cell proliferation index（PI）and increase cell viability,while low dose(10^-10M)of DP exposure could significantly downregulate the PI and significantly decrease cell viability.DP-induced stem cell sphere formation in OVCAR-3 cells was also dose-dependent.10^-7and 10^-8M of DP exposure significantly increased sphere number and size,while 10^-10M of DP exposure significantly reduced cell spheroidization.Quantitative analysis of CSCs biomarkers and Wnt/β-catenin pathway reveal the molecular mechanism of DP on the stem cells properties in OVCAR-3.10^-7M of DP exposure upregulated the transcriptions of ALDH1,CD133,NANOG,OCT4 and SOX2 genes,increased the protein levels of NANOG and SOX2,induced CSC of OVCAR-3 cells by actiating the Wnt/β-catenin pathway.On the contrary,10^-10M of DP exposure downregulated the transcriptions and protein levels of CSCs biomarkers,decreased CSC of OVCAR-3 cells by inhibiting Wnt/β-catenin pathway.To sum up,our findings demonstrated the complex biological effects of DP.Environmental relevant concentration of DP would have potential effects on both cell viability and stem cells properties in human ovarian cancer cells.These basic data will provide a scientific basis for a comprehensive understanding of the toxicity of DP and for objective evaluation of its ecological risk.