| Background and objective:Ovarian cancer is the most lethal one in gynecological cancers. The main reason is that most patients were diagnosed in advanced stage presenting with ascitic fluid and distant metastasis. Despite advances in treatment during the last decades, the prognosis of women with epithelial ovarian cancer (EOC) has not substantially improved since most patients would suffer from distant metastasis ultimately. Based on the well understanding of the mechanism by which ovarian cancer cells invade and migrate to distant parts, searching for drugs that can block this process has potential clinical significance for the therapy of ovarian cancer. Epithelial-mesenchymal transition (EMT) is a cell-biologic program, which plays a key role in embryonic development, organogenesis, wound healing and so on. Recently, researches in breast cancer, hepatic cancer, ovarian cancer have found that the carcinoma cells acquired several cell-biological traits that are needed to generate macroscopic metastases passing through EMT. Considering the key role that the EMT program played in steps of the invasion-metastasis cascade in various cancer cells, inhibition of EMT may be an attractive cancer therapy that could improve the outcome of malignant diseases. Salinomycin, a monocarboxylic polyether antibiotic which was originally used to eradicate bacteria, fungi and parasites in poultry, has been identified as the most selective inhibitor for breast CSCs. The relation between EMT and CSCs suggested that EMT may be a vital pathway upon which salinomycin could affect CSCs. Then subsequent publications demonstrated that salinomycin could repress the abilities of proliferation, invasion and EMT in several cancer cells. Our research aimed to investigate the therapeutic potential of salinomycin for EOC, especially its effect on EMT, and to clarify the underlying mechanism.Methods:The CCK-8 assay was performed to evaluate the capacity of proliferation of A2780 and SK-OV-3 cells after treating with a range of concentrations of salinomycin for 24h,48h,72h, respectively. Transwell chambers were performed to evaluate the migration and invasion abilities of ovarian cancer cells treated with salinomycin. Western-blot and RT-PCR were carried out to detect the expression of the EMT related markers (E-cadherin, Keratin, N-cadherin and Vimentin) and the Wnt/β-catenin pathway associated molecules in EOC cells treated with salinomycin. Immunofluorescence assay was used to localize the distribution of β-catenin in EOC cells. GSK-3β inhibitor (SB216763) was used to activate Wnt/β-catenin pathway in EOC cells.Results:(1) Salinomycin could repress the proliferation of EOC cells. CCK-8 assay was used to measure the cell viability of A2780 and SK-OV-3 cells treated with a range of concentration of salinomycin for different hours and found that salinomycin could effectively reduce the ability of proliferation of EOC cells in a time- and dose-dependent manner.(2) Salinomycin could repress the invasion and migration of EOC cells. Then we use transwell chambers to measure the invasion and migration abilities of A2780 and SK-OV-3 cells treated with salinomycin and found that treatment with salinomycin for 48h could repress the invasion and migration in both A2780 and SK-OV-3 cells (P≤0.001).(3) Salinomycin could repress the EMT program in EOC cells. Western-blot and RT-PCR were carried out to detect the expression of the epithelial-mesenchymal transition (EMT) related markers (E-cadherin, Keratin, N-cadherin and Vimentin) in A2780 and SK-OV-3 cells. The Western-blot showed that salinomycin could significantly increase the expression of epithelial markers (E-cadherin and Keratin) while decrease the expression of mesenchymal markers (N-cadherin and Vimentin) in a dose-dependent manner. Such results were ascertained by RT-PCR.(4) Salinomycin could block the Wnt/β-catenin pathway in EOC cells. Salinomycin could downregulate the expression of molecules associated with Wnt/β-catenin pathway (p-GSK-3β-ser9, β-catenin, slug, Axin2 and CCND1) measured by Western-blot and RT-PCR and reduce the nuclear distribution of β-catenin.(5) Activation of Wnt/β-catenin pathway could promote EMT in EOC cells and salinomycin could reverse the activation. Treatment with GSK-3β inhibitor (SB216763) could upregulate the expression of Wnt/β-catenin pathway related molecules. Meanwhile, the epithelial markers were downregulated while the mesenchymal markers were upregulated after treatment with SB216763, suggesting that the aberrant activation of Wnt/β-catenin pathway do contribute to the EMT program in EOC cells. After prepared with SB216763 for 6h, the EOC cells were treated with respective dose of salinomycin. As we expected, the p-GSK-3β-ser9, β-catenin and slug were all down-regulated compared with the prepared group without following salinomycin treatment, indicating that salinomycin could reverse the activation of the pathway by SB216763.Conclusions:(1) Salinomycin could inhibit the proliferation, migration and invasion in EOC cells by repressing the EMT program.(3) The inhibitive effect of salinomycin on the EMT program in EOC cells was achieved by blocking the Wnt/β-catenin pathway. |
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