| Background:Ovarian cancer is the most lethal gynecologic malignancy, and more than 90% is classified as epithelial ovarian cancer (EOC). The majority of EOC patients are diagnosed at advanced stages and the overall 5-year survival rate is approximately 40%. It has been reported that invasion and metastasis process remains the leading cause of recurrence and death from EOC and the molecular mechanisms of invasiveness and the metastatic property are not clearly understood. Accumulating evidence reveals that epithelial to mesenchymal transition (EMT) plays an essential role in the regulation of cancer metastasis. Emerging evidence indicates that EMT is of vital importance in obtaining invasive and migratory ability in EOC. Thus, EOC patients may benefit from targeted therapies that inhibit EMT.Emodin (1,3,8-trihydroxy-6-methylanthraquinone; EMO) is a natural anthraquinone derivative in the roots and rhizomes of Polygonum cuspidatum and Rheum palmatum. EMO displays anticancer activities in several types of cancers, including EOC. In one study it was elucidated that EMO exerted antiproliferative and apoptosis-inducing effects in A2780 (paclitaxel-sensitive) and A2780/taxol (paclitaxel-resistant) cells by reducing the expression of anti-apoptotic molecules, such as survivin and X-linked inhibitor of apoptosis (XIAP), whereas, EMO had potential to inhibit tumor invasion and metastasis of HO-8910PM cells via repression of the production of MMP-9. Studies in vitro also demonstrated that EMO was effective on restraining SKOV3 and HO8910 cell invasion. However, the molecular mechanisms of the anti-invasive and antimetastatic functions are unknown. In previous studies, EMO was shown to exert inhibitory effects on cell invasion and migration of colorectal and cervical cancer, and head and neck squamous cell carcinoma (HNSCC), which is associated with the inhibition of EMT and deregulation of WNT/β-catenin signaling pathway. Therefore, we speculated that EMO may inhibit the EOC cells to undergo the EMT progress by regulation of WNT/β-catenin signaling pathway, consequently weakening the invasiveness and metastatic ability.Objective:To investigate the effects of EMO on invasion, metastasis and EMT of EOC and explore its molecular mechanism.Methods:1. Inverted microscope was carried out to observe the cell morphology of A2780, OVCAR-3 and SK-OV-3 cells.2. Cell counting Kit-8(CCK-8) assay was used to explore the effect of EMO on A2780 and SK-OV-3 cell proliferation ability.3. Transwell assay was conducted to evaluate the relative cell invasion ability of A2780, OVCAR-3 and SK-OV-3 cells and the influence of EMO on A2780 and SK-OV-3 cell invasion ability.4. Western blot was performed to detect the expression levels of protein for epithelial markers like E-cadherin, keratin and mesenchymal markers like N-cadherin, vimentin, MMP-2, MMP-9, in addition, EMT-related pathway makers like glycogen synthase kinase 3(3 (GSK-3β), phospho-Ser9-GSK3β (p-GSK-3pSer9), β-catenin and transcription factor ZEB1 after A2780 and SK-OV-3 cells were treated with EMO (0,10,20,40 μM) and pretreated with 10 μM GSK-3β kinase inhibitor SB216763.5. To investigate the effects of EMO on cell invasion ability and EMT after ZEB1 siRNA was utilized to target A2780 and SK-OV-3 cells.Results:1. The cell morphology and the relative cell invasion ability of A2780, OVCAR-3 and SK-OV-3 cells:low invasive cells OVCAR-3 and high invasive cells A2780, SK-OV-3 shows epithelial and mesenchymal cell morphology and phenotype expression respectively.2. The effects of EMO on cell proliferation ability, cell invasion ability and EMT of A2780 and SK-OV-3 cells:20 μM EMO failed to regulate cell proliferation ability (P>0.05).20 μM EMO significantly inhibited the cell invasion ability (P<0.01). After the treatment of cells with EMO (0,10,20,40 μM) for 48 h, upregulation of E-cadherin and keratin and downregulation of vimentin, N-cadherin, MMP-2 and MMP-9 in a concentration-dependent manner were observed.3. To investigate the functional mechanism of EMO inhibition of EMT on A2780 and SK-OV-3 cells:EMO (0,10,20,40 μM) treatment dose-dependently increased levels of p-GSK-3βSer9, β-catenin, occurred in the absence of any changes in total GSK-3βlevels. EMO treatment also significantly decreased ZEB1 protein levels in a concentration-dependent. Cells were pretreated with 10 μM SB216763, a selective GSK-3β kinase inhibitor, and we found that the effects of 20 μM EMO were blocked significantly.4. To further study the mechanism of EMO inhibition of EMT on A2780 and SK-OV-3 cells:Either EMO treatment alone or treatment of ZEB1-knockdown cells with EMO almost equally played the same role in regulating the activity of GSK-3β and the expression of β-catenin. Upon ZEB1 knockdown, A2780 and SK-OV-3 cells treated with EMO exhibited more decreased invasion capacity, decreased vimentin protein levels and consistently more increased keratin expression levels compared to ZEB1 knockdown or EMO treatment alone. Accordingly, ZEB1 protein levels were lowest in the EMO-treated ZEB1 knockdown cells (P<0.01).Conclusion:1. EMO could significantly inhibit EOC cells invasion, metastasis and EMT.2. EMO inhibits EMT in EOC cells by regulation of GSK-3β/β-catenin/ZEBl signaling pathway. |
PDF Full Text Request