Effects Of Huaier On The Biological Behavior Of Ovarian Cancer Cells And Its Molecular Mechanism

Epithelial ovarian cancer accounts for the vast majority of ovarian cancer. Most patients present with advanced disease. The standard initial management of epithelial ovarian cancer consists of surgery and administration of chemotherapy with carboplatin. Although most patients with advanced disease could get completely remission after initial treatment, tumor recurrence occurs in almost all these patients. Recurrent ovarian cancer is hard to be managed. Recently, promising novel therapy targets in epithelial ovarian cancer are PARP inhibitor and EGFR antibody, which can inhibit tumor cells to repair single-strand damage and block tumor angiogenesis respectively. Clinical trials showed that they can prolong the median progression-free survival. However, the overall survival is similar to conventional therapy. Because none of these strategies can completely protect ovarian cancer patients from recurrence and metastasis, new drugs are urgently needed.In recent years, the traditional Chinese medicine (TCM) has gained popularity for its ability to kill tumor cells with less harm to normal cells. Studies have showed that TCM have anticancer ability against various tumor through inhibition of tumor growth, induction of apoptosis, inhibition the invasiveness and promotion chemotherapeutic drugs sensitivity.Trametes robiniophila murr (Huaier) is a type of fungus from China that been approved by the Chinese Food and Drug Administration. It has been used in hepatocellular cancer patients and showed unique effect. Therefore, this study will use in vitro and in vivo culture model of ovarian cancer to observe Huaier’s affection on the biological behavior of ovarian cancer and to investigate the mechanisms underlying Huaier’s function. Through above study, we want to get a comprehensive understanding on the clinical value of Huaier in treatment of ovarian cancer.These experiments were divided into 3 parts, as follows:1. The effects of Huaier on the biological behavior of ovarian cancer cells.2. Detecting protein expression in Huaier-treated ovarian cancer cells by RPPA and initial study on Huaier’s function.3. The effects of Huaier on human ovarian xenografts tumor growth in mice.Part I The effects of Huaier on the biological behavior of ovarian cancer cellsObjective:To investigate the effects of Huaier on the biological behavior of ovarian cancer cells.Methods:Ovarian cancer cells were treated with different concentrations of Huaier, at the indicated time, cells proliferation was evaluated by sulforhodamine proliferation assay (SRB) method. Ovarian cancer cells were treated with different concentrations of Huaier for 48 hours, cell apoptosis was detected by Annexin V-PI staining. Ovarian cancer cells were treated with 5 mg/ml Huaier for 24 hours, invasive ability was evaluated by transwell assay and migration ability was detected by in vitro scratch assay.Results:(1) Huaier inhibited ovarian cancer cell viability, this growth inhibition phenomenon showed a time-and dose-dependent manner. (2) Huaier induced ovarian cancer cell apoptosis, this pro-apoptotic effect showed a dose-dependent manner. (3) Huaier inhibited migration of ovarian cancer cells. (4) Huaier inhibited invasion of ovarian cancer cells.Conclusion:Huaier can greatly influence the biological behavior of ovarian cancer cells. It can inhibit proliferation, invasion and migration of ovarian cancer cells, induce apoptosis in ovarian cancer cells. Ovarian cancer cells, which have a higher metastatic potential, might be more sensitive to the Huaier treatment.Part II Detecting protein expression in Huaier-treated ovarian cancer cells by RPPA and initial study on Huaier’s functionObjective:Analysis the gene expression profile in Huaier-treated ovarian cancer cells to investigate the signaling pathways that might contribute to Huaier’s regulation effect. To investigate Huaier’s effect on pAKT/mT0R/S6 pathway and canonical Wnt pathway.Methods:Reverse phase protein assay (RPPA) was used to detect and make comparison of 153 important genes in the proliferation、apoptosis、adhesion or invasion-related pathway in Huaier-treated and control ovarian cancer cells. Protein expression of pAKT、AKT、pS6 (S235-236)、pS6 (S240-244)、GSK3β、GSK3β pS9、β-catenin、E-cadherin and Cyclin D1 was measured by western blot. The expression of DIXDC1, LRP6 and WNT5 were detected by RT-PCR. After transfection of GSK3β specific siRNA, western blot was applied to detect β-catenin expression in ovarian cancer cells with or without Huaier treatment.Results:(1) Cell proliferation, apoptosis and cell adhesion or invasion signaling proteins were down-regulated by Huaier treatment. (2) Huaier repressed AKT and S6 phosphorylation. (3) Huaier repressed cellular β-catenin protein expression and its nuclear translocation. (4) Huaier treatment enhanced GSK3β protein expression and decreased GSK3β S9 phosphorylation. (5) Huaier cannot repress β-catenin in the GSK3β-silenced ovarian cancer cells. (6) Huaier treatment represses the expression of DIXDC1、LRP6 and WNT5.Conclusion:Cell proliferation, apoptosis and cell adhesion or invasion signaling proteins were down-regulated by Huaier treatment.Huaier might induce ovarian cancer cell growth inhibition through pAKT/S6 pathway, and inhibit cellular invasion via the Wnt/GSK3β/β-catenin pathway. Part Ⅲ The effects of Huaier on the human ovarian xenografts tumor growth in miceObjective:The ovarian cancer xenograft model was used to evaluate Huaier’s effect in vivo.Methods:SKOV3 cells (4×106) were injected subcutaneously into the right flank of each mouse. Then, the mice were randomly divided into four groups (n= 6 mice/group) to receive either (1) normal saline (NS) as a control, (2) Huaier (4 g/kg body weight), (3) cisplatin (DDP) (5 mg/kg bodyweight) or (4) Huaier plus DDP. Huaier was administered orally daily. DDP was injected once a week for three weeks. The control mice were treated with the same volume of NS only. Tumor volumes were measured twice a week using a digital caliper.Results:There was no difference on the weight of nude mice in 4 groups. Compared with control, the tumor volumes in DDP group were much smaller from the 22th day (P＜0.05); the tumor volumes in Huaier group and Huaier plus DDP group were much smaller from the 34th day (P＜0.05).Conclusion:Huaier inhibited human ovarian xenograft tumor growth in mice, but exerted no synergistic effect with cisplatin treatment.The results showed that Huaier can inhibit proliferation, invasion and migration of ovarian cancer cells, induce apoptosis in ovarian cancer cells and inhibit tumor growth in nude mice. Given its low toxicity, Huaier’s effect on GSK3 β/β-catenin signaling makes it an attractive way to target invasion in epithelial ovarian cancer cells.