Experimental Study On The Effect Of Ampelopsin On Migration And Invasion Of Ovarian Cancer And Its Mechanism

Background:In USA,the fifth cause of cancer death in women is ovarian cancer. In about 25% of women diagnosed with ovarian cancer confined to one side or both sides, about 75%-90% live more than five years. However, in about 75% of women who were diagnosed as ovary cancer in phase Ⅲ/Ⅳ, the long-term disease-free survival is possibly about 15%-20%. There are two distinct types of ovarian cancer-type Ⅰ and type II, and the proportion of two types may be originated from pelvic organizations outside the ovary. Ovarian cancer is considered to initially originate from ovarian surface epithelium developed from mesoderm epithelial. Ⅰ type of ovarian cancer (such as clear cell carcinoma, mucinous carcinoma and low-grade serous and endometrial cancer) at the time of diagnosis is usually confined to the ovary and developed from precursor of boundary tumor.The typical somatic mutations are relatively stable, KRAS,BRAF,ERBB2 and PIK3CA included except TP53. Ⅱ type of ovarian cancer (such as advanced serous, endometrioid, advanced malignant mixed mesodermal tumors and undifferentiated carcinoma) has higher malignant degree and thought to be by oviduct epithelial cancer development, and genetic instability characterized by somatic TP53 mutations, but type I tumor rarely appears sudden change characteristic.Nuclear factor kappaB (NF-κB) refers to a family of transcription factors (also known as the Rel family) regulating the expression of more than 400 genes. The genes are crucial in many cell processes,The cytokines, many proinflammatory genes, adhesion molecules, complement factor, stress response genes, apoptosis genes, enzymes, growth factors and other transcription factors and so on.Because of this role, NF-κB is considered to be associated with several diseases, including cardiovascular disease and cancer.Evidence suggests that NF-κB in many tumor cell lines constitutively activated,the characteristics of signal pathways that affect cancer progression and response to chemotherapy of NF-κB mediated through inhibition of apoptosis, angiogenesis, proliferation and inflammation to promote the survival of tumor.Ovarian cancer can enhance the function of NF-κB, to avoid apoptosis pathway and resistance to external environmental damage, such as anti-tumor immune effector or chemotherapy.In vitro or human cell line xenograft in inhibiting the activity of NF-κB can increase the sensitivity of cells to cisplatin and paclitaxel induced apoptosis,And platinum combined with paclitaxel for first-line treatment of ovarian cancer is the most commonly used.Tumor necrosis factor alpha (TNFa) can induce cell apoptosis,however,ovarian cancer is cell tolerance, mainly cancer cells can up regulate the signaling pathway induced by NF-κB activation and caused apoptosis proteins.In addition to the direct role in tumor cells, NF-κB mediated events can also lead to the occurrence of associated with ovarian cancer microenvironment and peritonitis.The core of the role of NF-κB in inflammation has aroused widespread concern, because the anti tumor immune response can improve the prognosis of the patients.Ovarian cancer is a variety of immune effector T cell infiltration, including anti tumor.Types of immune infiltration can affect the progress of the disease.Chemokines and cytokines control anti tumor immune response type, these cytokines produced by tumor infiltrating macrophages, dendritic cells and tumor. The characteristics of the NF-κB mediated signal pathway by inhibiting apoptosis, angiogenesis, proliferation and inflammation to promote survival of tumor.All these functions of NF-κB seem to play a role in ovarian cancer, thus further understanding the biological activity mediated by NF-κB could better identify the risk of ovarian cancer and promote a more effective strategy. Various evidences support that NF-κB is crucial in ovarian cancer and the anti tumor immune response.Thus we can assume that the unknown risk of inflammation-related cancer is due to genetic variations of NF-κB pathway. NF-κB is the expression and regulation of hundreds of genes with normal cell function and carcinogenesis related cellular processe.Its signal pathway includes a complex cascade of events started by the immune and other signal protein, consisting of some inhibitory molecules regulating, eventually leading to control the expression of NF-κB mediated gene.((2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one), also called dihydromyricetin, is one type of flavonoid, it’s isolated from young stems and leaves of Ampelopsis grossedentata. AMPLS is not soluble in water also in aqueous and organic solvent, and decomposes easily exposing to light and thus forms a colorful photolysis product. Low water soluble, low intestinal permeability and easy to degradation in aqueous solution limit AMPLS as drug and the potential as experiment compounds. Ampelopsis has lots of activities of pharmacology just as anti-inflammatory, antimicrobial, relieving a cough, antioxidant, depressurization, liver protection, anti-tumor effect also included. A recent study reported the effect of AMPLS anti human immunodeficiency virus (HIV) and HIV-1 receptor CXCR4. The antiviral effect of AMPLS cuold be the result of its antioxidant property. AMPLS makes melanoma cells in vitro and melanoma in vivo develop slowly.AMPLS inhibits the growth of lung cancer by reducing tumor cell proliferation.AMPLS generates anti-angiogenesis through reducing the secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).AMPLS also could change multi-drug resistance of leukemia cells in vitro by reducing the expression of p glycoprotein. Therefore, this research is to find the effect of ampelopsis on ovarian cancer and the possible signaling pathway mechanism.Objective:1. To investigate the impact of ampelopsis on proliferation, migration and invasion in A2780 cells;2. To investigate the impact of ampelopsis on epithelial-mesenchymal transition;3. To research ampelopsis’s specific effect on ovarian cancer and the related signaling pathways.Methods:1.Cell cultureA2780 cell line,also called human ovarian cancer cell line, came from American Type Culture Collection, fostered by the containing 10% fetal bovine serum RPMI1640 to 37 degrees Celsius,5%CO2 incubation box.2. The MTT experimentA2780 cells plated at the density of 5000/hole in the 96 hole plate, after stimulation of cells,using Ampelopsis japonica of different concentrations and different time points, , MTT (5 mg/ml,20μl) was added into each well for 4 h incubation. Then,80μl of dimethyl sulfoxide was added for another 15min to fully solubilize formazan. At last the liberated purple product was detected using a microplate luminometer at 490nm.3. Scratch Wound Healing AssayA2780 cells were cultured in six-well plates (1×106/well). Straight scratches of the same width were made in the monolayer of A2780 cells with a pipette tip. After incubation with ampelopsin of 25μM for 24 h, photo images were taken to measure the wound healing under a microscope4. Transwell assayA2780 cells were seeded in 24 hole plates and the upper chamber for 1% fetal bovine serum RPMI1640 contained, the lower chamber for 10% fetal bovine serum RPMI1640 contained. Cells were re-suspended in the upper chamber at 37℃ and 5% CO2. Cells (1 x 105/hole) put on cultivating incubator chamber at the temperature of 37,5%CO2,24 hours later, the cells of lower methanol fixed about 30 minutes and stained by crystal violet, upper wiped away. Western blot analysisAfter stimulation, A2780 cells were gatherd and lysed,Using specific antibodies to test the protein concentration, the expression of E-cadherin, N-cadherin, Vimentin, GAPDH, NF-κB, p-IkBa and Snail, and band gray analysis with Photoshop software.6. Statistical analysisAll data in the study were evaluated with predictive analytics software (PASW) Statistics 18.0 (SPSS Inc, Chicago, IL). Analysis of normal distribution data of single factor variance analysis, non parameter variable test by Mann-Whitney U. P< 0.05 was thought statistically significant.Results:1.Effect of ampelopsin to the ovarian cancer cells proliferationDifferent concentrations (for 0,5,10,25,50μM) of ampelopsin were added into the cultured A2780 cells. After 24h-incubation at 50μM, MTT assay detected that ampelopsin obviously inhibited cell viability. However, at concentrations below 50μM (5,10,25μM), the inhibition was not so significant. Subsequently,25μM and 50μM of ampelopsin were selected to stimulate the cells for different time (Oh, 1h,3h, 6h,12h,24h and 48h). Ampelopsin of 50μM significantly inhibited the proliferation after 24h stimulation, while ampelopsin of 25μM didn’t inhibit the proliferation after 48h stimulation.2.Ampelopsin influent the ovarian cancer cells’s migration and invasionWound healing assay demonstrated that healing over the scratch was obviously reduced after treated by ampelopsin. Meanwhile, the results of transwell assay demonstrated that the invading cells transferred from upper surfaces to lower surfaces were also significantly decreased after treated by ampelopsin.3. Ampelopsin’influence on EMT marker expressionVarious concentrations of ampelopsin (for 5,10,25 and 50μM) were respectively added into the cells and the cancer cells were cultured for another 12h. Ampelopsin treatment could obviously increase the epithelial marker (E cadherin) expression and decrease mesenchymal marker N (E-cadherin and vimentin) expression.The results above suggested that ampelopsin could change the expressions of EMT markers in a concentration-dependent manner and remaindered that the inhibitory effect of ampelopsin on A2780 cells invasion and migration might be associated with EMT.4. NF-κB Pathway is involved in the anti-metastatic mechanism of AmpelopsinWith various concentrations (for 5,10,25 and 50 M) ampelopsin stimulated 12h,Then cytosol and nucleus proteins were extracted respectively, following with the detection of the expression of P65 and IκBα. Ampelopsin obviously increased the p65’s expression in cytosol but decreased its expression in nucleus with concentration-dependent manner. Meanwhile, the phosphorylation of IkBα in the cytosol was decreased after ampelopsin treatment. After that, to further determine the effect of NF-κB on the expression of EMT markers, we blocked the NF-κB pathway by its inhibitor:BAY11-7082 (20p.M), Before joining the ampelopsin before, with the NF-B pathway inhibitor BAY11-7082 (20 M) pretreatment for 2 hours. The results showed, reduced with BAY 11-7082’s increase obviously reversed ampelopsin induced E cadherin and N of E-cadherin and vimentin。5. Ampelopsin-induced Snail upregulation via NF-kB activationIn a concentration gradient (5,10,25 and 50 M) of ampelopsin stimulation of A278024h, ampelopsin increases with increasing concentration can inhibit the expression of Snail at the concentration of 50 μM, the maximum activity. BAY 11-7082 (20 M) 2H pretreatment can significantly eliminate the ampelopsin’s inhibitory effect. The above results show that, NF-kappa B is crucial for the Snail regulation.Conclusion:1.Ampelopsin can inhibit the proliferation, migration and invasion of ovarian cancer cell:2.Ampelopsin could change the expressions of EMT markers in a concentration-dependent manner;3.Ampelopsin induced the expression of Snail via NF-κB pathway.