Mechanisms Of Inhibited Proliferation And Peritoneal Metastasis Of IFN-a On Serous Ovarian Cancer

IntroductionOvarian cancer is one of the most common cancers in the female and the leading cause of death from gynaecological malignancy. The largely unchanged mortality rate from ovarian carcinoma results from its late clinical appearance,with two-thirds of the patients being diagnosed with stageâ…¢orâ…£disease. By the time patients are diagnosed with ovarian carcinoma, peritoneal dissemination of the tumor often has occurred. Despite aninitial 70-80% responserate,current therapy is frequently followed by recurrence,which is often resistant to chemotherapy, as demonstrated by the 30% long-term survivors. Formation of ascites is related to the cancer cell peritoneal dissemination, and is the leading cause of death of serous ovarian cancer. The rapid tumor cell growth and early transmission may be due to low oxygen microenvironment. Cellular adaptation to hypoxia represents an essential step in tumor progression. Peritoneal dissemination involves several steps, including tumor-cell attachment, invasion, and growth in the peritoneum. Many cytokines, growth factors, matrix metalloproteinases (MMPs), and angiogenic factors play important roles in these steps.Angiogenesis and cellular adaptation to hypoxia represent key step in tumor progression. This is because of the fact that the limited diffusion capacity of does not allow tumor growth beyond several mm without neoangiogenesis. Also,cancer cell proliferation may outpace the rate of angiogenesis,and tumor cells will have to adapt to tissue hypoxia in this situation.One of the key factors regulating cellular O₂ homeotasis is HIF-1α. HIF-1αmay increase O₂ availability or metabolicadaptation to O₂ deprivation by influencing a number of genes which in part play a role in tumor progression including erythropoietin, transferrin, endothelin-1,inducible nitricoxide synthetase,hemeoxygenasel, VEGF, insulin-like growth factor-2, insulin-like growth factor binding proteins 2 and 3,and 13 different glucose transporter sandglycolytic-enzymes.Furthermore,HIF-1αprotein may also influence cell cycle progression/proliferation and the rate of apoptosis,thus influencing tumor progression. Also independent from its regulation of VEGF expression.Growth and metastatic dissemination of solid tumours requires vascular support for nutritive supply and access. For a tumour to get larger than 1 mm,a process of angiogenesis or neovascularisation must occur, To produce ablood vessel, endothelial cells must divide, migrate,degrade the extracellular matrix, differentiate,and survive. This multistep process is controlled by a large number of distinct positive and negative regulatory molecules. Of these molecules, VEGF and bFGF,are potent mitogen for endothelial cells.Antiangiogenic therapy is a promising strategy for inhibiting tumor growth and metastasis. IFN-αis a multifunctional cytokine capable of interfering with viral infection of cells,as well as inhibting tumor cell proliferation,regulating cell differentition,and modulating immune response. Earlier published results proved IFN-αcould be used as an agent exhibiting strong antitumor effects by suppression of tumor angiogenesis.However,the molecular mechanisms of this inhibition were still not elucidated.We determined the expression of HIF-1α,VEGF,bFGF,CD34 protein of human serous ovarian carcinomas, and expression of these agents of effusion and metastaticsites of 31 advanced patients. The aim of the present study was to clarify the significance of HIF-1α,VEGF and bFGF in prognosis and peritoneal metastasis and asctic formation of serous ovarian cancer. To elucidate the molecular mechanisms of antiangigenic effects of IFN-αⅡb in OVCAR₃ cell,we screened HIF-1α,VEGF and bFGF, in OVCAR₃ cell before and after IFN-αⅡb treatment, demonstrate the mechanisms of inhibited peritoneal metastasis of IFN-αⅡb on serous ovarian cancer. Materials and Methods一,Patients52 consecutive cases of serous ovarian cancer, FIGO stagesâ… -â…£,were retrieved from our files,as well as 9 cases of ovarian LMP tumors (so-called borderline tumors), and 20 cases of ovarian cystadenomas. They were initially evaluated by clinical and ultrasound examination, chest X-ray,and computerized tomography of the abdomen. Treatment of cancer patients consisted of radical surgery with total abdominal hysterectomy,bilateral salpingo-oopherectomy,pelvic lymph node dissection,and omentectomy.All patients,except those with gradel, stageIA,were given adjuvant chemotherapy. Patients received six cycles of a platinum/Taxol containing multiple drug chemotherapy. There were 21 patients with peritoneal recurrence.二,methods1,The expression of HIF-1α,VEGF,bFGF,CD34,PCNA protein in primary and metastaticsites was determined immunohisto-chemically (S-P) in paraffin-embedded specimens.2,The expression of HIF-1α,VEGF,bFGF protein in effusion was determined immuno-chemically(EnVision) in cellblock specimens.3,Enzyme-linked immunosorbent assay (ELISA) commercial kits were used to determine the serum and effusion concentrations of VEGF and bFGF.4,CoCL₂ was used to estabolished hypoxia model.5,Effect of IFN-αon the Proliferation of Ovarian OVCAR₃ Cell Lines was investigated with colorimetric assays with MTT cell growth assay kits.6,Cell Cycle Analysis with a FACScan.7,Expression of HIF-1α,VEGF,bFGF and survivin of Ovarian OVCAR₃ Cell Lines were analyzed by Western blot.8,Statisticalanalysis: x² test were used to analyze the data for significant differences.The results were presented as means±SD. Data were analyzed using SPSS13.0 for windows oftware.Spearman's-rank correlation was used for theVEGF and HIF-1αprotein expressions. The significance of any difference between the survival curves was determined using the Cox-Mantel test. Differences were considered significant at P＜0.05.Results1,HIF-1α,VEGF,bFGF,PCNA expression of normal and cystadenomas was significantly lower than in LMP tumors and carcinomas.There was significant correlation of HIF-1αexpression with expression of VEGF,bFGF,CD34,PCNA in invasive cancers, LMP tumors, and cystadenomas (P＜0.001).2,HIF-1αexpression was significantly stronger in low grade or stagesâ…¢andâ…£or presence of ascites invasive ovarian cancer when compared with high grade or stagesâ… andâ…¡or absence of ascites tumors (P＜0.05). There were no significant differences in HIF-1αexpression based on age(P＞0.05).3,HIF-1αexpression showed correlation with VEGF, bFGF, and PCNA expressions and MVD, 150μmol/L CoCL₂ increased the expressions of HIF-1αand VEGF.4,HIF-1αand VEGF expression showed a marked downregulation in effusions,when compared with primary and metastaticsites (P＜0.05). Incontrast, constantly high expression of bFGF was observed in all three compartments.5,respectively, these concentrations of VEGF and bFGF were significantly elevated (P＜0.05) compared to normal and serous cystadenomas and LMP tumors serum concentrations, the mean ascitic concentration of VEGF and bFGF was greater than the mean serum level (P＜0.01).6,Residual disease mass and expressions of HIF-1α,VEGF,bFGF and MVD in tumor cells is correlated with disease recurrence (P＜0.05). VEGF and MVD were independent prognostic parameters in patients with advanced ovarian serous cancer. (P＜0.05). 7,under hypoxia condition, HIF-1αis a induction of the inhibition of cell cycle progression at the G₀/G₁ phase via the down-regulation of survivin protein. expressions of survivin in tumor cells is negative correlated with HIF-1α(P＜0.001). And no apoptosis.8,under hypoxia condition,VEGF and HIF-1αin OVCAR₃ cells treated for 24 hours with IFN-αⅡb were suppressed by 10²,10³ and 10⁴IU/ml IFN-αⅡb, OVCAR₃ cells treated by 10²IU/ml IFN-αⅡb for 6,12,24 and 48 hours, VEGF and HIF-1αwere suppressed respectly. the effects suppressed in a time-and dose-dependent manner(P＜0.05).9,under normal oxygen condition, HIF-1αin OVCAR₃ cells treated for 24 hours with bFGF were increased by 25,50,100ng/ml bFGF, OVCAR₃ cells treated by 50ng/ml bFGF for 6,12,24 and 48 hours, HIF-1αwere increased respectly. the effects increased in a time-and dose-dependent manner(P＜0.01).10,under hypoxia condition,The proliferation of OVCAR₃ cells was suppressed with 10⁴IU/ ml of IFN-αⅡb,and it was more remarkable suppression was observed 24 hours or later (P＜0.05).11,under hypoxia condition,In OVCAR₃,the ratio of cells in the S phase tended to increased,and that in the G₂/M phase tended to decreased slightly under the IFN-αⅡb treatment compared with the control at timepoint during the 24 hours after the addition of 10³ and 10⁴IU/ml of IFN-αⅡb to the cultures. under hypoxia condition,survivin in OVCAR₃ cells treated for 24 hours with IFN-αⅡb were suppressed by 10²,10³ and 10⁴IU/ml IFN-αⅡb(P＜0.001),And no apoptosis.DiscussionThe rapid tumor cell growth and early transmission may be due to low oxygen microenvironment. Cellular adaptation to hypoxia represents an essential step in tumor progression. One of the key factors regulating cellular hypoxia responses via transcription is hypoxia-inducible factor 1α. When cells are exposed to low ambient oxygen, HIF-1αis stabilized and translocated to the nucleus,where it activates angiogenesis naerobic metabolism. Previous studies indicated that HIF-1αacts as a positive regulator of tumor growth in many solid tumors,including ovarian carcinoma.The HIF-1α-express in cells may promote transformation and invasion of tumor.The growth and spread of neoplasms depend,in part,on the formation of adequate vascular support,i.e.,angiogenesis, the extent of which is determined by the balance between positive and negative regulatory molecules. The process of angiogenesis can be divided into four distinct phases: 1)the degradation of extra cellular matrix,2)cell migration,3)cell proliferation,and 4)structural reorganization. These distinct steps are likely to be differentially regulated. Many angiogenic molecules have been identified. Among the major molecules identified is basic fibroblast growth factor(bFGF),which induces the proliferation,migration,proteolytic activity,and differentiation of endothelial cells. Vascular endothelial growth factor(VEGF) has been shown to induce the proliferation of endothelialcells,to increase the cells' vascula rpermeability and to induce the production of plasminogen activator by these cells.HIF-1α,VEGF,bFGF,PCNA expression of normal and cystadenomas was significantly lower than in LMP tumors and carcinomas. significant correlations between HIF-1αexpression and VEGF,bFGF,MVD,PCNA was found in invasive cancers pecimens.HIF-1αis considered to support tumor growth through induction of angiogenesis via e.g., transactivation of the VEGF gene. This finding can be explained by the notion that angiogenesis was promoted by HIF-1αinduced expression of VEGF.HIF-1αwas reported to influence the expression of the cell cycle regulator and apoptosis.We found that HIF-1αinduced blockage of cell cycle progression at the G₀/G₁ phase.Prognostic studies of angiogenic gene expression in ovarian carcinoma have concentrated upon the role of VEGF. Using enzyme-linked immunosorbent assay(ELISA),three studies demonstrated an association between high levels of VEGF and disease-free survival(DFS) or overall survival(OS).Four additional reports, analyzing VEGF expression on both protein and mRNA level,led to similarly inconclusive results. Interestingly,high cytoplasmic content of bFGF,as measured by immunoassay,correlated with prolonged survival in stageâ… -â…¢tumors in the prognostic study.The authors hypothesized that high expression of bFGF may improve surviv althrough the induction of stromal response to tumors. In our studies,residual disease mass and expressions of HIF-1α,VEGF,bFGF and MVD in tumor cells is correlated with disease recurrence,high VEGF and MVD is associated with poor prognosis in serous ovarian cancers. HIF-1α,VEGF and bFGF play important role in tumor angiogenesis and growth and peritoneal metastasis of serous ovarian cancer.Ovarian carcinoma shows are markably characteristic pattern for tumor progression,centering on a consistent predilection for the peritoneal cavity and its bordering organs,most often accompanied by the presence of tumor cells in the ascitic fluid. The study of some molecules participating in the metastatic processis therefore central to the understanding of this disease.Despite this fact,large-scale comparative studies of effusions and solid tumors are not available. Expressions of HIF-1αand VEGF is more pronounced in primary tumors and metastaticsites than effusions,Our present findings of a down-regulation in VEGF and HIF-1αprotein levels in carcinoma cells in effusions provide further evidence of the extensive alterations in the cellular profile of these cells.This alteration may result from changes in the microenvironment of the cells,namely the loss of interaction with stromal cells and possibly with systemic signals facilitating angiogenesis and invasion. It may thus reflect the superfluous nature of angiogenic gene expression in a compartment where the formation of new vessels is not necessary.The subsequent up-regulation in expression in metatstatic tumors supports this hypothesis,especially if one accepts the metastatic sequence of primary tumor-effusion-solid metastasis. In our study,consistent expression of bFGF protein was found in carcinoma cells in both serous effusions and solid tumors,it suggest that this cytokine is the major angiogenic factor in advanced-stage ovarian carcinoma.Local administration of VEGF has been shown to transiently increase vascular permeability through activation of vesicular-vacuola rorganelles present in the cytoplasm of endothelial cells and through the induction of interendothelial cell gaps and endothelial fenestration. Increased permeability of blood vessels facilitates the extravasation of proteins and,thus,the formation of ascites. Although bFGF does not modify vessel permeability, bFGF induces the expression of VEGF via the up-regulation of HIF-1α.. The release of VEGF, in addition to promoting ascites, may also enhance metastatic potential, not only by circulating cancer cells throughout the peritoneal cavity but by an intrinsic mechanism.This study showed that the HIF-1αwas strongly expressed in ovarian cancer. It could upregulate the expression of protein of VEGF,thus increasing tumor growth and angiogenesis. we show that the expression of different genes that regulate angiogenesis in human ovarian carcinomas is associated with the pattern of the disease and its progression: Expression of bFGF reflects tumorigenicity,expression of VEGF is directly associated with the development of peritoneal ascites and carcinomatosis. Targeting specific genes such as these that regulate angiogenesis could offer new approaches to the therapy of ovarian cancer.under hypoxia condition, HIF-1αis a induction of the inhibition of cell cycle progression at the G₀/G₁ phase via the down-regulation of survivin protein. expressions of survivin in tumor cells is negative correlated with HIF-1α(P＜0.001). And no apoptosis.IFN-αpossesses various biological activities in addition to antiviral effects,and it is clinically indicated for renal cancer and leukemia.Reported mechanisms of the antitumor effects of IFN-αare the direct suppressive effect on tumor proliferation and indirect effects such as the enhancement of tumor killing through the activation of immune cells and inhibition of tumor growth through the suppression of angiogenesis. These results implied that IFN-αⅡb was able to downregulate the transcriptional activity of HIF-1α,a well-characterized regulator of VEGF expression that regulated the transcription of VEGF by binding to the HIF-1αin dingsite at the VEGF promoter. the decrease of VEGF synthesis and secretion in IFN-αⅡb treated cells might be a consequence of the downregulation of HIF-1αexpression. These findings suggest that IFN-αⅡb directly inhibits the proliferation of OVCAR₃ by inducing blockage of cell cycle progression at the S phase. under hypoxia condition,survivin in OVCAR₃ cells treated for 24 hours with IFN-αⅡb were suppressed by 10²,10³ and 10⁴IU/ml IFN-αⅡb(P＜0.001),And no apoptosis.These observations may have important implications for our understanding of the complicated effects of IFN-αand may help to better understand and exploit the effects of this cytokine in the cancer therapy.Conclusion1,HIF-1α,VEGF and bFGF play important role in tumor angiogenesis and growth and peritoneal metastasis of serous ovarian cancer.2,VEGF and MVD were independent prognostic parameters in patients with advanced ovarian serous cancer.3,HIF-1αinduces blockage of cell cycle progression at the G₀/G₁ phase via the down-regulation of survivin protein.And inhibites angiogenesis via the up-regulation of VEGF protein.4,bFGF increase the expression of VEGF via the up-regulation of HIF-1α.bFGF is the major angiogenic factor expressed in ovarian carcinoma at the protein level.5,IFN-αⅡb directly inhibits the proliferation of OVCAR₃ by inducing blockage of cell cycle progression at the S phase via the down-regulation of survivin protein, and inhibits peritoneal metastasis and the formation of ascites by downregulating the expression of HIF-1αregulated the transcription of VEGF.