Dissertation
Dissertation > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors

Relationship of Lysophosphatidic Acid Up-regulated CXCL12-CXCR4 Biological Axis and Metastasis of Epithelial Ovarian Cancer in the Peritoneal Cavity

Author WangHui
Tutor WuXiaoHua;DanBaoEn
School Hebei Medical University
Course Surgery
Keywords ovarian neoplasms lysophosphatidic acid CXCL12-CXCR4 biology axis metastasis peritoneal cavity
CLC R737.31
Type PhD thesis
Year 2007
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Ovarian carcinoma, predominantly derived from ovarian surface epithelium, is most lethal gynecologic cancer. More than two thirds of patients have late-stage metastatic disease at initial diagnosis with a 5-year survival rate of 20% to 30%. Conversely, at early stages, the long-term survival rate approaches 90%. There is currently no proven effective method for early detection of ovarian cancer through biomarkers, imaging, or other means. Ovary cancer is characterized by early spreading to peritoneal wall, diaphragm and omental structures. At last, widely tumor spreading and metastasis in pelvic and peritoneal cavity result in death. The related mechanism remains poorly understood. The most likely way to identify ovarian cancer at an early, curable stage and to develop new, effective therapies for advanced ovarian cancers is to improve our understanding of the processes leading to the initiation and progression of this disease. Lysophosphatidic acid (LPA) is one kind of intercellular phospolipid signal molecule which recently discovered, it causes many kinds of biology effect through the G protein-coupled receptors. LPA is detected at significantly high levels in the plasma and ascitic fluids of many ovarian cancer patients. Recent studies demonstrated that LPA can be synthesized by ovarian cancer cells, LPA induces the expression of a number of genes which are involved in the promotion of angiogenesis and metastasis to stimulate growth, prevents apoptosis and anoikis, decreases sensitivity to chemotherapeutic drugs, increases production of neovascularization mediators, and increases invasiveness of ovarian cancer cells. The tumor metastasis is one complex, continual, multi-stepped process, study shows that the selective expression of the chemokine receptor CXCR4 and its ligand CXCL12 by malignant ovarian epithelium has a number of important consequences to tumor development and spread. CXCR4, with its ligand CXCL12 constituted CXCL12-CXCR4 biological axis may stimulate directed migration and invasion of tumor cells, as well as promoting their growth and the establishment of a tumor-promoting cytokine network in suboptimal conditions and subverting tumor immunity. Objective:We measured LPA in plasma and in ascetic fluid from ovarian epithelial cancer, and discussed LPA-mediated the CXCL12-CXCR4 biology axis in tumor proliferation, migration, invasion and secretion of ovarian cancer cell lines in vitro, and evaluated inhibition of Gi protein antagonist pertussis toxin(PTX) in LPA-mediated the CXCL12-CXCR4 biology axis. Finally, the animal model further confirmed the conclusion.Method: This subject was divided into three parts:1 A diagnostic and monitoring value of lysophosphatidic acid for ovarian epithelial cancer. We measured LPA and CA125 in plasma and in ascitic fluid from ovarian epithelial cancer (n=40) with preoperative and postoperative and benign ovarian neoplasm (n=20). We try to evaluate the diagnostic and monitoring value of lysophosphatidic acid for ovarian epithelial cancer and correlation between LPA and clinicopathological features such as age, histological type, clinical stage, tumor grade, as well as CA125 level.2 The effect and mechanism of LPA-mediated the CXCL12-CXCR4 biology axis in tumor proliferation, migration, invasion and secretion of ovarian cancer cell lines. Humen ovarian cancer cell lines CAOV3 and SKOV3 were cultured in vitro. Two ovarian cancer cell lines were detected CXCR4 protein expression by immunocytochemistry. Ovarian cancer cell lines were cultured for LPA different dose and different time .The expressions of cell membrane CXCR4 protein was investigated by Reverse transcriptaseolymerase chain reaction (RT-PCR), immunocytochemistry and Flow Cytometry. MTT was used to analyze effect of LPA, CXCL12 and LPA-mediated CXCL12 on ovarian cancer cells proliferation. Migration of cancer cells was assayed using Transwell invasion chamber with 24mm diameter chambers and 8-μm pore filters. Transwell and Matrigel were used to evaluate effect of LPA, CXCL12 and LPA-mediated CXCL12 on ovarian cancer cells migration and invasion. The expression of VEGF and changes stimulated by LPA, CXCL12 and LPA-mediated CXCL12 were detected in ovarian cancer cell lines CAOV3 and SKOV3 by ELISA, as well as inhibition of Gi protein antagonist PTX.3 Effect of Lysophosphatidic Acid on metastasis of transplanted tumor and expression of various molecules of ovarian cancer in nude mice. 20 nude mouse were randomly divided into two groups, control group and LPA-treated group. 6×106 untreated and LPA-treated human ovarian cancer cells SKOV3 were transplanted intraperitoneally to nude mice. The LPA-treatment group was treated with intraperitoneal injection of 0.134mg/20g LPA daily, while the control group was treated with physiological saline. When every nude mouse naturally died, the weight of the transplanted tumors and the number of the metastatic tumors were observed and measured. Expression of VEGF、MMP2、COX-2 and CXCR4 in transplantated tumors were assay by immunohistochemistry and Flow Cytometry.Result:1①the preoperative LPA levels in the plasma of patients with human epithelium ovarian cancer were 5.89±1.98mol/L, while the LPA levels of patients with benign tumor patients were 1.65±0.71μmol/L(P<0.01), the difference was prominent.②The group of ovarian cancer LPA level in the plasma and the CA125 level relevance comparison: the two has the relevance , also presents positive correlation (P<0.01).③The difference of preoperative LPA levels in the plasma were not prominent in different types, stages, and grades of human epithelium ovarian cancer (P>0.05).④the preoperative LPA levels in the plasma were 5.89±1.98μmol/L, while the postoperative LPA levels were 2.87±1.34μmol/L, the distinction was prominent (P<0.01).⑤The LPA levels in the ascetic fluid were 5.68±1.75μmol/L, which were obviously higher than the LPA levels in the benign abdominal cavity rinse solution 1.34±0.77μmol/L, the difference was prominent (P<0.01).⑥13 of 20 cases LPA levels in the ascetic fluid were higher than LPA levels in the plasma ,and LPA levels in the ascetic fluid were 5.68±1.75μmol/L, while LPA levels in the plasma were 5.346±1.93μmol/L, but the difference was not prominent (P>0.05).2 The immunocytochemistry result showed that, ovary cancer cell CAOV3 and SKOV3 expressed CXCR4 protein in the cell membrane and the cytoplasm.②RT-PCR, immunocytochemistry and Flow Cytometry showed that expression of CXCR4 mRNA and the protein increased in ovary cancer cells with LPA different doses (0, 5, 20μmol/L) respectively and different time (0, 12h, 24h) ,compared with control groups(P<0.05). The expression of CXCR4 increased significantly compared with control group, after cultured with 20μmol/L LPA for 24 hours (P<0.05).③The ELISA method showed that expression of CXCL12 protein increased in ovary cancer cells with LPA different doses (0, 5, 20μmol/L) respectively and different time (0, 12h, 24h), compared with control groups (P<0.05).④Under serum-free suboptimal culture conditions, 100ng/ml CXCL12 enhanced ovary cancer cell CAOV3 and SKOV3 proliferation (P<0.05), LPA-mediated CXCL12 greatly enhanced ovary cancer cells proliferation compared with 100ng/ml CXCL12 (P<0.05), after stimulated by Gi protein antagonist PTX, the proliferational function tend to weaken (P<0.05).⑤Ovary cancer cells displayed minimal invasiveness through Matrigel when no CXCL12 was present in the lower of chamber of transwall. 100ng/ml CXCL12 promoted Ovary cancer cells invasion across Matrigel (P<0.05). The number of invading cells through Matrigel in the presence of LPA-mediated CXCL12 was significantly higher than that of 100ng/ml CXCL12 (P<0.05), and was strongly inhibited by treated with Gi protein antagonist PTX (P<0.05).⑥1 00ng/ml CXCL12 promoted Ovary cancer cells CAOV3 and SKOV3 secrete VEGF,LPA-mediated CXCL12 greatly enhanced ovary cancer cells proliferation compared with 100ng/ml CXCL12 (P<0.05).3①Days of tumor formation were not prominent in different groups (P>0.05). Days of Survival period were 27.63±1.92 in control group, while days of Survival period were 23.57±2.37 in LPA-treatd group, the distinction was prominent (P<0.05).②When every nude mouse naturally died, we dissected the abdominal cavity to demonstrate that, the transplanted tumors localized in abdominal cavity under the left mainly. The rate of tumor formation was 100% in two groups. Metastatic tumors were localized in peritoneum and septum mainly. But the number of the metastatic tumors in LPA group was 16.3±4.06, which of significantly higher than that of control group12.3±4.56 (P<0.01).③the weight of the transplanted tumors in LPA group were 3.81±1.04g significantly higher than that of control group2.91±0.87g (P<0.05).④E xpression of VEGF、MMP2、COX-2 and CXCR4 increased significantly in LPA- treated group, compared with control group (P<0.05).Conclusion:LPA levels may be a new biomarker for detection ovarian epithelial cancer, LPA levels appear useful as monitoring biomarkers of ovarian epithelial cancer. LPA is detected at significantly high levels in the ascitic fluids of ovarian cancer patients, LPA induces the expression of CXCL12 and CXCR4 in ovarian cancer cells to promote proliferation, migration, invasion and secretion. LPA promotes invasiveness of ovarian cancer in the peritoneal cavity by up-regulating CXCL12-CXCR4 biology axis expression.

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