Proinvasive properties of ovarian cancer ascites-derived membrane vesicles.

Malignant ovarian ascites are rich in cellular components, membrane-bound vesicles, and soluble proteins. This study focused on the structure of membrane-bound vesicles and their ability to promote invasion in cultured malignant ovarian epithelium. Membrane vesicles were derived from women with stage I-IV malignant ovarian ascites and from nonmalignant gynecologic ascites. Isolated vesicles were characterized by immunofluorescence and Western blot analysis. Using gel zymography for matrix metalloproteinase (MMP) detection and a colorimetric assay for urokinase-type plasminogen activator (uPA) analysis, we analyzed the proteinase activities of MMP-2, MMP-9, and uPA from the prepared vesicles, whole cells isolated from ascites, and the cell-free ultracentrifuged supernatant. The invasiveness of established cultured malignant ovarian epithelium on addition of ascites-derived vesicles was tested using a Matrigel-based invasion assay. Fractionation of malignant ascites revealed that extracellular matrix-degrading proteinases including MMPs and uPA are localized preferentially in membrane vesicles. All malignant vesicles tested, regardless of cancer stage, stimulated invasion. Furthermore, the combination of ovarian cancer cells and membrane vesicles resulted in greater uPA activation than that of cells or vesicles alone. Membrane vesicles from malignant ascites were also found to contain activated MMP-2, MMP-9, and uPA. Our data suggest that vesicle-stimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.

Methylation of TFPI-2 gene is not the sole cause of its silencing.

TFPI-2 (tissue factor pathway inhibitor-2) is a serine protease inhibitor that may suppress tumor cell invasion and metastasis. TFPI-2 expression is often lost in cells derived from tumors of diverse organs. We have examined whether aberrant hypermethylation of the 5' end of the TFPI-2 gene is associated with its loss of expression. After 5-azacytidine treatment of three cell lines lacking TFPI-2 expression (HT1080 fibrosarcoma cells, MCF-7 breast carcinoma cells, and LNCaP prostate carcinoma cells), TFPI-2 transcripts could be detected by RT-PCR. In these three cell lines, methylation of the 5' end of the TFPI-2 gene was detected, while two prostate carcinoma cell lines in which the TFPI-2 gene was expressed, PC-3 and DU-145, showed no methylation. However, all the three cell lines which lacked TFPI-2 expression also contained unmethylated TFPI-2 alleles. Furthermore, a transiently transfected TFPI-2 promoter was non-functional in the three cell lines, but function was attained following treatment with 5-azacytidine. Our results indicate that while methylation of the TFPI-2 gene is associated with its silencing, it is not the sole cause, and we suggest that one or more components of pathways regulating TFPI-2 expression have also undergone methylation-associated silencing in these cell lines.

LPA-induced epithelial ovarian cancer (EOC) in vitro invasion and migration are mediated by VEGF receptor-2 (VEGF-R2).

OBJECTIVE: Lysophosphatidic acid (LPA) stimulates ovarian tumor growth partially via induction of VEGF expression through transcriptional activation. Previous studies have shown that LPA induces epithelial ovarian cancer (EOC) in vitro metastasis. In this study, we examined the role of VEGF in LPA-induced EOC invasion and migration and underlying mechanisms. METHODS: The invasiveness of DOV13 cells was determined by in vitro basement membrane Matrigel invasion assay. Ovarian carcinoma cellular migration was quantified by the colloidal gold migration assay. Matrix metalloproteinase (MMP)-2 secretion and activation were detected by gelatin zymography. Urokinase type plasminogen activator (uPA) activity was determined by a coupled colorimetric assay measuring the activity of generated plasmin. Student's t test and one-way ANOVA were used for statistical analysis. RESULTS: Using a VEGF neutralizing monoclonal antibody (mAb), we show that LPA-induced EOC invasion is dependent upon VEGF. Using the selective VEGF receptor (VEGFR)-2 inhibitor, SU1498, LPA-induced EOC invasion and migration were significantly inhibited in a concentration-dependent manner. In addition, SU1498 inhibits MMP-2 secretion and uPA activity in ovarian cancer DOV13 cells. At 5 and 20 microM, SU1498 almost completely inhibited the activity of MMP-2 and uPA. SU1498 also decreases the LPA-induced increase of uPA activity in DOV13 cells. CONCLUSIONS: Our results show that LPA-induced EOC invasion is at least partially mediated by VEGF. Further, the VEGFR-2-mediated signaling transduction pathway may be involved in LPA-induced EOC invasion and migration by regulating the secretion and activation of MMP-2 and uPA.

Lysophosphatidic acid enhances epithelial ovarian carcinoma invasion through the increased expression of interleukin-8.

OBJECTIVE: We previously reported that lysophosphatidic acid (LPA) stimulates cellular invasion of ovarian cancer (OVCA) cells by enhancing membrane-type-1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP2. Here, we investigate a second mechanism in which LPA enhances cellular invasion through the increased expression of IL-8, independent of the expression or activation of MMP2. METHODS: Epithelial ovarian carcinoma cells (DOV 13) were exposed to LPA (80 microM) and IL-8 (100 ng/ml) for 24 h. IL-8 expression was quantified by enzyme-linked immunosorbent assay (ELISA). Cellular invasion (Matrigel invasion), migration (colloidal gold), and urinary-type plasminogen activator (uPA) activity (colorimetric assay) were quantified. Conditioned medium was also assayed for MMP activation and expression by SDS-PAGE gelatin zymography, ELISA, and Western blotting. In addition, IL-8 neutralizing antibody and MMP inhibitors were employed. RESULTS: Our results found LPA to increase IL-8 expression threefold. IL-8 did not affect cellular migration, MMP2 activation, or uPA expression. However, exposure to various concentrations of IL-8 increased cellular invasiveness. Using an IL-8 blocking antibody and various MMP inhibitors, we determined that the increase in invasion was IL-8-dependent, while independent of the activation of MMP2 or MMP9. We further determined IL-8 exposure increased the expression of matrilysin (MMP7). Cells exposed to LPA and IL-8 resulted in a synergistic effect on cellular invasion. Adding the IL-8 blocking antibody, slightly decreased cellular invasion, indicating LPA in part, increases cellular invasion through the increased expression of IL-8. CONCLUSION: We have identified a separate mechanism of enhanced cellular invasion, which is independent of MMP2 activation and involves the increased expression of IL-8 and subsequent increased expression of MMP7.