Independent anti-angiogenic capacities of coagulation factors X and Xa.

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation.

Hypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.

Characterization and phenotypic variation with passage number of cultured human endometrial adenocarcinoma cells.

Despite numerous endometrial cancer cell lines, little is know about the progression and transition of primary cultured endometrial tumours. Herein, a stage I grade III endometrial adenocarcinoma was maintained in primary culture and the phenotypic and protein expression changes were observed in relation to passage number. At early passage numbers, cultured human endometrial cancer (CHEC) cells displayed classic epithelial cell morphology, growing in groups in a glandular structure and staining positive for cytokeratin. However, with increasing passage number, CHEC cells changed in morphology to display a stromal phenotype which was accompanied by a significant reduction in cytokeratin and increases in alpha-actin and vimentin expression. Simultaneous culture of stromal cells isolated from the original tumour failed to show the same morphological characteristics or protein expression patterns. We further characterised CHEC cells through a screening of cancer related proteins, among others, caveolin-1 and Tissue factor in comparison with established cancer cell lines and corresponding non-cancerous cells. This report demonstrates that endometrial adenocarcinoma cells in culture can undergo phenotypic and protein expression changes reminiscent of epithelial-mesenchymal transition. This work suggests that primary tumours and cell lines displaying stromal morphologies may have undergone epithelial-mesenchymal transition from an adenocarcinoma origin.

The oestrogen metabolite 2-methoxyoestradiol alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand mediates apoptosis in cancerous but not healthy cells of the human endometrium.

Cancers of the reproductive tract account for 12% of all malignancies in women. As previous studies have shown that oestrogen metabolites can cause apoptosis, we characterised the effect of oestrogen and oestrogen metabolites on non-cancerous and cancerous human endometrial cells. Herein, we demonstrate that 2-methoxyoestradiol (2ME), but not 17beta-oestradiol, induces apoptosis in cancer cell lines and primary cultured tumours of endometrial origin. In contrast, 2ME had no effect on cell viability of corresponding normal tissue. This ability of 2ME to induce apoptosis does not require oestrogen receptor activation, but is associated with increased entry into the G2/M phases of the cell cycle and the activation of both the intrinsic and the extrinsic apoptotic pathways. The selective behaviour of 2ME on cancerous as opposed to normal tissue may be due to a reduction in 17beta-hydroxysteroid dehydrogenase type II levels in cancer cells and to a differential down-regulation of superoxide dismutase. Furthermore, we demonstrate that pre-treatment with 2ME enhances the sensitivity of reproductive tract cancer cells to the apoptotic drug tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), without the loss in cell viability to normal cells incurred by currently chemotherapeutic drugs. In conclusion, 2ME, alone or in combination with TRAIL, may be an effective treatment for cancers of uterine origin with minimal toxicity to corresponding healthy female reproductive tissue.

Involvement of the ANGPTs/Tie-2 system in ovarian hyperstimulation syndrome (OHSS).

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-ß. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS.

Antiangiogenic, antimigratory and antiinflammatory effects of 2-methoxyestradiol in zebrafish larvae.

2-Methoxyestradiol (2ME), an endogenous metabolite of 17ß-estradiol, has been previously reported to possess antiangiogenic and antitumor properties. Herein, we demonstrate that the effects of this antiangiogenic steroid can be readily assayed in live zebrafish, introducing a convenient and robust new model system as a screening tool for both single cell and collective cell migration assays. Using the in vitro mammalian endothelial cell line EA.hy926, we first show that cell migration and angiogenesis, as estimated by wound assay and tube formation respectively, are antagonized by 2ME. In zebrafish (Danio rerio) larvae, dose-dependent exposure to 2ME diminishes (1) larval angiogenesis, (2) leukocyte recruitment to damaged lateral line neuromasts and (3) retards the lateral line primordium in its migration along the body. Our results indicate that 2ME has an effect on collective cell migration in vivo as well as previously reported anti-tumorigenic activity and suggests that the molecular mechanisms governing cell migration in a variety of contexts are conserved between fish and mammals. Moreover, we exemplify the versatility of the zebrafish larvae for testing diverse physiological processes and screening for antiangiogenic and antimigratory drugs in vivo.

Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

2-methoxyestradiol mediates apoptosis through caspase-dependent and independent mechanisms in ovarian cancer cells but not in normal counterparts.

OBJECTIVE: The estrogen metabolite 2-methoxyestradiol has shown antitumorigenic action in some epithelial tumors. In the present work we investigate its effects in ovarian cancer used alone or in combination with other apoptotic-inducing reagents such as tumor necrosis factor-related apoptosis-inducing ligand. METHODS: To assess the effect of 2-methoxyestradiol, dose response and time courses in ovarian cancer and normal cells were conducted. Apoptosis was confirmed through DNA laddering, by flow cytometry, and Western blotting of proteins involved in the apoptotic cascade. RESULTS: 2-Methoxyestradiol induced apoptosis in ovarian cancer cells but not in normal counterparts. 2-Methoxyestradiol activates both the intrinsic and extrinsic apoptotic pathways. 2-Methoxyestradiol-mediated apoptosis involves reactive oxygen species generation and caspase-dependent and caspase-independent mechanisms. We also demonstrate that 2-methoxyestradiol selectively induces an additive/synergistic apoptotic response in ovarian cancer cells when used in combination with tumor necrosis factor-related apoptosis-inducing ligand. CONCLUSIONS: 2-Methoxyestradiol, alone or in combination with tumor necrosis factor-related apoptosis-inducing ligand, should be considered as a potential treatment for ovarian cancer.

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract.

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2.Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue.