Serum levels of vascular endothelial growth factor as a prognostic factor in bladder cancer.

PURPOSE: Vascular endothelial growth factor is an overriding growth factor mediating tumor angiogenesis. We correlated serum vascular endothelial growth factor in patients with bladder cancer with clinical parameters. MATERIALS AND METHODS: Serum vascular endothelial growth factor in 58 patients with bladder cancer, including superficial and invasive tumors in 42 and 16, respectively, and 41 healthy controls was measured by sandwich enzyme immunoassay. RESULTS: Significant differences in serum vascular endothelial growth factor were observed in healthy controls and patients with bladder cancer (mean 248 versus 100 pg./ml., p <0.001). The serum level was significantly associated with tumor stage (p <0.0001), grade (p <0.002), vascular invasion (p <0.001) and carcinoma in situ (p <0.01). Patients with metastasis had a significantly higher levels than those with localized diseases (mean 582 versus 194 pg./ml., p <0.0001). At a cut-off of 400 pg./ml. the sensitivity and specificity of the test for differentiating patients with and without metastatic diseases was 87.5% and 98%, respectively (p <0.0001). Univariate statistical analysis showed that an increase in serum vascular endothelial growth factor level greater than 400 pg./ml. was significantly related to disease-free survival. On multivariate analysis only stage remained as an independent prognostic factor. CONCLUSIONS: Although vascular endothelial growth factor did not remain an independent prognostic factor on multivariate analysis, our data indicate that the level of vascular endothelial growth factor may be a valuable angiogenic marker for identifying metastatic bladder cancer. It may be used as a new predictor of disease.

Protein kinase C signalling pathway is involved in the regulation of vascular endothelial growth factor expression in human bladder transitional carcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the growth and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF(189), VEGF(165), VEGF(121)). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10(-7) M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

Insulin up-regulates vascular endothelial growth factor and stabilizes its messengers in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes vascular growth and therefore tumoral growth and metastasis. Overweight, frequently associated with hyperinsulinemia, constitutes the major risk factor for endometrial carcinoma. Thus, elevated insulin levels may partly explain the increased risk of endometrial cancer observed in obese postmenopausal women. The aim of the present work was to test the role of insulin in the control of VEGF expression in endometrial carcinoma cells (HEC-1A). We have shown that insulin induced a biphasic expression of VEGF messenger ribonucleic acid, with an early, but low, induction (4 h of stimulation) and a delayed, but high, induction (24 h). The delayed effect of insulin on VEGF expression involved transcriptional and posttranscriptional regulation, as evidenced by the increased rate of VEGF transcription and the prolonged half-life of VEGF messenger ribonucleic acid. Simultaneously we observed higher levels of VEGF protein in the conditioned medium of stimulated cells compared with unstimulated ones. Therefore, insulin could contribute to the increased risk of endometrial carcinoma due to its ability to induce VEGF expression and thus participate in the maintenance of an angiogenic phenotype.

Cell-associated insulin-like growth factor-binding proteins inhibit insulin-like growth factor-I-induced endometrial cancer cell proliferation.

Insulin-like growth factor I (IGF-I) is a peptidic growth factor implicated in the proliferation of a wide variety of cell types, and especially endometrial epithelial cells. Its action is modulated by the presence of IGF-binding proteins (IGFBPs) which are secreted by IGF-I target cells. The partition of IGFBPs between cell-associated and soluble form determines the potentiation or the inhibition of IGF-I action. It is commonly accepted that cell-associated IGFBPs potentiate the IGF-I action while the soluble form of IGFBPs has an inhibitory effect. In endometrial adenocarcinoma, IGF-I is involved in tumoral progression and IGFBPs may be key modulators of the IGF-I-induced cell proliferation. Here we showed that the responsiveness of human endometrial adenocarcinoma cells (HEC-IA cell line) to the mitogenic activity of IGF-I was dependent on the pre-incubation conditions. This responsiveness to IGF-I was conditioned by a differential expression of the IGF system components (IGFBPs and IGF-I receptor) and particularly of the IGFBPs. Indeed, the IGF-I-induced proliferation of the HEC-1A cells was attenuated by the presence of cell-associated IGFBPs. Moreover, the IGF-I incubation induced a release of IGFBP-3 in the culture media as the consequence of an interaction between IGF-I and the cell-associated IGFBP-3. This effect was dose-dependent and was associated with the attenuation of the IGF-I action on cellular proliferation. Thus, IGFBP-3 might be initially expressed as a cell-associated form and then released in the interstitial fluid after a direct interaction with IGF-I. Therefore, in HEC-IA endometrial adenocarcinoma cells responsive to IGF-I, the IGFBP-3 is the main binding protein expressed and both soluble and cell-associated forms act as inhibitors of IGF-I-induced cellular proliferation.

Regulation of vascular endothelial growth factor expression by insulin-like growth factor-I in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes endothelial cell proliferation and chemotaxis. VEGF occurs as 5 isoforms, as a result of an alternatively spliced transcript that originates from one gene, of which the 2 majors are the VEGF 121 and 165 isoforms. Our aim was firstly to determine the role of Insulin-like Growth Factor-I (IGF-I) in the regulation of VEGF expression in endometrial adenocarcinoma cells and then the mechanism by which this regulation occurs. IGF-I treatment of HEC-1A cells provoked an increase of VEGF mRNA expression that peaked at 48 hr with a 165 isoform mRNA more abundant than the 121 isoform. The IGF-I action was confirmed at the protein level, whose concentration was increased in the conditioned media. In experiments using transient transfection of VEGF promoter-luciferase constructs, the IGF-I failed to increase the activity of the VEGF promoter after a 24-hr period of IGF-I treatment, while the addition of Actinomycin D showed an increase of the VEGF mRNA half-life. Most interestingly, Northern blot analysis showed a different stability of the 2 major VEGF isoform mRNAs (VEGF 121 and 165), of which the 121 isoform was more stable than the 165 isoform. The IGF-I treatment prolonged the half-life of both of the VEGF isoform mRNAs. Our results suggest that IGF-I regulates VEGF expression in endometrial adenocarcinoma cells at the post-transcriptional level by enhancing the stabilization of the 2 major VEGF isoform mRNAs (VEGF(121) and VEGF(165)). In addition to its proliferative functions, IGF-I induces VEGF expression and participates in the maintenance of an angiogenic phenotype.

Effect of progesterone on proteins vectorially secreted by glandular epithelial cells of guinea-pig endometrium: modulation by homologous stroma.

Endometrial glandular epithelial cells were subcultured on matrix-coated filters in bicameral chambers in a serum-free chemically defined medium. The cells were untreated or treated with 50 nmol progesterone l-1 or 10 nmol oestradiol l-1 or 10 nmol oestradiol l-1 plus 50 nmol progesterone l-1 and the proteins secreted into the basal or apical compartment were analysed after [35S]methionine labelling. Compared with the untreated cells, oestradiol treatment did not affect the electrophoretic profiles of proteins secreted by the glandular epithelial cells in either compartment. Progesterone treatment induced a decrease in the labelling of 88 and 53 kDa proteins secreted in the apical and basal compartments and an increase in the labelling of a 28 kDa protein. Moreover, progesterone specifically induced the apical secretion of a 137 kDa protein. Interaction between the epithelial and stromal cells was also investigated. When stromal cells were cultured in the basal compartment under the epithelial monolayer, the progesterone effect on the apical secretion of the 137 kDa protein and basal secretion of the 88 and 28 kDa proteins were altered, whereas this progesterone effect was not altered when the epithelial cells were cultured alone in media conditioned with stromal cells. Interactions between epithelial and stromal cells modified the effect of progesterone on protein secretion by the epithelial cells.

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions.

Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and growth to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5-7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial-specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]-methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated.