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.

17beta-estradiol inhibits forskolin-induced vascular endothelial growth factor promoter in MCF-7 breast adenocarcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is induced by the cAMP-dependent signalling pathway in several cell types, and by estrogens in some human breast cancer cells. Here, we investigated the cross-talk between estrogens and cAMP/PKA-dependent signalling pathway in human breast cancer MCF-7 cells. The results show that, in the absence of any CRE and ERE, forskolin induces whereas estrogens have no effect on VEGF promoter. Moreover, estrogens, through estrogen receptors, partly inhibit the forskolin-induced VEGF promoter in MCF-7 human breast cancer cells. Therefore, in breast cancers, estrogens could partly inhibit the effect of ligand-activated G protein-coupled receptors on VEGF expression.

Relationship between expansion of the CAG repeat in exon 1 of the androgen receptor gene and idiopathic male infertility.

OBJECTIVE: To determine whether expansion of CAG repeats in exon 1 of the androgen receptor is correlated with impaired spermatogenesis in patients with male idiopathic infertility. DESIGN: A retrospective study. SETTING: Medical school in Besançon, France. PARTICIPANT(S): Thirty-seven infertile patients with azoospermia or oligospermia and 50 fertile controls. INTERVENTION(S): History, physical, hormonal assays, semen analysis, and collection of blood samples in order to study the androgen receptor's gene. MAIN OUTCOME MEASURE(S): Blood samples were collected from each infertile patient and control. The length of the CAG repeat segment was evaluated by using polymerase chain reaction (PCR) electrophoresis in exon 1 and PCR single-strand conformation polymorphism in exons 2-8. RESULT(S): The mean length of the CAG repeats was significantly different between infertile and fertile patients (23.91 +/- 0.5 vs. 22.20 +/- 0.4). No mutation was detected in exons 2-8 of the androgen receptor gene in infertile patients. CONCLUSION(S): Expansion of the CAG repeat segment of the androgen receptor is correlated with male idiopathic infertility. The number of CAG repeats may therefore have a modulatory effect on normal androgen receptor function.

Comparison of the effects of epidermal growth factor and fetal calf serum in human endometrial carcinoma RL95-2 cells.

This study was designed to evaluate if c-myc expression could influence RL95-2 cell proliferation after EGF or fetal calf serum (FCS) stimulation. Upon FCS addition, c-myc mRNAs slightly increased in the first 30 minutes, peaked at 75 minutes (2.2 fold induction) and returned to the control value within 24 hours. This slight and transient FCS effect on c-myc expression contrasted with the marked and long-lasting EGF effect (17 fold induction up to 48 hours). The increase of cell number by FCS was partially but significantly reduced in the presence of c-myc antisense oligodeoxynucleotides (ODNs). The EGF inhibitory effect on cell growth was not reversed by c-myc antisense ODNs whatever the backbone may be (phosphorothioate or phosphodiester). It appears that EGF and FCS have differential effects on c-myc and RL95-2 cell proliferation and that c-myc is necessary but insufficient for proliferation.

Substituted benzopyranobenzothiazinones. Synthesis and estrogenic activity on MCF-7 breast carcinoma cells.

In the search for new agents with estrogenic activity mediated by estrogen receptors (ER), six 6,12-dihydro-1-benzopyrano[3,4-b][1,4]benzothiazin-6-ones 3a-f were synthesized. These compounds were readily prepared by the addition of 2-aminothiophenol 2 to substituted 4-hydroxycoumarin derivatives 1a-e. The estrogenic effect has been evaluated on the proliferation of MCF-7 breast adenocarcinoma cells and the specificity of described compounds was evaluated by the inhibition of their effect by ICI 182,780, an antiestrogenic compound. Among the compounds tested, 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one 3e and 6,12-dihydro-3-hydroxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one 3f exhibited an ER-dependent proliferation and a high binding affinity to ER, but a moderate capacity to activate the transcription of a reporter gene. Their pharmacological profiles are defined by their binding properties and their mechanism of action by computational modelling studies.

Synthesis and cytotoxic activity of new 2,4-diaryl-4H,5H-pyrano[3,2-c]benzopyran-5-ones on MCF-7 cells.

A series of eight halogenated 2,4-diaryl-4H,5H-pyrano[3,2-c]benzopyran-5-ones have been synthesized, characterized and their stereochemistry determined. In a second stage of our work, the reported molecules were tested for their antiproliferative activity on MCF-7 breast carcinoma cells. Pharmacological results were compared with those of diethylstilbestrol (DES), an estrogen, as well as ICI 182,780, a pure antiestrogen. Then, these derivatives were evaluated for their capacity to activate the transcription of a reporter gene and for their affinity for human recombinant estrogen receptors alpha (hER alpha). These results were compared with those of coumestrol, a phytoestrogen structurally close to 2,4-diaryl-4H,5H-pyrano[3,2-c]benzopyran-5-ones, and with RU 58668, a pure antiestrogen. Although these derivatives exhibit a significant antiproliferative activity higher than that of ICI 182,780, neither of them displayed a significant estrogenicity or an affinity for hER alpha. Such results may suggest that their antiproliferative activity is not dependent of an antiestrogenic response.

Circulating vascular endothelial growth factor in cutaneous malignant melanoma.

BACKGROUND: Angiogenesis has been reported as a parameter of potential prognostic value in solid tumours, as it may facilitate tumour growth and metastasis. One of the most important growth factors involved in angiogenesis is vascular endothelial growth factor (VEGF). OBJECTIVES: To determine the predictive value of circulating VEGF levels in a cohort of patients with melanoma. METHODS: In a prospective cohort study, 324 patients with cutaneous melanoma at different clinical stages were investigated over 2 years (2002-04). VEGF was measured in plasma using enzyme-linked immunosorbent assay. Two hundred and eight patients were able to be followed up for progression of their disease and for blood sample collection (mean +/- SD follow-up 13.4 +/- 0.8 months). Data were compared with the extent of the disease and the clinical course. RESULTS: A significant increase in plasma VEGF levels was found in patients with melanoma compared with healthy controls, with statistically significant differences between patients in stages I, II and III vs. those in stage IV, but not between patients in stages I, II and III. When considering the 237 patients in stages I and II, no statistical correlation was found between plasma VEGF levels and tumour thickness. Baseline plasma VEGF levels were not significantly higher in patients who relapsed compared with nonprogressing patients. Among the 35 patients (two stage I, eight stage II and 25 stage III) who experienced a progression during follow-up, an increase in plasma VEGF level to > 100 pg mL(-1) was found in 20 (sensitivity 57.1%), while 38 of the 173 remaining nonprogressing patients demonstrated an increase in VEGF level, indicating a specificity of 78%. In addition, an increase in plasma VEGF level was found in 58 patients during follow-up, of whom 20 showed evidence of progression, indicating a positive predictive value of 34.5%. However, among the 150 remaining patients who did not demonstrate any increase in plasma VEGF level during follow-up, only 15 experienced a progression, indicating a negative predictive value of 90%. CONCLUSIONS: Our data confirm that blood VEGF levels are significantly increased in patients with melanoma and, more interestingly, that the absence of plasma VEGF level increase during follow-up appears to be associated with remission.

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.

Relevance of p185 HER-2/neu oncoprotein quantification in human primary breast carcinoma.

The c-erbB-2 proto-oncogene encodes a transmembrane protein tyrosine kinase receptor of 185 kDa (p185) and has been associated with several types of human cancers. In human breast cancer, overexpression of p185 occurs in 15-30% of cases, correlates with poor prognostic factors and characterizes breast cancers with a more aggressive behavior. Overexpression of p185 is usually associated with c-erbB-2 amplification, though it may occur independently and thus define subpopulations of breast cancers which might be of clinical interest. p185 expression is usually detected by immunohistochemistry (IHC) and few studies have been carried out to evaluate the p185 content of breast cancers with an ELISA technique. In this context, we showed, in 106 breast cancer samples, that p185 was expressed at high levels in 13.2%, intermediate levels in 55.7% and negative ones in 31.1% of cases. All p185 positive samples showed a c-erbB-2 oncogene amplification while none of the p185 negative samples and only 4% of p185 imtermediate samples had an amplification of c-erbB-2. p185 expression is significantly correlated with the negativity of estrogen and progestrone receptors, with high levels of cathepsin D and in some conditions with axillary nodal involvement. Thus, using the p185 ELISA assay, the c-erbB-2 status of breast cancers can be defined and moreover a subset can be discriminated which is characterized by intermediate levels of p185 and absence of c-erbB-2 amplification. The quantitative approach towards p185 in breast cancers affords the possibility of identifying more appropriately patients with high or low risk and thus permits adaptation of therapeutic regimens.