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.

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.

Influence of cigarette smoking on P53 gene mutations in bladder carcinomas.

BACKGROUND: The aim of this study was to analyse the influence of cigarette smoking on bladder carcinomas. PATIENTS AND METHODS: 98 cases of bladder cancers were examined by single strand conformation polymorphism analysis of exons 5 to 9, followed by DNA direct sequencing. RESULTS: The incidence of p53 gene mutations was not significantly influenced by habitual smoking. However, the p53 mutation spectrum of current smokers differed significantly from the pattern for non-smokers and ex-smokers. Differences between the two populations included multiple mutations in the current-smokers and an absence in non- and ex-smokers (p<0.01), with the predominance of G:C to A:T transitions at CpG sites in non-smokers (60.0%) in comparison with current smokers (7.6%) (p<0.02). Moreover, G:C to T:A and G:C to C:G transversions were found solely in current smokers. CONCLUSION: It would appear that, in current-smokers, the spectrum of p53 gene mutations is related to tobacco-smoke carcinogens and that the habit of smoking increases the extent of DNA damage.

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.

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.

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.

Early detection of pancreatic cancer in patients with chronic pancreatitis: diagnostic utility of a K-ras point mutation in the pancreatic juice.

OBJECTIVE: Point mutations of the K-ras oncogene at codon 12 have been described several months before the onset of pancreatic cancer in isolated cases of chronic pancreatitis (CP). The aim of this study was to evaluate the interest of a prospective follow-up of patients with CP and K-ras mutations at codon 12 in the detection of early pancreatic cancer. METHODS: From February 1996 to March 1998, 36 patients (mean age 52.6 yr, 31 men, five women) with CP (alcoholic: 61.1%, pancreas divisum: 5.6%, autoimmune: 5.6%, unknown origin: 27.7%) were included and then prospectively monitored (median duration of 22 months) for detection of pancreatic carcinoma. K-ras point mutations were examined by two-step polymerase chain reaction combined with restriction enzyme digestion in pancreatic juice collected during endoscopic retrograde pancreatography. RESULTS: Ten patients (27.8%) were positive for K-ras mutation. Patients with and without the mutation were not different with respect to age and sex ratio. K-ras mutations were homogeneously distributed according to the etiology (alcoholic vs nonalcoholic) and morphological characteristics (ductal stricture or mass vs none) of CP. A pancreatic carcinoma was discovered at an invasive stage in two patients, respectively at 7 and 17 months after disclosure of a K-ras mutation, versus none in patients without the mutation (p < 0.02). CONCLUSIONS: Presence of a K-ras gene mutation is not rare in patients with CP and represents an increased risk of developing pancreatic cancer. However, its utility for the detection of early pancreatic cancer remains doubtful in clinical practice.

The predictive value of muscularis mucosae invasion and p53 over expression on progression of stage T1 bladder carcinoma.

PURPOSE: We determine the significance of muscularis mucosae invasion and nuclear p53 over expression on the progression of stage T1 transitional cell bladder cancer. MATERIALS AND METHODS: The pathological findings in 149 cases of T1 tumors diagnosed between 1973 and 1996 were reviewed. Diagnosis was stage T1 in 94 tumors in which the muscular layer was clearly identifiable and disease-free. Mean followup was 64.9 months (range 5 to 288). T1 bladder cancers were subclassified into 2 groups, with (T1b) or without (T1a) muscularis mucosae invasion. The p53 nuclear antibody immunoreactivity was determined with antibody D07 and a cutoff point at 15%. RESULTS: T1 subclassification was possible in all 94 patients. Of all tumors 37.2% expressed p53 nuclear over expression. Univariate statistical analysis showed that p53 expression (p <0.05) and tumor invasion depth (p <0.001) significantly correlated with progression. However, on multivariate analysis only invasion depth (p <0.0001) and associated carcinoma in situ (p <0.03) remained independently significant as predictors of progression. CONCLUSIONS: In our study the depth of tumor invasion was a significant independent predictor of progression in patients with T1 bladder cancer. This result suggests that the depth of invasion in stage T1 should be included in the histopathological report.

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.

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.

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.

Immunohistochemical detection of p53 protein overexpression versus gene sequencing in urinary bladder carcinomas.

PURPOSE: Mutations of p53 tumor suppressor gene and nuclear accumulation of p53 protein are common in bladder tumors. The prognostic significance of p53 alterations in bladder tumors has not been established. The aim of the present study was to evaluate an immunohistochemical (IHC) method for the routine determination of p53 protein overexpression in human bladder tumors and to determine the relation between nuclear accumulation of p53 with the traditional prognostic indicators and patient survival. MATERIALS AND METHODS: 104 transitional cell carcinomas of the bladder were analyzed simultaneously by immunohistochemistry for p53 protein overexpression and direct DNA sequencing for p53 gene mutations. RESULTS: The overexpression of p53 protein was reported in 30.8% of the cases and mutations of p53 gene in 23.0%. A significant association was observed between p53 alterations established either by IHC or direct DNA sequencing and stage (p<0.0001), grade (p<0.001), vascular invasion (p = 0.0005), DNA ploidy (p = 0.0002) and carcinoma in situ (p<0.0001). The correlation between the p53 gene mutations and p53 nuclear reactivity as detected by IHC was highly significant (p<0.0001). Univariate statistical analysis showed that the expression of p53 was significantly correlated to poor prognosis (p<0.0001). However, in multivariate analysis, only stage was significantly correlated to prognosis (p<0.0001). CONCLUSIONS: The IHC method was highly sensitive and specific and simple to apply for the routine examination of p53 overexpression in bladder tumors. However, overexpression of p53 as determined immunohistochemically, does not appear to have a better predictive prognostic value than stage in bladder tumors.

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Competitive RT-PCR to quantify CFTR mRNA in human endometrium.

Because the down-regulation by progesterone of cystic fibrosis transmembrane conductance regulator (CFTR) expression could be a useful specific marker to define the state of implant receptivity in endometrium, a competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed for quantifying the CFTR mRNA concentration in human endometrial samples. A competitor RNA was constructed with the same sequence as the CFTR sequence except for a 20-nucleotide insertion in the middle. The amplified products were separated by polyacrylamide gel electrophoresis. The ratio of CFTR band areas to competitor band areas provided the basis of quantification. Using this competitive RT-PCR, we measured CFTR mRNA in human endometrial samples taken at different periods of the menstrual cycle, in endometriosis, and in hyperplasia. Results show that the method is suitable for measuring the concentration of CFTR mRNA.

Gene transfer into subcultured endometrial cells using lipofection.

Lipofection using the Lipofectin reagent was optimized to transiently transfect subcultured guinea pig endometrial stromal cells with a beta-galactosidase gene driven by a simian virus 40 promoter. Efficient transfection was obtained in the following conditions: a value of six for the ratio of lipofectin to DNA, a low cellular density (10(5) cells per 35-mm well) at the time of subculture (48 h before lipofection) and a lipofection duration of 12 hours. Lipofection was compared to calcium phosphate precipitation previously optimized in the same culture model. At a low cellular density, the lipofection method was found to be more efficient than the calcium phosphate precipitation. This result gives a great relevance to lipofection since the cultured cells available in an experiment are often limited. Then, using cells at low density and a plasmid containing the chloramphenicol acetyltransferase (cat) gene linked to an estrogen response element, it was shown that the lipofection procedure is a suitable tool for the evaluation of gene regulation by estrogen.

Differential EGF action on nuclear protooncogenes in human endometrial carcinoma RL95-2 cells.

Our aim was to analyze EGF action on nuclear protooncogenes in RL95-2 since it has not been documented so far. Synchronization and partial' growth arrest were obtained by maintaining cells for 15 hours in L-methionine-free medium. After this depletion, EGF transiently increased fos and jun mRNAs: the expression peaked at 45 minutes for c-fos (5.5 fold induction) and at 60 minutes for c-jun and jun-B (3 fold induction) and the mRNA levels returned to the basal value within 3 hours. Upon EGF addition, c-myc mRNAs peaked at 12 hours (7.6 fold induction) and surprisingly remained higher than the control up to 48 hours. Unlike fetal calf serum, EGF did not increase the cell number and this could be linked to steadily induced c-myc expression. These data provide evidence for a differential EGF action on fos/jun and c-myc in RL95-2 cells.

Down-regulation by progesterone of CFTR expression in endometrial epithelial cells: a study by competitive RT-PCR.

To determine the effect of progesterone on the CFTR mRNA level in glandular epithelial cells of guinea-pig endometrium, a competitive RT-PCR was developed using an an internal standard a competitor with the same sequence as CFTR RNA except for a 20 nucleotide insertion. Using this method, the results showed that the CFTR mRNA level decreased in cells treated with estradiol plus progesterone compared to cells receiving estradiol alone. The decrease in CFTR mRNA level was maximal at 12 h incubation and was 27.3% of the CFTR mRNA level in estradiol-treated cells. The effect of progesterone was mimicked by the progestagen, R5020 and was inhibited by antiprogestins, RU 38,486 and ZK 98,299. Results are discussed in relation to the changes taking place in the endometrium in preparation for implantation of the embryo.

Adenosine 3',5'-monophosphate mediates progesterone effect on sulfate uptake in endometrial epithelial cells.

We have previously shown that progesterone increased sulfate uptake in glandular epithelial cells of guinea pig endometrium. To investigate whether cAMP might be the cause of the progesterone effect on sulfate uptake, cAMP accumulation and the effect of cAMP on sulfate uptake were evaluated in cells treated with 17 beta-estradiol alone or with progesterone. Progesterone provoked an increase in the intracellular cAMP accumulation in cells treated with 17 beta-estradiol. Moreover, cAMP or forskolin elicited the same marked increase in sulfate uptake as that observed with progesterone. The effect of progesterone on sulfate uptake was abolished by blocking either the cAMP pathway or the genomic action of progesterone and was independent of the cAMP-activatable apical chloride channel. This study is the first evidence of cAMP activation of sulfate uptake and suggests a genomic effect of progesterone on the production of cAMP which activates the sulfate transport system in a short term activation and a long term activation independent of transcriptional or translational events. The endometrium is a unique tissue that undergoes profound highly regulated modifications during the secretory phase in providing a suitable environment for embryo implantation. The regulation of sulfate uptake could participate in this process.

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.