Biomedical subjects

S M Hyder

Publications and source records attributed to S M Hyder.

At least 19 recordsLinked to original sources

PubMed2006

Sex-steroid regulation of vascular endothelial growth factor in breast cancer.

Angiogenesis is a key event, which occurs in both normal and pathological expansion of tissues and provides the nourishment necessary for growth. The role of angiogenic growth factors in breast pathology is rapidly gaining recognition since scientists and clinicians realized that these factors could function as molecular targets event 550 209822 for controlling tumor expansion. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. Although significant advances have been made in understanding the sex-steroid-dependent regulation of this key factor, the role of VEGF in controlling breast tumors is not well understood. In this review, I discuss recent studies describing the role of the female sex steroids estrogens and progestins in the regulation of VEGF in breast cancer cells. Furthermore, I present a summary of recent studies from other biological systems (mainly focused on tumor biology) directed towards providing us with a better understanding of the regulation of classical VEGF and VEGF receptors. I propose that by extending such studies we will gain deeper insights into how we might combat the progression of breast cancer by controlling hormone-dependent angiogenesis within tumor tissue. I believe that information gained from such studies will permit us to target angiogenic growth factors and their initiated signal transduction pathways in a more precise and selective manner, and thereby to control the formation of new blood vessels that fuel the rapid growth of breast tumors. Finally, it is my hope that the concepts discussed here will help elucidate molecular targets in the hormone-dependent angiogenesis pathway that will ultimately allow us to overcome anti-hormone resistance and to provide insights into how we might pursue the concept of chemoprevention by considering 'angioprevention' as the end point.

Breast Neoplasms↗

PubMed2001

Pharmacological and endogenous progestins induce vascular endothelial growth factor expression in human breast cancer cells.

Tumor expansion is dependent on angiogenesis, which is regulated by peptide growth factors of which vascular endothelial growth factor (VEGF) is one of the most selective and potent. VEGF expression is regulated by steroid hormones in a number of systems, including T47-D human breast cancer cells in which VEGF protein levels are elevated by progestins. In the present study, we investigated the effect of progestins on VEGF mRNA levels in human breast cancer cells. For these experiments, T47-D cells were exposed to progestins, RNA was prepared for measurement of VEGF transcript levels by Northern blot analysis and VEGF protein in the cell culture media was measured by enzyme-linked immunosorbent assay. Basal expression of VEGF mRNA is low in these cells, and is rapidly induced following exposure to progestins, reaching a maximum induction of 2- to 5-fold between 3 and 6 hr after hormone addition. This induction was inhibited by the antiprogestin RU-486 indicating that it is progesterone receptor (PR) dependent. Transcripts for VEGF165 and VEGF121 were the two major spliced forms of VEGF mRNA that were detected by reverse transcription-polymerase chain reaction in basal and progestin-stimulated T47-D cells. Maximum induction of VEGF mRNA was achieved with 10(-8) M progesterone, and induction was hormone specific, as estrogens, glucocorticoids, and androgens were without effect. Actinomycin D completely abolished the induction of VEGF transcript levels by progestins, suggesting that this response involves de novo mRNA synthesis, but puromycin did not inhibit induction, suggesting that this effect does not require protein synthesis. This report demonstrates that progestins stimulate VEGF mRNA levels and raises the possibility that anti-progestins may be useful to inhibit proliferation and metastasis in some human breast cancers by blocking VEGF production.

Alternative Splicing↗

PubMed2001

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus.

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Animals↗

PubMed2000

Induction of the angiogenic factor VEGF in the uterus by the antiprogestin onapristone.

Onapristone (also referred to as ZK 98,299) is an antiprogestin that shares a number of structural similarities to mifepristone (RU-486) and other drugs in this class. While investigating the actions of antiprogestins on steroid hormone induced gene expression of angiogenic factors such as vascular endothelial growth factor (VEGF), we noted that onapristone alone induces VEGF transcript levels in the immature, ovariectomized rat uterus. In addition, onapristone induces expression of c-fos mRNA, which is induced by estrogens but not progestins in this target tissue. This induction of VEGF and c-fos by onapristone is inhibited by the antiestrogen ICI 182,780, but not by the antiprogestin RU-486. Both transcripts are very rapidly induced by onapristone, with maximal mRNA levels observed 3-6 h after in vivo administration of the drug. This time course is similar to that for induction of these genes by estrogenic hormones. Dose-response studies show that both these genes are maximally induced by a 2.5 mg/kg dose of onapristone following intra peritoneal injection. These results indicate that onapristone rapidly upregulates several genes normally under estrogenic regulation in the immature rat uterus. Importantly, this is the first report of the induction of a major angiogenic factor by an antiprogestin. Since an increase in vascularity increases tumor expansion and metastasis, the induction of angiogenesis and its regulatory factors such VEGF may be an important end-point to consider in the development and use of antiprogestins for the treatment of neoplastic disease.

Animals↗

PubMed2000

Identification of functional estrogen response elements in the gene coding for the potent angiogenic factor vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and a prognostic factor for many tumors including those of endocrine-responsive tissues such as the breast and uterus. We and others have previously shown that VEGF is regulated by estradiol and tamoxifen in the uterus and by estradiol in human breast cancer cells, and pharmacological evidence has suggested that this regulation was mediated by transcriptional activation of the estrogen receptor (ER). This prompted us to investigate whether the VEGF gene contains sequences that bind the ER and confer hormonal inducibility to reporter constructs in the presence of the two ER subtypes. These studies identified two sequences homologous to the consensus estrogen response element, GGTCAnnnTGACC, which bind both ER-alpha and ER-beta. One of these elements is located in the 5'-untranslated region of the VEGF gene (GGGCAaagTGACT), and the other is located in the 3'-untranslated region (GAGCAcccTGCCC). Competition with excess unlabeled oligonucleotides indicates that these two elements bind both ERs specifically, mutations in either half-site of the two elements abolish receptor binding, and ER-alpha- and ER-beta-specific antibodies interact with complexes formed with the corresponding receptor subtypes. In cells containing either ER-alpha or ER-beta, the 3'-element behaves as a traditional enhancer that confers hormone inducibility to reporter constructs in an orientation-independent manner, and transcriptional activity is blocked by the pure antiestrogen ICI 182,780. The pattern of transcriptional activity of the element located in the 5'-flanking region is more complex. In the orientation found in the endogenous gene, this element is nonresponsive to ER-beta but confers estrogen-dependent inhibition of transcription with ER-alpha that is blunted by ICI 182,780. In the opposite orientation, the 5'-element confers hormone inducibility with either ER-alpha or -beta, and ICI 182,780 blocks activation by ER-alpha but not by ER-beta. These findings support the hypotheses that estrogens directly regulate VEGF transcription in target tissues and tumors, although such regulation appears likely to involve a complex interplay of cis- and trans-acting elements not previously observed for other hormone-responsive genes.

3' Untranslated Regions↗

PubMed2000

Expression of estrogen receptor coactivators in the rat uterus.

Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17 beta or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17 beta, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Regulation of vascular endothelial growth factor expression by estrogens and progestins.

Estrogens increase the expression of vascular endothelial growth factor (VEGF) mRNA in the rodent uterus. This regulatory effect is rapid, beginning within 1 hr after hormone treatment, dose dependent, and blocked by the pure antiestrogen ICI 182,780. The induction of the transcript is blocked by inhibitors of RNA but not of protein synthesis, and we have recently identified estrogen response elements in the VEGF gene. Collectively, these findings indicate that estrogens regulate uterine VEGF expression at the transcriptional level via the classical nuclear estrogen receptor pathway. Estrogen induction of VEGF occurs in the stromal layer of the rodent uterus, and estradiol induces expression of VEGF transcript levels in cultured human uterine stromal cells. Progestins also induce VEGF expression in the rodent uterus, although the effect is less marked and slower in onset than estrogenic effects. The effect of progestins is blocked by the antiprogestin mifepristone (RU-486), suggesting that it is also mediated by a classical nuclear receptor pathway. In addition, progestins regulate expression of VEGF mRNA and protein in cultured human T47-D breast cancer cells. The development of uterine leiomyomas is associated with exposure to ovarian sex steroids, abnormal uterine bleeding is commonly seen in patients with leiomyomas, and fibroids require an increased vascular supply for their growth. These observations suggest that VEGF and other angiogenic factors may represent potential targets for the treatment and prevention of uterine fibroids.

Breast Neoplasms↗

PubMed2000

Regulation of VEGF in the reproductive tract by sex-steroid hormones.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. In adults, angiogenesis is an infrequent event in the normal tissue except in the female reproductive tract where angiogenesis occurs frequently during the cyclical repair and regeneration of the endometrium as well as in the ovary. Little is known about angiogenesis in the male reproductive tract. The role of VEGF in controlling reproductive tract physiology and the role of hormones in regulating this key regulator of angiogenesis is not well understood. Since reproductive tract physiology is largely under sex-steroid regulation, we have reviewed some recent studies describing the role of sex-steroid hormones in regulating VEGF. We have also included studies on the role of sex-steroids in regulating VEGF and angiogenesis in endometrial, breast and prostate pathologies. We have provided an extensive review of the classical VEGF and VEGF receptors with examples drawn from numerous studies in the literature using diverse biological systems to encourage similar studies in the area of reproductive tract physiology. It is speculated that such studies will provide insights into understanding the role of VEGF in reproductive tract development, causes of infertility, and cancer. Such knowledge would allow us to target VEGF for improving human reproductive tract abnormalities, for enhancing implantation and fertility, and for designing drugs for treatment of endocrine dependent cancers.

Adult↗

PubMed1999

Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements.

Estrogen receptors are derived from two different gene products referred to as estrogen receptor-alpha (ER-alpha) and ER-beta. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-beta binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-alpha and confer estrogen inducibility to reporter constructs. Binding of ER-alpha and ER-beta occurred at similar DNA concentrations for some EREs, but different DNA concentrations were required to form complexes of the two receptors with other elements. These results illustrate for the first time by direct receptor-DNA binding studies that both ER-alpha and ER-beta bind to a number of EREs present in endogenous hormone regulated genes, and further suggest that the two forms of the receptor display different patterns of affinities for naturally occurring hormone response elements.

Animals↗

PubMed1999

The pure antiestrogen ICI 182,780 inhibits progestin-induced transcription.

The pure antiestrogen ICI 182,780 binds to the estrogen receptor with high affinity and is currently in clinical trials for the treatment of human breast cancer. We now show for the first time that ICI 182,780 also exhibits potent antiprogestin activity at doses frequently used in laboratory investigations. The antiprogestin activity of ICI 182,780 was detected in HeLa, HepG2, and CV1 cells transiently transfected with either the A or B forms of the human progesterone receptor and a luciferase construct driven by a progesterone-response element. ICI 182,780 inhibited progesterone-induced gene transcription in a dose-dependent fashion with maximum inhibition obtained at 10(-6) M and an IC50 of approximately 2 x 10(-7) M. The ICI compound produced the same degree of inhibition as that obtained with the antiprogestin RU-486. The antiestrogen tamoxifen did not display antiprogestin activity in this test system, and ICI 182,780 did not inhibit the activity of transfected androgen or glucocorticoid receptors. These results clearly establish that ICI 182,780 has significant antiprogestin activity in addition to its well-documented antiestrogenic activity and raises the possibility that both antihormonal properties of this compound are exhibited in laboratory studies and in the course of treatment of human breast tumors.

Antineoplastic Agents↗

PubMed1999

Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain.

We report that a small population of neurons expresses both choline acetyltransferase and classical estrogen receptor immunoreactivity and they are found primarily in the bed nucleus of the stria terminalis. In short-term ovariectomized ageing mice (24 months, n = 5) there were 41.0 +/- 4.1% fewer of these double-labeled cells than in young (five months, n = 5) short-term ovariectomized C57BL/6J mice. To study cholinergic neuron estrogen responsiveness, young mice (n = 8) were ovariectomized at puberty (five weeks). After three months half of the mice (n = 4) were given physiological levels of 17beta estradiol for 10 days. Bed nucleus double-labeled neurons increased by 32.9% (P < or = 0.003) in the young mice given estrogen. In a gel shift assay, double-stranded oligonucleotides with putative estrogen response elements from the choline acetyltransferase gene were used as competitors against estrogen receptor binding to consensus estrogen response elements. A sequence with 60% homology to the vitellogenin estrogen response element was found to compete at 500- and 1000-fold excess. Young mice (five months) with ovaries demonstrated significantly (P < or = 0.04) better performance in the spontaneous alternation T-maze test than did old (19 month) mice with ovaries (young = 66.3 +/- 3.3% correct choices; vs old = 55.0 +/- 4.0% in old mice with ovaries). Young mice (five months old), ovariectomized for one month and treated with estrogen, showed significantly more spontaneous alternation than ovariectomized controls (69.1 +/- 2.8% vs 58.3 +/- 3.9%; P < or = 0.04). Estrogen also increased spontaneous alternation in old, short-term ovariectomized mice (61.5 +/- 2.7% vs 48 +/- 3.3%; P < or = 0.005). In either young or old ovariectomized mice, estrogen increased spontaneous alternation to levels seen in young animals with ovaries. Estrogen increases the number of choline acetyltransferase-immunoreactive and choline acetyltransferase/estrogen receptor-immunoreactive cells in old or young mice lacking estrogen, and enhances working memory in old or young mice lacking estrogen. Our data suggest that estrogen may act at the level of the choline acetyltransferase gene, but in view of the limited distribution of cholinergic cells expressing the classical estrogen receptor, it is unlikely that these cells can account for a memory enhancing effect of estrogen replacement.

Aging↗

PubMed1999

Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins.

Proper regulation of angiogenesis and vascular permeability is essential for the physiological functioning of the female reproductive tract, and major health problems in women, such as dysfunctional uterine bleeding, endometriosis, and uterine cancer, involve a vascular component. There is a large body of literature that describes the effects of sex steroids on the vasculature of the reproductive tract, but far less is known about the molecular mechanisms that regulate these important actions. We hope that this minireview will help emphasize the need for mechanistic studies in this area to improve treatment and prevention of these major health problems in women. Specifically, we believe it will be important to 1) define the exact roles of FGF, VEGF, and other factors in physiological and pathological events in the reproductive tract and the cell types and receptors involved; 2) identify estrogen and progesterone receptor subtypes, the DNA elements, nuclear protein factors, and signaling pathways that mediate regulation of these genes by sex steroids; 3) elucidate any mechanisms of cross-talk between sex steroids and other regulatory factors in the overall regulation of FGF, VEGF, and other angiogenic/permeability factors; and 4) eventually understand how genetic polymorphisms of key regulatory elements affect angiogenesis and the regulation of vascular function in the female reproductive tract.

Animals↗

PubMed1999

Synthetic estrogen 17alpha-ethinyl estradiol induces pattern of uterine gene expression similar to endogenous estrogen 17beta-estradiol.

17alpha-Ethinyl estradiol is one of most widely prescribed estrogens. We compared the effects of this synthetic estrogen to those of the endogenous ovarian hormone 17beta-estradiol on the expression of four estrogen-inducible genes in the rat uterus. The genes examined include c-fos, c-jun, vascular endothelial growth factor, and creatine kinase B, which are all known to be primary responses to estrogen administration. Both estrogens induced the four target genes with similar time courses and produced the same pattern of cell-specific expression of c-fos and vascular endothelial growth factor in the uterine epithelium and stroma, respectively. Dose-response studies established that the potency and efficacy of both estrogens in the uterus were the same for all four hormone-regulated genes. These studies suggest that 17alpha-ethinyl and 17beta-estradiol produce similar if not identical patterns of gene expression in the uterus.

Animals↗

PubMed1998

Progestin regulation of vascular endothelial growth factor in human breast cancer cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the degree of vascularity, progression, and metastasis of breast cancer, and cases of this disease with increased vascular density have a poor prognosis. We show that in T47-D human breast cancer cells, progesterone induces a dose-dependent increase of 3-4-fold in media VEGF levels, with a maximum response occurring at a concentration of 10 nM. This effect is blocked by the antiprogestin RU 486. In addition to progesterone, a number of synthetic progestins used in oral contraceptives (e.g., norethindrone, norgestrel, and norethynodrel), hormone replacement therapy (medroxyprogesterone acetate), and high-dose progestin treatment of breast cancer (megestrol acetate) also increase VEGF in the media of cultured T47-D cells. This effect is hormone specific and is not produced by estrogens, androgens, or glucocorticoids. Collectively, these observations suggest that the increase in VEGF caused by progestins is mediated by progesterone receptors present in T47-D cells. The induction of VEGF by progestins is also cell type specific and does not occur in human breast cancer cell lines MCF-7, ZR-75, or MDA-MB-231, nor in Ishikawa cells derived from a human endometrial carcinoma. This is the first report that progestins regulate VEGF expression in human breast cancer cells and raises the possibility that increased angiogenesis in response to endogenous progesterone or its therapeutically used analogues may play a role in cell growth or metastasis in a subset of human breast tumors.

Breast Neoplasms↗

PubMed1998

The 3-flanking region of the mouse c-fos gene contains a cluster of GGTCA hormone response-like elements.

We previously identified an estrogen response element in the 3'-flanking region of the c-fos protooncogene [1, 2]. This element, GGTCAnnnCAGCC, has one half-site identical to that of the consensus ERE (GGTCAnnnTGACC) but only limited homology to the second half-site. Because of this non-canonical sequence and atypical location in the 3'-untranslated region of an estrogen target gene, we decided to analyze sequences adjacent to this element for the possible presence of other regulatory elements. We now report that the 635 base pairs downstream of the c-fos ERE contain: (i) an unusual cluster of 7 GGTCA hormone response-like elements; (ii) potential binding sites for other known DNA binding proteins; and (iii) a sequence specific binding site for a non-estrogen receptor protein present in hormone target tissues.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Phorbol ester inhibition of estrogen-induced uterine deoxyribonucleic acid synthesis.

Protein kinase C (PKC) is a key regulatory enzyme in the control of growth and differentiated function in many cell types. Recently it has become clear that cross talk occurs between PKC and steroid hormone-activated signaling pathways. In this work we have thus used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to investigate the relationship between PKC activation and estrogen-induced proliferation in an in vivo model of hormone action, the immature rat uterus. A single injection of estradiol (E2) to immature female animals increases DNA synthesis in all major uterine cell types. Administration of TPA alone, simultaneous administration of TPA and E2, or administration of TPA 12 h after E2 did not alter uterine DNA synthesis. However, administration of TPA 6 h after E2 markedly decreased [3H]thymidine incorporation and the labeling indices in uterine epithelial, stromal, and myometrial cells. This inhibition represents a decrease in DNA synthesis per se rather than a change in the time course of the tissue response to the hormone. The inhibitory effect of TPA was reversible within 72 h and did not appear to be due to a decrease in the level or degree of occupancy of uterine estrogen receptors. These results suggest that a discrete regulatory event(s) in the pathway of estradiol-induced proliferation is inhibited by PKC activation approximately 6 h after hormonal stimulation.

Animals↗

PubMed1997

Triphenylethylene antiestrogens induce uterine vascular endothelial growth factor expression via their partial estrogen agonist activity.

Estradiol induces vascular endothelial growth factor (VEGF) expression in the rat uterus and this may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue. Triphenylethylene antiestrogens such as tamoxifen have mixed agonist/antagonist activity and their specific effects are tissue and gene specific. These drugs exhibit primarily antiestrogenic actions in mammary tissue and are thus used for the treatment of breast cancer. These drugs are also suggested to be inhibitors of angiogenesis. However, uterine side effects of tamoxifen are thought to stem largely from the agonist activity of the drug in this tissue. Since side effects of tamoxifen such as uterine bleeding and endometrial cancer seem likely to have an angiogenic component, we have examined the effects of this drug, its metabolite, 4-hydroxy-tamoxifen and two additional triphenylethylene antiestrogens, nafoxidine and clomiphene, on the expression of VEGF and another estrogen regulated gene, c-fos, using the rat uterus as an experimental system. All four compounds increase uterine VEGF and c-fos mRNA levels indicating that the triphenylethylene class of antiestrogens are predominantly agonists for the induction of these genes in the uterus.

Animals↗

PubMed1997

Selective inhibition of estrogen-regulated gene expression in vivo by the pure antiestrogen ICI 182,780.

Estrogen-stimulated cell proliferation is thought to be mediated by the induction of growth-regulatory factors. Abnormal regulation of such factors may be associated with tumorigenesis. Two such factors, vascular endothelial growth factor and c-fos, are rapidly induced in the uterus by estrogens. We show that the induction of these transcripts by 17beta-estradiol or tamoxifen is selectively blocked by the pure antiestrogen ICI 182,780 in a dose-dependent manner. This indicates that induction of the two genes requires different levels of estrogen receptor or that their induction occurs by different mechanisms. This suggests that selective dose-dependent antagonism of estrogen-dependent transcription may be possible in target tissues and tumors.

Animals↗