A B S T R A C T Ketoconazole has recently been shown to interfere with steroidogenesis in patients and rat in vitro systems. In this study we attempted to elucidate the site of inhibition in the adrenal gland. Although ketoconazole impaired adrenocorticotropic hormone stimulated cyclic (c)AMP production, dibutyryl cAMP addition did not bypass the steroidogenic blockade indicating that the critical ketoconazole-inhibited step was distal to cAMP. Addition of radiolabeled substrates to isolated adrenal cells and analysis of products by high performance liquid chromatography demonstrated a ketoconazole block between deoxycorticosterone (DOC) and corticosterone. This 11-hydroxylase step is carried out by a P450-dependent mitochondrial enzyme. No restriction of progesterone or pregnenolone conversion to DOC was detected, steps carried out by non-P450-dependent microsomal enzymes. Inhibition of cholesterol conversion to pregnenolone by mitochondrial fractions indicated a second block at the side chain cleavage step, another mitochondrial P450-dependent enzyme. Adrenal malate dehydrogenase, a non-P450-dependent mitochondrial enzyme was not inhibited while renal 24-hydroxylase, a P450-dependent mitochondrial enzyme in another organ, was blocked by ketoconazole. We conclude that ketoconazole may be a general inhibitor of mitochondrial P450 enzymes. This finding suggests that patients receiving ketoconazole be monitored for side effects relevant to P450 enzyme inhibition. Further, we raise the possibility that this drug action may be beneficially exploited in situations where inhibition of steroidogenesis is a therapeutic goal.

Ketoconazole, a broad-spectrum, antifungal drug that is administered orally, has been shown to inhibit sterol synthesis in fungi. When gynecomastia developed in some patients taking this drug, we investigated the effects of ketoconazole on steroid synthesis in humans and in isolated adrenal cells from rats. In healthy humans, the cortisol response to adrenocorticotropic hormone was significantly blunted 4 hours after a 400-mg or 600-mg dose. The inhibition persisted for up to 8 hours and was absent by 16 hours. This finding indicated that adrenal androgen response was reduced. Easily achieved therapeutic concentrations of ketoconazole virtually eliminated corticosterone production by isolated adrenal cells from rats. Although ketoconazole at currently used doses has never been documented to cause clinical hypoadrenalism, caution is urged in high- or multiple-dose trials. The drug may prove useful as an agent to block steroid synthesis.

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate B-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH 2 -terminal kinase -responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.

Purpose: Although there is considerable information on the molecular aberrations associated with endometrial cancer, very little is known of the changes in gene expression associated with endometrial hyperplasia. Experimental Design: To address this, we have compared the level of expression of estrogenregulated genes and components of the insulin-like growth factor I (IGF-I) signaling pathway in endometrial biopsies from subjects with normal endometrium, complex atypical endometrial hyperplasia, and endometrial adenocarcinoma (type I).Results: There was a significant increase in the expression of the IGF-I receptor (IGF-IR) in biopsies from hyperplastic endometrium and endometrial carcinoma compared with the proliferative endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in endometrial hyperplasia and carcinoma, the downstream components of the IGF-IR pathway are activated, as reflected in increased Akt phosphorylation. Loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in endometrial hyperplasia did not correlate with increased activation of IGF-IR. However, the simultaneous loss of PTEN expression and increased IGF-IR activation in hyperplasia was associated with an increased incidence of endometrial carcinoma. Conclusions: These results suggest that up-regulation of IGF-IR and loss of PTEN may be independent events that give rise to complementary activation of the IGF-I pathway and increase the probability of the development of cancer. These studies suggest that increased expression of IGF-IR may be an important contributor to the risk of endometrial hyperplasia and cancer.

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK-inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds which inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2-inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 hours, after which total RNA was isolated and genome-wide gene expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor (CTGF), a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and CTGF mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced CTGF mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have anti-fibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.

Paracoccidioidomycosis, a disease caused by Paracoccdioides brasilensis, which is endemic to Latin America, is much more common in men than women, suggesting a role for hormonal factors. We recently showed that two other yeasts possess steroid binding proteins and postulated that these receptorlike molecules represented a mechanism by which the hormonal milieu of the host might influence an infecting pathogen. Therefore, we examined P. bralensis for a sex steroid binding protein. Mr of =60,000. Competition experiments revealed that estrone, estriol, and progesterone had 25% of the affinity of estradiol, whereas diethylstilbestrol, androgens, and corticosteroids had low affinity. Investigation of steroid hormone actions in P. brasiienis indicated that estradiol inhibited the fungal transformation from mycelial form to yeast form, the initial step of infection. This suppressive effect was dose-dependent and not found with testosterone. We hypothesize that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelialto-yeast transformation, thus explaining the resistance of women *to paracoccidioidomycosis.Paracoccidioides brasiliensis is a pathogenic fungus, the etiologic agent of paracoccidioidomycosis (South American blastomycosis). Although paracoccidioidomycosis is 13-87 times more common in men than women (1), contact with P. brasiliensis is essentially the same for the two sexes (2). The possibility has been considered that the hormonal milieu of the host might directly influence P. brasiliensis, affecting its pathogenicity (1, 3). Although this hypothesis is speculative, our recent findings (4-6) suggest a basis for a mechanism by which host hormones could affect an invading pathogen and alter its infectious properties. We have demonstrated the existence of intracellular proteins in two fungal genera that bind vertebrate steroid hormones with high affinity and specificity. Candida albicans possesses a protein that binds corticosterone and progesterone (4, 5), and Saccharomyces cerevisiae contains a different protein that selectively binds estrogens (6). We have postulated that these macromolecules may represent primitive hormone receptors in fungi. These proteins appear not only to bind endogenous fungal ligands but also to bind vertebrate steroid hormones, a fact we have exploited in selecting the radioprobes for these studies. Therefore, we considered the possibility that P. brasiliensis could possess either an androgen or an estrogen binding protein which, when occupied by the appropriate host sex steroid, might result in altered pathogenicity, thus explaining the predominance of infections in males. The data to be presented in this report demonstrate that P. brasiliensis does contain an estrogen binding protein. We also show that estradiol inhibits the fungal, transformation from mycelial form to yeast form (mycelial-to-yeast transformation), the initial step in the establishment of infection (7). Although the concordance of binding in vitro and bi...

The molecular progression of endometrial cancer is poorly understood, and both genetic and epigenetic factors play a role. Survivin is a member of the inhibitor of apoptosis (IAP) gene family and contains a canonical CpG island that has been described as epigenetically regulated. As survivin is overexpressed in endometrial tumors, we hypothesized that hypomethylation could explain this expression pattern. Surprisingly, methylation-specific PCR and pyrosequencing showed that survivin was hypermethylated in endometrial tumors and correlated with increased survivin expression. We speculated that methylation could inhibit the binding of p53, a repressor of survivin expression. Our data indicates that demethylation of the survivin promoter by decitabine results in p53-dependent survivin repression and that p53 binding can be inhibited by DNA methylation. We are the first to report survivin de-repression by DNA methylation. We also present microarray data, which suggest that de-repression by methylation is a general mechanism of p53 regulation. Demethylation induced by decitabine is traditionally thought to be active in tumors by allowing the reexpression of tumor suppressor genes. However, our results indicate that an additional important mechanism is to decrease the expression of oncogenes.

A B S T R A C TWe then examined the functional response of this binding by measuring tyrosine aminotransferase (TAT) activity in HTC cells. The antimycotics did not exhibit TAT agonist activity and inhibition of basal enzyme levels was not detected. However, the drugs were potent antagonists of dexamethasone-induced TAT activity and the effect was temporally reversible. This antagonist activity was in the same sequence and closely correlated with the binding potency of the three drugs. We conclude that ketoconazole and other imidazole antimycotic drugs possess glucocorticoid antagonist activity by virtue of occupancy of glucocorticoid receptor sites in target tissues.