Steroid Sulfatase mRNA Expression Research Articles

Steroid sulfatase is increased in the placentas and whole blood of women with early-onset preeclampsia

IntroductionPreeclampsia is a serious complication of pregnancy affecting 5% of pregnancies. Our team identified 137 genes highly expressed in placenta relative to other human tissues. Here, we have explored a role for steroid sulfatase (STS) in preeclampsia by characterising STS expression and the functional effects of STS on primary placental trophoblasts. MethodsCharacterisation of STS was performed on preterm preeclamptic and gestation-matched normotensive preterm controls who delivered at <34 weeks gestation. We characterised placental and maternal whole blood STS mRNA and placental protein expression via qRT-PCR, immunohistochemistry and Western Blot. To assess whether STS is involved in sFlt1 secretion and syncytialisation, we administered siRNA to silence STS in primary trophoblasts before measuring sFlt1 and hCG secretion and E-Cadherin expression. ResultsA custom array containing 45 placental specific genes identified 10 genes significantly altered in the placentas of preeclamptic patients relative to normotensive gestation-matched controls. Of these genes, qRT-PCR and western blot on a larger cohort confirmed that the expression of STS was significantly elevated in preeclamptic placentas (n = 44) relative to gestation matched controls (n = 26). Given placental RNA leaks in to the maternal circulation, we also assessed STS mRNA expression in the whole blood of patients with preeclampsia and found it was significantly increased relative to normotensive controls. siRNA knockdown of STS in primary trophoblast resulted in a modest but significant reduction in sFlt1 secretion, but had no affect on hCG secretion or E-Cadherin protein expression. DiscussionSTS is increased in preeclamptic placentas and maternal whole blood. Our data suggests that STS may affect sFlt1 secretion by regulating sFlt1-i13 transcription, and not via alterations in syncytialisation.

Effects of steroid hormone on estrogen sulfotransferase and on steroid sulfatase expression in endometriosis tissue and stromal cells

Endometriosis is an estrogen-dependent disease that afflicts about 10% of women in their reproductive age, causing severe pain and infertility. The potential roles of female steroid hormones in modulating key estrogen-metabolizing enzymes, steroid sulfatase (STS) and estrogen sulfotransferase (SULT1E1), were investigated. The expression of STS and SULT1E1 mRNA in biopsy samples (n=78) of superficial and deep endometriotic lesions, eutopic endometrium of women with endometriosis and endometrium from control patients were compared according to the menstrual cycle phase. Increased STS gene expression was detected in superficial and deep-infiltrating lesions and a reduced SULT1E1 expression was also observed in the eutopic endometrium relative to the superficial lesions. Additionally, a significantly positive correlation was detected between STS and SULT1E1 mRNA expression levels in biopsy specimens collected from the endometriosis patients, and not in control individuals. The actions of female steroid hormones on SULT1E1 and STS expression were evidenced in endometriosis, revealed by increased expression levels in the luteal phase of the cycle. There was an increased STS expression in primary eutopic and ectopic endometrial stromal cells treated with estradiol and progesterone (representative of the luteal phase, n=3). Although an increased STS mRNA expression was observed in hormone-induced endometrial stromal cells in vitro, no difference could be detected between the hormone treatment groups in estradiol formation from estradiol sulfate measured by LC–MS–MS. Interestingly, a greater expression of STS was observed in stromal cells from eutopic endometrium with an agreement in estradiol formation originated from estradiol sulfate. The differential regulation of STS and SULT1E1 could provide insights for novel studies of the therapeutic use of STS inhibitors.

Structure Based Design, Synthesis, and Evaluation of Potential Inhibitors of Steroid Sulfatase

The activity of the enzyme steroid sulfatase (STS) is high in breast tumors and elevated levels of STS mRNA expression have been associated with a poor prognosis. Potent STS irreversible inhibitors have been developed, paving the way to use this new type of therapy for the treatment of breast cancer. Several small molecules belonging to a natural products-inspired library of previously obtained inhibitors of tumor cell growth and new molecules planned to be reversible inhibitors of this enzyme were docked into STS. Some of the synthesized xanthone derivatives, which revealed high scores against STS, namely oxo-9H-xanthene-3,6-diyl bis(3-chlorobenzoate) (5), 9-oxo-9H-xanthene-3,6-diyl bis(4-tertbutylbenzoate) (6) and 9-oxo-9H-xanthene-3,6-diyl bis(4-methoxybenzoate) (7) showed poor water solubility. Therefore, formulations of these derivatives with cyclodextrins were prepared and characterized. The compounds were evaluated regarding their effect on the in vitro growth of various human tumor cell lines, as well as the effect in STS inhibition, for the compounds with the most favorable ΔG values. Additionally, the capacity of these derivatives and of some prenyl and acetoxy-benzophenone and xanthones to inhibit the in vitro growth of MCF-7 ER(+) and/or to inhibit STS in a micromolar range was also assessed. Some compounds developed in the present work were shown to be potential STS inhibitors.

Induction of steroid sulfatase expression in PC-3 human prostate cancer cells by insulin-like growth factor II

Human steroid sulfatase (STS) plays an important role in regulating the formation of biologically active estrogens and may be a promising target for treating estrogen-mediated carcinogenesis. The molecular mechanism of STS gene expression, however, is still not clear. Growth factors are known to increase STS activity but the changes in STS expression have not been completely understood. To determine whether insulin-like growth factor (IGF)-II can induce STS gene expression, the effects of IGF-II on STS expression were studied in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that IGF-II treatment significantly increased the expression of STS mRNA and protein in concentration- and time-dependent manners. To understand the signaling pathway by which IGF-II induces STS gene expression, the effects of specific PI3-kinase/Akt and NF-κB inhibitors were determined. When the cells were treated with IGF-II and PI3-kinase/Akt inhibitors, such as LY294002, wortmannin, or Akt inhibitor IV, STS expression induced by IGF-II was significantly blocked. Moreover, we found that NF-κB inhibitors, such as MG-132, bortezomib, Bay 11-7082 or Nemo binding domain (NBD) binding peptide, also strongly prevented IGF-II from inducing STS gene expression. We assessed whether IGF-II activates STS promoter activity using transient transfection with a luciferase reporter. IGF-II significantly stimulated STS reporter activity. Furthermore, IGF-II induced expression of 17β-hydroxysteroid dehydrogenase (HSD) 1 and 3, whereas it reduced estrone sulfotransferase (EST) gene expression, causing enhanced estrone and β-estradiol production. Taken together, these results strongly suggest that IGF-II induces STS expression via a PI3-kinase/Akt–NF-κB signaling pathway in PC-3 cells and may induce estrogen production and estrogen-mediated carcinogenesis.

Aromatase and steroid sulfatase mRNA expression in fine needle aspirates from benign and malignant breast disorders

The fine needle aspiration (FNA) test is a convenient and tolerable technique with minimal invasion that is accepted by most women. Local estrogen synthesis depends mainly on the aromatase and steroid sulfatase pathways that are believed to play important roles in breast carcinogenesis. However, little is known about the level of aromatase and steroid sulfatase mRNA expression in FNA samples which contain only small amounts of tissue. The nested Q-PCR assay has been proven to be a highly sensitive and specific method to assess the aromatase expression of breast tissue. In this study, aromatase and steroid sulfatase mRNA expression in 74 patients with benign or malignant disorders was evaluated and compared using nested Q-PCR and non-nested Q-PCR assays. The expression levels were analyzed and correlated with clinical parameters. No difference in the aromatase expression levels between nested and non-nested Q-PCR was noticed. Age and aromatase mRNA expression level were two independent risk factors for breast cancer (P=0.04 and P=0.00, respectively), while menopausal status and steroid sulfatase mRNA expression levels were not associated with breast cancer. This study showed that both nested and non-nested Q-PCR assays were effective methods for research using FNA breast samples.

Induction of steroid sulfatase expression by tumor necrosis factor-α through phosphatidylinositol 3-kinase/Akt signaling pathway in PC-3 human prostate cancer cells

Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and has a pivotal role in regulating the formation of biologically active estrogens. STS may be considered a new promising drug target for treating estrogen-mediated carcinogenesis. However, the molecular mechanism of STS expression is not well-known. To investigate whether tumor necrosis factor (TNF)-α is able to regulate gene transcription of STS, we studied the effect of TNF-α on STS expression in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that TNF-α significantly induced the expression of STS mRNA and protein in a concentration- and time-dependent manner. Treatment with TNF-α resulted in a strong increase in the phosphorylation of Akt on Ser-473 and when cells were treated with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, or Akt inhibitor (Akt inhibitor IV), induction of STS mRNA expression by TNF-α was significantly prevented. Moreover, activation of Akt1 by expressing the constitutively active form of Akt1 increased STS expression whereas dominant-negative Akt suppressed TNF-α-mediated STS induction. We also found that TNF-α is able to increase STS mRNA expression in other human cancer cells such as LNCaP, MDA-MB-231, and MCF-7 as well as PC-3 cells. Taken together, our results strongly suggest that PI 3-kinase/Akt activation mediates induction of human STS gene expression by TNF-α in human cancer cells.

SR16157 Is a Potent Dual-Acting Inhibitor of Estrogen Biosynthesis and Estrogen Receptor.

Abstract Background: SR16157 is a novel dual-acting antiestrogen with promise as a treatment for breast and other estrogen-modulated cancers. SR16157 inhibits the hydrolysis of estrone sulfate to estrone, and is cleaved by estrone sulfatase (ES) to SR16137, a tissue-selective estrogen receptor modulator, with potent antiestrogen effects in breast and uterus and protective estrogen-like agonist effects on bone and cardiovascular systems. Steroid sulfatase (STS) is much higher than aromatase activity in breast cancer, and high levels of STS mRNA expression in tumors is associated with a poor prognosis. Using in vitro and xenograft MCF-7 breast cancer models, we demonstrated that SR16157 inhibited STS activity, cell growth, and estrogenic functions and that SR16137 reduced expression of estrogen-regulated genes with greater potency than tamoxifen.Methods: We conducted preclinical toxicity and pharmacokinetic evaluations of SR16157 as part of an investigational new drug application. SR16157 was administered to female Sprague-Dawley rats or beagle dogs by oral gavage or capsule. In rats, the maximum tolerated dose (MTD; highest dose that did not cause lethality, but produced toxicity) was 33 mg/kg/day (198 mg/m2/day), and the no observed adverse effect level (NOAEL) was &amp;lt;10 mg/kg/day (60 mg/m2/day). In dogs, the MTD was estimated to be &amp;gt;10 mg/kg/day (200 mg/m2/day), and the NOAEL was estimated to be ≥2.5 mg/kg/day (50 mg/m2/day). In a GLP toxicity study, female dogs received SR16157 by oral capsule for 28 days at 0.5, 2.5, or 10 mpk (10, 50, 200 mpm2) with 14 days recovery (Day 42). ES activity was determined in peripheral blood mononuclear cells (PBMC) before and during the study, and in liver at necropsy. Plasma drug levels and urinary metabolites were evaluated by LC-MS/MS.Results: All dogs survived to scheduled sacrifice with no test article-related clinical observations or body weight changes. Reversible changes related to white blood cells were observed in SR16157-treated dogs, including reduced white blood cell count, absolute lymphocytes, absolute eosinophils, and absolute monocytes. Mean thymus weights for high dose dogs were decreased 30% compared with controls at Day 29 and remained 20% lower at Day 42. Tissues showed no drug-related findings. Hepatic ES activity was completely inhibited in SR16157 groups by Day 29. PBMC ES activity was completely inhibited by Day 6, remained low through Day 29, and increased by Day 42. SR16137 levels were highest in plasma samples that also contained higher concentrations of SR16157.Discussion: SR16157 has excellent bioavailability and a favorable safety profile. Daily oral administration of SR16157 for 28 days in dogs was not associated with toxicologically significant effects that would limit clinical use. ES inhibition in PBMC is a sensitive indicator of the pharmacologic action of SR16157 and a potential biomarker of efficacy in humans. Based on these results, the recommended Phase I starting dose of SR16157 given orally once daily for 28 days is 20 mg/m2/day, which is 10% of the MTD in the more sensitive preclinical species (the rat). This work was supported by NCI Contract N01-CM-42203. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5124.

In vivo Efficacy of STX213, A Second-Generation Steroid Sulfatase Inhibitor, for Hormone-Dependent Breast Cancer Therapy

Steroid sulfatase (STS) inhibitors that can decrease or prevent the biosynthesis of estrogenic steroids via the sulfatase route may play an important role in the treatment of breast cancer. We compare the in vivo efficacy of two potent STS inhibitors, STX64 and STX213, in a xenograft breast cancer model. MCF-7 cells stably expressing STS cDNA (MCF-7STS) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of estradiol sulfate (E2S) and bearing both MCF-7STS and wild-type MCF-7 (MCF-7WT) tumors were orally treated with STX64 and STX213. Treatment was given for 49 days followed by a recovery period of 35 days in which animals received only E2S. Mice were weighed, and tumor measurements were taken weekly. STX64 and STX213 exhibited potent STS inhibition in vivo. However, STX213 showed a greater duration of activity. In vehicle-treated nude mice receiving E2S, tumor volumes increased 5.5-fold for MCF-7WT and 3.8-fold for MCF-7STS after 49 days compared with day 0. MCF-7WT tumor growth was reduced by 56% by STX213 over the dosing period, and subsequent growth was retarded during the recovery period. All treatments fully inhibited growth of MCF-7STS tumors, and recovery of these tumors was significantly retarded (P<0.01). All compounds completely inhibited liver and tumor STS activity. Additionally, STS mRNA expression in the MCF-7STS tumors directly correlated with the corresponding STS enzyme activity. This study indicates that STS inhibitors attenuate hormone-dependent human breast cancer growth and therefore offer a potentially novel treatment for this condition.

Strong expression of steroid sulfatase in human cumulus cells in patients with endometriosis

To investigate the localization and expression of steroid sulfatase (STS) in cumulus cells obtained from subjects with and without endometriosis. Case-control study. In vitro fertilization program at the Showa University School of Medicine. Cumulus cells (142 samples) were obtained from 49 patients for whom IVF was indicated. Some of these samples were taken from cases complicated by endometriosis (35 samples), polycystic ovary syndrome (PCOS; 16 samples), or latent hyperprolactinemia (16 samples). Immunohistochemical staining for STS. Measurement of STS mRNA expression by real-time polymerase chain reaction (PCR). Expression of STS mRNA and localization of STS. Steroid sulfatase was localized in the cytoplasm of the cumulus cells, and expression of STS mRNA was observed. The expression level of STS mRNA from patients with endometriosis was significantly higher (11.8-fold) than that of patients without endometriosis. These results suggest a local steroidal regulation mechanism in cumulus cells. Although the physiological role of STS in cumulus cells remains unclear, STS may be involved in the quality of eggs in patients with endometriosis.

Localization and gene expression of steroid sulfatase by RT-PCR in cumulus cells and relationship to serum FSH levels observed during in vitro fertilization

BackgroundThe purpose of this study was to localize the expression of steroid sulfatase (STS) in cumulus cells and to determine the relationship between STS mRNA expression and the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol and progesterone.MethodsThe subject group included 49 women (29 to 44 years old) for whom in vitro fertilization treatment was indicated. All subjects gave informed consent. One hundred fourteen samples of cumulus-oocyte complex (COC) were obtained under microscopic observation. Part of the COC was stained by STS antibody. RNA was extracted by phenol-chloroform method and real-time PCR was performed. Serum of each patient was collected and was measured by ELISA.ResultsSome of the cumulus samples were stained by STS antibody. The expression of STS mRNA in all samples was confirmed by quantitative RT-PCR. Although there was no significant correlation between the level of STS mRNA and the serum levels of estradiol, progesterone and LH, there was a statistically significant negative correlation between the level of STS mRNA expression and the serum level of FSH (n = 105, p = 0.018, r = -0.22).ConclusionThese results have demonstrated for the first time the expression of STS in cumulus cells by immunohistological stainings and real-time RT-PCR. STS expression in cumulus cells may be related to the control of the local steroidal environment in the oocyte. Serum FSH may control STS mRNA expression from the results of RT-PCR, although the correlation was low.

Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer

The great majority of breast cancers are in their early stage hormone-dependent and it is well accepted that estradiol (E 2) plays an important role in the genesis and evolution of this tumor. Human breast cancer tissues contain all the enzymes: estrone sulfatase, 17β-hydroxysteroid dehydrogenase, aromatase involved in the last steps of E 2 bioformation. Sulfotransferases which convert estrogens into the biologically inactive estrogen sulfates are also present in this tissue. Quantitative data show that the ‘sulfatase pathway’, which transforms estrogen sulfates into the bioactive unconjugated E 2, is 100–500 times higher than the ‘aromatase pathway’, which converts androgens into estrogens. The treatment of breast cancer patients with anti-aromatases is largely developed with very positive results. However, the formation of E 2 via the ‘sulfatase pathway’ is very important in the breast cancer tissue. In recent years it was found that antiestrogens (e.g. tamoxifen, 4-hydroxytamoxifen), various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, dydrogesterone, norelgestromin), tibolone and its metabolites, as well as other steroidal (e.g. sulfamates) and non-steroidal compounds, are potent sulfatase inhibitors. In another series of studies, it was found that E 2 itself has a strong anti-sulfatase action. This paradoxical effect of E 2 adds a new biological response of this hormone and could be related to estrogen replacement therapy in which it was observed to have either no effect or to decrease breast cancer mortality in postmenopausal women. Interesting information is that high expression of steroid sulfatase mRNA predicts a poor prognosis in patients with +ER. These progestins, as well as tibolone, can also block the conversion of estrone to estradiol by the inhibition of the 17β-hydroxysteroid dehydrogenase type I (17β-HSD-1). High expressison of 17β-HSD-1 can be an indicator of adverse prognosis in ER-positive patients. It was shown that nomegestrol acetate, medrogestone, promegestone or tibolone, could stimulate the sulfotransferase activity for the local production of estrogen sulfates. This is an important point in the physiopathology of this disease, as it is well known that estrogen sulfates are biologically inactive. A possible correlation between this stimulatory effect on sulfotransferase activity and breast cancer cell proliferation is presented. In agreement with all this information, we have proposed the concept of selective estrogen enzyme modulators (SEEM). In conclusion, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity in combination with anti-aromatases can open interesting and new possibilities in clinical applications in breast cancer.

Steroid Sulfatase and Estrogen Sulfotransferase in the Atherosclerotic Human Aorta

Various epidemiological studies have demonstrated a relatively low incidence of cardiovascular events in premenopausal women and its marked increment after menopause. In addition, estrogens have been postulated to exert direct anti-atherogenic effects via binding to estrogen receptors in vascular smooth muscle cells (VSMCs). However, not all postmenopausal women develop atherosclerosis despite decreased levels of serum estrogen. Therefore, we believe it is important to examine the status of estrogen metabolism in situ in the human cardiovascular system. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1) by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of estrogen-dependent human tissues and various sex steroid-dependent tumors. STS and EST, however, have not been studied in detail in the human vascular system associated with atherosclerotic changes. In the present study, we evaluated the relative abundance of STS- and EST-immunoreactive protein and mRNA expression in human aorta using immunohistochemistry and reverse transcription followed by quantitative polymerase chain reaction in addition to enzyme activity. STS expression levels were found to be significantly higher in the VSMCs obtained from female aortas with mild atherosclerotic changes than in those with severe atherosclerotic changes and in male aortas regardless of atherosclerotic changes. EST expression levels in the VSMCs of these aortas, however, were significantly higher in female aortas with severe atherosclerotic changes and in male aortas than in female aortas with mild atherosclerotic changes. We believe it is important to examine factors regulating the expression and activity of these estrogen-metabolizing enzymes in the human aorta. Various cytokines have been proposed to function as regulators of these enzymes in other tissues. In the present study, we studied the effects of interleukin (IL)-1beta, known to be produced in human atherosclerotic lesions, on the expression of these enzymes using cultured human VSMCs originally obtained from a female patient. IL-1beta markedly inhibited the expression of STS mRNA and enzyme activity, but stimulated the expression of EST mRNA and enzyme activity. In addition, IL-1beta also reduced E2 production from E1S and E1 in VSMCs. Results from the present study seem to suggest that the expression levels of both STS and EST mRNA and activity may be significantly associated with the degree of atherosclerotic changes in the female aorta, which may be related to cytokines produced in situ, such as IL-1beta, in human atherosclerotic lesions.

Regulation of estrogen activity in human endometrium: effect of IL-1beta on steroid sulfatase activity in human endometrial stromal cells.

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.