Regulations of expressions of rat/human sulfotransferases by anticancer drug, nolatrexed, and micronutrients.

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; ß-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 µM) were applied for 10 days. Folic acid (0-10 µM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P < 0.05/mRNA, AST-IV, F = 6.702, P < 0.014; Student's t test, P < 0.01-0.05) and HepG2 cells. Folic acid increased sulfotransferases activity/protein in gender-dependant manner. Both folic and folinic acid increased several human sulfotransferases isoforms with varied level of significance (least or no increase at highest dose) in HepG2 cells pointing its dose-dependent multiphasic responses. The clinical importance of this study may be furthered in the verification of sulfation metabolism of several exogenous/endogenous molecules, drug-drug interaction and their influences on cancer pathophysiological processes. Further studies are necessary.

Oxidant stress induction and signalling in xenografted (human breast cancer-tissues) plus estradiol treated or N-ethyl-N-nitrosourea treated female rats via altered estrogen sulfotransferase (rSULT1E1) expressions and SOD1/catalase regulations.

N-ethyl-N-nitrosourea (ENU) is highly used in rodent models of tumerogenesis/carcinogenesis. Xenografting human-cancer tissues/cells with estradiol (E2) treatment is also used to generate rodent-models of gynaecological cancers. The altered metabolic-redox environment leading to establishment of pre-tumorigenesis condition and their mechanism are less studied. Here, female Wister rats were treated with these drugs at their pre-tumerogenic dosage (one group ENU single intra-peritoneal dose of 90 mg/kg b.w. and another group were implanted with human breast tumor (stage-IIIB) and fed with 2.5 mg of 17ß-estradiol once in a week for 4 months). After 4 months, animals were sacrificed; their serum and liver tissues were tested. A brief comparison was made with a rat model (regarded as positive control) of toxicity induced by mutagenic environmental pollutant arsenic (0.6 ppm daily/4 weeks). The increase in serum alkaline phosphatase and glutamate-pyruvate transaminase suggests the possible organ toxicity is favoured by the increase in hepatic/systemic free radicals and oxidative stress in all drug application models. But the increase in the serum E2 level as noted in the ELISA data with impairment in the hepatic estrogen sulfotransferase (SULT1E1) protein expression (immuno-blot data) were noticed with interfered hepatic free-thiols only in ENU and xenograft-E2 group compared to arsenic group. It is also evident in the in vitro result from E2/GSH/NAC added hepatic slices with altered antioxidant regulations. Moreover, impairment in hepatic SOD1, catalase and glutathiole peroxidase activities (PAGEzymographic data), especially in the ENU-treated group makes them more vulnerable to the oxidative threat in creating pre-tumerogenic microenvironment. This is evident in the result of their higher DNA-damage and histological abnormalities. The Bioinformatics study revealed an important role of rSULT1E1 in the regulations of E2 metabolism. This study is important for the exploration of the pre-tumerogenic condition by ENU and E2 by impairing SULT1E1 expression and E2 regulations via oxidant-stress signalling. The finding may help to find new therapeutic-targets to treat gynaecological-cancers more effectively.

Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Intermediates of the Cholesterol Biosynthetic Pathway in Primary Cultured Rat Hepatocytes.

Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 µM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 µM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1ß. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.

In vitro-assessment of putative antiprogestin activities of phytochemicals and synthetic UV absorbers in human endometrial Ishikawa cells.

Critical steps of embryo implantation are controlled by progesterone. These processes can be interrupted by progesterone receptor (PR) antagonists, e.g. drugs used for abortion. Antiprogestin effects induced by natural compounds and environmental chemicals have been rarely addressed. In our in vitro study, we investigated putative antiprogestin activities of the plant compounds apigenin (API) and trans-ferulic acid (t-FA) as well as the UV absorbers octyl methoxycinnamate (OMC) and 4-methylbenzylidene camphor (4-MBC). They were compared with the selective progesterone receptor modulators (SPRMs) mifepristone (RU486) and ulipristal acetate (UPA) as well as the full PR-antagonist ZK137316. Effects of test compounds in combination with progesterone on the progesterone-sensitive target gene estrogen sulfotransferase (SULT1E1) were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The agonistic effect of progesterone on SULT1E1 mRNA levels was concentration-dependently antagonized by RU486, UPA and ZK137316 as well as, with lower potency, apigenin. t-FA, OMC and 4-MBC had no effect on SULT1E1 mRNA levels. We demonstrated that apigenin, although at higher concentrations, exerts a similar effect as the well-characterized SPRMs RU486 and UPA or the progesterone antagonist ZK137316 in this model. Our endometrium-specific Ishikawa cell assay is a useful complement to artificial transactivation assays for the identification of environmental substances with antiprogestin activities.

Epigenetics: methylation-associated repression of heparan sulfate 3-O-sulfotransferase gene expression contributes to the invasive phenotype of H-EMC-SS chondrosarcoma cells.

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H-HEMC-SS chondrosarcoma cells showed a typical hypermethylation profile of 3-OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA-methyltransferase inhibitor, up-regulated expression of 3-OST1, 3-OST2, and 3-OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5-Aza-dc treatment or reintroduction of 3-OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion-exchange and gel-filtration chromatography. Notably, 5-Aza-dc treatment of HEMC cells or expression of 3-OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3-OST genes resulting in altered HSPG sulfation and point to a defect of HS-3-O-sulfation as a factor in cancer progression.

Curcumin suppresses growth of mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis.

Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although, platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis pathways are frequently altered in MPM and thus remain attractive targets for intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. In this study, we investigated the biological and molecular responses of MPM cells to curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose- and time-dependent manner while pretreatment of MPM cells with curcumin enhanced cisplatin efficacy. Curcumin activated the stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in effective management of resistant MPM.

Why does oxamniquine kill Schistosoma mansoni and not S. haematobium and S. japonicum?

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.

Molecular basis for hycanthone drug action in schistosome parasites.

Hycanthone (HYC) is a retired drug formerly used to treat schistosomiasis caused by infection from Schistosoma mansoni and S. haematobium. Resistance to HYC was first observed in S. mansoni laboratory strains and in patients in the 1970s and the use of this drug was subsequently discontinued with the substitution of praziquantel (PZQ) as the single antischistosomal drug in the worldwide formulary. In endemic regions, multiple organizations have partnered with the World Health Organization to deliver PZQ for morbidity control and prevention. While the monotherapy reduces the disease burden, additional drugs are needed to use in combination with PZQ to stay ahead of potential drug resistance. HYC will not be reintroduced into the schistosomiasis drug formulary as a combination drug because it was shown to have adverse properties including mutagenic, teratogenic and carcinogenic activities. Oxamniquine (OXA) was used to treat S. mansoni infection in Brazil during the brief period of HYC use, until the 1990s. Its antischistosomal efficacy has been shown to work through the same mechanism as HYC and it does not possess the undesirable properties linked to HYC. OXA demonstrates cross-resistance in Schistosoma strains with HYC resistance and both are prodrugs requiring metabolic activation in the worm to toxic sulfated forms. The target activating enzyme has been identified as a sulfotransferase enzyme and is currently used as the basis for a structure-guided drug design program. Here, we characterize the sulfotransferases from S. mansoni and S. haematobium in complexes with HYC to compare and contrast with OXA-bound sulfotransferase crystal structures. Although HYC is discontinued for antischistosomal treatment, it can serve as a resource for design of derivative compounds without contraindication.

Effects of endocrine disruptors on dehydroepiandrosterone sulfotransferase and enzymes involved in PAPS synthesis: genomic and nongenomic pathways.

BACKGROUND: Sulfation plays an important role both in detoxification and in the control of steroid activity. Studies in rodents have shown that the conversion of dehydroepiandrosterone (DHEA) to DHEA-sulfate is involved in learning and the memory process. METHODS: The effects of a range of plasticizers and related compounds commonly encountered in the environment were evaluated kinetically against human DHEA sulfotransferase (SULT 2A1) and by reverse transcriptase-polymerase chain reaction (RT-PCR) against several enzymes involved in the synthesis of the sulfotransferase cofactor adenosine 3'-phosphate 5'-phosphosulfate (PAPS). RESULTS: We found that several of the chemicals acted as competitive inhibitors of SULT 2A1 (K(i) for 4-tert-octylphenol is 2.8 microM). Additionally, after treatment of TE 671 cells with 0.005-0.5 microM 4-n-octylphenol, bis(2-ethylhexyl)phthalate, and diisodecyl phthalate, real-time RT-PCR showed dose-dependent decreases in the steady-state mRNA levels of cysteine dioxygenase type I, sulfite oxidase, and 3'-phosphate 5'-phosphosulfate synthase I. CONCLUSIONS: These data suggest that environmental contaminants may exert effects on neuronal function both by direct inhibition of sulfotransferase enzymes and by interrupting the supply of PAPS, which has wider implications for endocrine disruption and xenobiotic metabolism.

A role for sulfation-desulfation in the uptake of bisphenol a into breast tumor cells.

Bisphenol A (BPA) is a widely used plasticizer whose estrogenic properties may impact hormone-responsive disorders and fetal development. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. This may be a result of BPA sulfation/desulfation providing a pathway for selective uptake into hormone-responsive cells. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Treatment of MCF-7 cells with BPAS leads to desulfation and uptake of BPA. No BPAS is found inside the cells. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. Therefore, sulfation may increase the estrogenic potential of xenobiotics.

Induction of human sulfotransferase 1A3 (SULT1A3) by glucocorticoids.

Sulfotransferases (SULTs) play an important role in the detoxification and bioactivation of endogenous compounds and xenobiotics. Studies on rat sulfotransferases had shown that SULT genes, like cytochrome P450 genes, can be regulated by ligands that bind nuclear receptors. For human SULT genes, the regulation of human SULT2A1 expression is currently the best characterized. In this study, we systematically examined the regulation of human SULT1A genes by glucocorticoids. Treatment of the human hepatocellular carcinoma derived HepG2 cells with 10(-7) M dexamethasone did not affect the SULT1A1 activity toward p-nitrophenol. In contrast, SULT1A3 activity toward dopamine was significantly induced. Transient transfection of the SULT1A3 5'-flanking region/luciferase reporter construct showed that SULT1A3 was responsive to dexamethasone and prednisolone in a concentration-dependent manner with maximal induction at 10(-7) M dexamethasone or 1 microM prednisolone. In addition, induction by dexamethasone was dependent on the level of expression of the glucocorticoid receptor. Analysis of the 5'-flanking region led to the identification of a putative glucocorticoid response element at position (-1211 to -1193) upstream of the transcription start site and deletion or mutation of this element resulted in a loss of response. In summary, the data from this study shows that the human SULT1A3 gene is inducible by glucocorticoids through a glucocorticoid receptor-mediated mechanism and the glucocorticoid response element at position (-1211 to -1193) is necessary for this induction.

Sulfonation of 17beta-estradiol and inhibition of sulfotransferase activity by polychlorobiphenylols and celecoxib in channel catfish, Ictalurus punctatus.

The sulfonation of 17beta-estradiol (E2) by human liver and recombinant sulfotransferases is influenced by environmental contaminants such as hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs), which are potent inhibitors, and the therapeutic drug, celecoxib, which affects positional sulfonation of E2. In some locations, the aquatic environment is contaminated by PCBs, OH-PCBs and widely used therapeutic drugs. The objectives of this study were to investigate the sulfonation kinetics of E2 in liver cytosol from channel catfish (Ictalurus punctatus); to examine the effect of OH-PCBs on E2 sulfonation; and to determine if celecoxib altered the position of E2 sulfonation, as it does with human liver cytosol. E2 was converted to both 3- and 17-sulfates by catfish liver cytosol. At E2 concentrations below 1 microM, formation of E2-3-sulfate (E2-3-S) predominated, but substrate inhibition was observed at higher concentrations. Rates of E2-3-S formation at different E2 concentrations were fit to a substrate inhibition model, with K'm and V'max values of 0.40 +/- 0.10 microM and 91.0 +/- 4.7 pmol/min/mg protein, respectively and K(i) of 1.08 +/- 0.09 microM. The formation of E2-17-S fit Michaelis-Menten kinetics over the concentration range 25 nM to 2.5 microM, with K(m) and V(max) values of 1.07 +/- 0.23 microM and 25.7 +/- 4.43 pmol/min/mg protein, respectively. The efficiency (V(max)/K(m)) of formation of E2-3-S was 9.8-fold higher than that of E2-17-S. Several OH-PCBs inhibited E2 3-sulfonation, measured at an E2 concentration of 1 nM. Of those tested, the most potent inhibitor was 4'-OH-CB79, with two chlorine atoms flanking the OH group (IC(50): 94 nM). The inhibition of estrogen sulfonation by OH-PCBs may disrupt the endocrine system and thus contribute to the known toxic effects of these compounds. Celecoxib did not stimulate E2-17-S formation, as is the case with human liver cytosol, but did inhibit the formation of E2-3-S (IC(50): 44 microM) and to a lesser extent, E2-17-S (IC(50): > 160 microM), suggesting the previously found effect of celecoxib on E2-17-S formation may be specific to human SULT2A1.

Gene expression and proliferation biomarkers for antidepressant treatment resistance.

The neurotrophic hypothesis of depression suggests an association between effects on neuroplasticity and clinical response to antidepressant drug therapy. We studied individual variability in antidepressant drug effects on cell proliferation in lymphoblastoid cell lines (LCLs) from n=25 therapy-resistant patients versus n=25 first-line therapy responders from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. Furthermore, the variability in gene expression of genes associated with cell proliferation was analyzed for tentative candidate genes for prediction of individual LCL donor's treatment response. Cell proliferation was quantified by EdU (5-ethynyl-2'-deoxyuridine) assays after 21-day incubation of LCLs with fluoxetine (0.5 ng µl-1) and citalopram (0.3 ng µl-1) as developed and described earlier. Gene expression of a panel of candidate genes derived from genome-wide expression analyses of antidepressant effects on cell proliferation of LCLs from the Munich Antidepressant Response Signature (MARS) study was analyzed by real-time PCR. Significant differences in in vitro cell proliferation effects were detected between the group of LCLs from first-line therapy responders and LCLs from treatment-resistant patients. Gene expression analysis of the candidate gene panel revealed and confirmed influence of the candidate genes ABCB1, FZD7 and WNT2B on antidepressant drug resistance. The potential of these genes as tentative biomarkers for antidepressant drug resistance was confirmed. In vitro cell proliferation testing may serve as functional biomarker for individual neuroplasticity effects of antidepressants.

Sulfation of tibolone metabolites by human postmenopausal liver and small intestinal sulfotransferases (SULTs).

Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3alpha-hydroxytibolone (3alpha-OH-tibolone) and 3beta-hydroxytibolone (3beta-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n=5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 +/- 0.3 units/mg protein, n = 8, mean +/- SEM), and activities with 3alpha-OH-tibolone (0.6 +/- 0.1, n = 8) and 3beta-OH-tibolone (0.9 +/- 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 +/- 0.01 with 3alpha-OH-tibolone and 0.04 +/- 0.01 with 3beta-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.

Steroid levels and steroid metabolism in the mussel Mytilus edulis: the modulating effect of dispersed crude oil and alkylphenols.

Significant amounts of oil and alkylphenols are released into the sea by petroleum installations as a result of discharges of produced water. Some of these pollutants elicit estrogenic responses in fish, but their effects on the endocrine system of molluscs are largely unknown. In this study, mussels Mytilus edulis were exposed to North Sea oil (O) and the mixture of North Sea oil+alkylphenols (OAP), and the effects on tissue steroid levels and steroid metabolism (P450-aromatase and estradiol-sulfotransferase) were monitored. Levels of free testosterone and free estradiol were much higher in gonad tissue than in peripheral tissue, whereas esterified steroids (released after saponification) were of the same order of magnitude in both tissues. Levels of free steroids determined in gonads were not affected by exposure, but esterified steroids significantly increased in OAP exposed mussels (up to 2.4-fold). The sulfation of estradiol was investigated as a conjugation pathway, and increased activities were observed in digestive gland cytosol of both O and OAP exposure groups (up to 2.8-fold). Additionally, increased P450-aromatase activity was determined in OAP exposed mussels (up to three-fold, both in gonad and digestive gland), but not in the O group. Altogether, the results indicate that North Sea oil leads to increased sulfation of estradiol, and that in combination with alkylphenols, additional alterations are observed: increased P450-aromatase, and increased levels of esterified-steroids in gonads. Nonetheless, mussels are able to maintain gonad concentrations of free steroids unaltered, possibly via homeostatic mechanisms such as the conjugation with fatty acid or the formation of sulphate conjugates.

Effects of detergents on the sulfation of chondroitin sulfate by sulfotransferase from chicken embryo epiphyseal cartilage.

Homogenates of chicken embryo epiphyseal cartilage were prepared in buffered saline. The bulk of the sulfotransferase was found in the supernatant. However, small amounts of sulfotransferase were consistently found in the particulate fraction. Detergents (Triton X-100 and C12E8) added to the incubation mixture activated the sulfation of exogenous sulfate acceptor by the particulate fraction, whereas detergent treatment during homogenization increased sulfotransferase activity in the supernatant at the expense of that in the particulate fraction. Since sulfotransferase activities of the supernatant and particulate fractions had similar properties concerning specificity, affinity for chondroitin with different degrees of sulfation, thermal stability and activation by protamine, we conclude that the same enzyme is present in both fractions and that detergent activates indirectly, by releasing it to the medium.

Sulfotransferase 2A1 forms estradiol-17-sulfate and celecoxib switches the dominant product from estradiol-3-sulfate to estradiol-17-sulfate.

Using recombinant sulfotransferases (SULTs) expressed in E. coli, beta-estradiol (E2) sulfonation was examined to determine which SULT enzyme is responsible for producing E2-17-sulfate (E2-17-S). SULTs 1A1*1, 1A1*2, 1A3, 1E1 and 2A1 all sulfated E2 to varying extents. No activity was observed with SULT1B1. Among the SULTs studied, SULT2A1 produced primarily E2-3-sulfate (E2-3-S), but also some E2-17-S and trace amounts of E2 disulfate. SULT2A1 had a K(m) value of 1.52 microM for formation of E2-3-S and 2.95 microM for formation of E2-17-S. SULT2A1 had the highest V(max) of 493 pmol/min/mg protein for formation of E2-3-S, which was 8.8- and 47-fold higher than the maximal rates of formation of E2-17-S and E2 disulfate, respectively. SULT2A1 formed E2-3-S more efficiently. However, when celecoxib (0-160 microM) was included in the incubation with either SULT2A1 or human liver cytosol, sulfonation switched from E2-3-S to E2-17-S in a concentration-dependent manner. The ratio of E2-17-S/E2-3-S went up to 15 with SULT2A1, and was saturated at 1 with human liver cytosol. In both cases, more E2-17-S was formed, with the unreacted E2 remained unchanged, suggesting celecoxib probably bound to a separate effector site to cause a conformational change in SULT2A1, which favored production of E2-17-S. The ability of celecoxib to alter the position of sulfonation of E2 may in part explain its success in the experimental prevention and treatment of breast cancer.

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells.

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.

Biochemistry and reproductive endocrinology of estrogen sulfotransferase.

Estrogen sulfotransferase is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. Significant progress has been made in the last few years regarding the structure, substrate specificity, tissue expression, and regulation of mammalian estrogen sulfotransferases. The enzyme has high affinity for estrogens and is expressed in a number of estrogen target tissues, including the male and female reproductive systems. Expression of the enzyme in the testis has been particularly well characterized. In the testis, estrogen sulfotransferase is localized selectively to Leydig cells and its expression in these cells is dependent on LH and androgen. It was concluded, from both in vitro and in vivo studies, that estrogen sulfotransferase can function as an effective modulator of local estrogen activity in target tissues. The finding that certain hydroxylated polychlorinated biphenyls are potent inhibitors of the human estrogen sulfotransferase enzyme raises the possibility that environmental chemicals can cause endocrine disruption by enhancing endogenous estrogen activity through inhibition of steroid transformation enzymes such as estrogen sulfotransferase. This provides a new paradigm in explaining the endocrine disrupting potential of environmental chemicals that have low or no binding affinities for steroid hormone receptors.

Effect of nomegestrol acetate on human estrogen sulfotransferase activity in the hormone-dependent MCF-7 and T-47D breast cancer cell lines.

Breast cancer cells possess all the enzymes involved in the last steps of estradiol (E2) bioformation, as well as in its transformation (e.g. sulfotransferases) for the conversion to estrogen sulfates (ES). As ES are biologically inactive, the formation of these conjugates is an important transformation pathway in the control of the hormone. In the present study, we explored the effect of nomegestrol acetate on the sulfotransferase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cells. After 24-h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5 x 10(-9) mol/l), it was observed that the sulfotransferase activity was present in both cell lines, since the concentrations of estrogen sulfates found were 9.40 +/- 1.10 in MCF-7 cells and 6.65 +/- 0.72 in the T-47D cells. The presence of ES was found exclusively in the culture medium, which suggests that as soon as the sulfate is biosynthesized it is secreted into the medium. Nomegestrol acetate has a stimulatory effect on sulfotransferase activity: at low doses (5 x 10(-8) and 5 x 10(-7) mol/l) this compound strongly increases the activity of this enzyme by 60.6% and 83%, respectively, in the MCF-7 cells and by 69.2% at 5 x 10(-7) mol/l in T-47D cells. At a high concentration (5 x 10(-5) mol/l) the stimulatory effect of nomegestrol acetate on the sulfotransferase activity was only 5.4% and 6.1%, respectively, in MCF-7 and T-47D cells. In conclusion, the stimulation provoked at low doses by nomegestrol acetate on the estrogen sulfotransferase activity involved in the biosynthesis of the biologically inactive estrogen sulfates in hormone-dependent breast cancer cells is an important effect of this progestin and can open attractive clinical applications.