Validation of genetic classifiers derived from mouse and human tumors to identify molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Standard treatment for advanced-stage CRC for decades has included 5-fluorouracil-based chemotherapy. More recently, targeted therapies for metastatic CRC are being used based on the individual cancer's molecular profile. In the past few years, several different molecular subtype schemes for human CRC have been developed. The molecular subtypes can be distinguished by gene expression signatures and have the potential to be used to guide treatment decisions. However, many subtyping classification methods were developed using mRNA expression levels of hundreds to thousands of genes, making them impractical for clinical use. In this study, we assessed whether an immunohistochemical approach could be used for molecular subtyping of CRCs. We validated two previously published, independent sets of immunohistochemistry classifiers and modified the published methods to improve the accuracy of the scoring methods. In addition, we evaluated whether protein and genetic signatures identified originally in the mouse were linked to clinical outcomes of patients with CRC. We found that low DDAH1 or low GAL3ST2 protein levels in human CRCs correlate with poor patient outcomes. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for patients with CRC.

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Development of novel monoclonal antibodies and immunoassays for sensitive and specific detection of SULF1 endosulfatase.

BACKGROUND: Cell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. METHODS: We have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. RESULTS: This study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. CONCLUSIONS: This study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. GENERAL SIGNIFICANCE: New tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application.

Regional Proteomic Quantification of Clinically Relevant Non-Cytochrome P450 Enzymes along the Human Small Intestine.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

HSulf­1 and palbociclib exert synergistic antitumor effects on RB­positive triple­negative breast cancer.

Human sulfatase­1 (HSulf­1) is emerging as a novel prognostic biomarker in breast cancer. Previous studies demonstrated HSulf­1 to function as a negative regulator of cyclin D1 in breast cancer. Accumulating preclinical evidence is supporting the efficacy of cyclin­dependent kinase (CDK) 4/6 inhibitors against the luminal androgen receptor sub­type of triple­negative breast cancer (TNBC). It was therefore hypothesized that HSulf­1 may cooperate with CDK4/6 inhibitors to control cell cycle progression in breast cancer cells. HSulf­1 expression was found to be downregulated in TNBC tissues and cell lines compared with that in healthy tissues and non­breast cancer cell lines, respectively. High levels of HSulf­1 expression was also found to be associated with increased progression­free survival and overall survival in patients with TNBC. Functionally, it was demonstrated that HSulf­1 served as tumor suppressor in TNBC by inducing cell cycle arrest and apoptosis whilst inhibiting proliferation, epithelial­mesenchymal transition, migration and invasion. Subsequent overexpression of HSulf­1 coupled with treatment with the CDK4/6 inhibitor palbociclib exhibited a synergistic antitumor effect on retinoblastoma (RB)­positive TNBC. Further studies revealed the mechanism underlying this cooperative antiproliferative effect involved to be due to the prohibitive effects of HSulf­1 on the palbociclib­induced accumulation of cyclin D1 through AKT/STAT3 and ERK1/2/STAT3 signaling. Taken together, findings from the present study not only suggest that HSulf­1 may be a potential therapeutic target for TNBC, but also indicate that combinatorial treatment could be an alternative therapeutic option for RB­positive TNBC, which may open novel perspectives.

Abnormal expression of chondroitin sulfate sulfotransferases in the articular cartilage of pediatric patients with Kashin-Beck disease.

The objective of this study is to investigate the expression of enzymes involved in the sulfation of articular cartilage from proximal metacarpophalangeal (PMC) joint cartilage and distal metacarpophalangeal (DMC) joint cartilage in children with Kashin-Beck disease (KBD). The finger cartilage samples of PMC and DMC were collected from KBD and normal children aged 5-14 years old. Hematoxylin and eosin staining as well as immunohistochemical staining were used to observe the morphology and quantitate the expression of carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), uronyl 2-O-sulfotransferase (UST), and aggrecan. In the results, the numbers of chondrocyte decreased in all three zones of PMC and DMC in the KBD group. Less positive staining cells for CHST-3, CHST-12, CHST-13, UST, and aggrecan were observed in almost all three zones of PMC and DMC in KBD. The positive staining cell rates of CHST-12 were higher in superficial and middle zones of PMC and DMC in KBD, and a significantly higher rate of CHST-13 was observed only in superficial zone of PMC in KBD. In conclusion, the abnormal expression of chondroitin sulfate sulfotransferases in chondrocytes of KBD children may provide an explanation for the cartilage damage, and provide therapeutic targets for the treatment.

High-throughput method for in process monitoring of 3-O-sulfotransferase catalyzed sulfonation in bioengineered heparin synthesis.

Bioengineered heparin (BEH) offers a potential alternative for the preparation of a safer pharmacological heparin. Construction of in-process control assays for tracking each enzymatic step during bioengineered heparin synthesis remains a challenge. Here, we report a high-throughput sensing platform based on enzyme-linked immunosorbent assay (ELISA) and enzymatic signal amplification that allows the rapid and accurate monitoring of the 3-OST sulfonation in BEH synthesis process. The anticoagulant activity of target BEH was measured to reflect the degree of sulfonation by testing its competitive antithrombin (AT) binding ability. BEH samples with different sulfonation degrees show different AT protein binding capacity and thus changes the UV response to a different extent. This BEH-induced signal can be conveniently and sensitively monitored by the plate sensing system, which benefits from its high sensitivity brought in by the enzymatic signal amplification. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This proposed strategy exhibits excellent analytical performance in both BEH activity analysis and 3-OST sulfonation evaluation. The simple and sensitive plate system shows great potential in developing on-chip, high-throughput methods for fundamental biochemical process research, drug discovery, and clinic diagnostics.

Ontogeny of Hepatic Sulfotransferases and Prediction of Age-Dependent Fractional Contribution of Sulfation in Acetaminophen Metabolism.

Cytosolic sulfotransferases (SULTs), including SULT1A, SULT1B, SULT1E, and SULT2A isoforms, play noteworthy roles in xenobiotic and endobiotic metabolism. We quantified the protein abundances of SULT1A1, SULT1A3, SULT1B1, and SULT2A1 in human liver cytosol samples (n = 194) by liquid chromatography-tandem mass spectrometry proteomics. The data were analyzed for their associations by age, sex, genotype, and ethnicity of the donors. SULT1A1, SULT1B1, and SULT2A1 showed significant age-dependent protein abundance, whereas SULT1A3 was invariable across 0-70 years. The respective mean abundances of SULT1A1, SULT1B1, and SULT2A1 in neonatal samples was 24%, 19%, and 38% of the adult levels. Interestingly, unlike UDP-glucuronosyltransferases and cytochrome P450 enzymes, SULT1A1 and SULT2A1 showed the highest abundance during early childhood (1 to <6 years), which gradually decreased by approx. 40% in adolescents and adults. SULT1A3 and SULT1B1 abundances were significantly lower in African Americans compared with Caucasians. Multiple linear regression analysis further confirmed the association of SULT abundances by age, ethnicity, and genotype. To demonstrate clinical application of the characteristic SULT ontogeny profiles, we developed and validated a proteomics-informed physiologically based pharmacokinetic model of acetaminophen. The latter confirmed the higher fractional contribution of sulfation over glucuronidation in the metabolism of acetaminophen in children. The study thus highlights that the ontogeny-based age-dependent fractional contribution (fm) of individual drug-metabolizing enzymes has better potential in prediction of drug-drug interactions and the effect of genetic polymorphisms in the pediatric population.

New tools for evaluating protein tyrosine sulfation and carbohydrate sulfation.

Sulfation is a common modification of extracelluar glycans and tyrosine residues on proteins, which is important in many signalling pathways and interactions. Existing methods for studying sulfotransferases, the enzymes that catalyse sulfation, are cumbersome and low-throughput. Recent studies published in the Biochemical Journal have repurposed established biochemical assays from the kinase field and applied them to the characterisation of sulfotransferases. Biochemical screening of a library of kinase inhibitors revealed that compounds that target RAF kinases may also be repurposed to inhibit sulfotransferases. Together with the available structures of sulfotransferases, these studies open the door to the development of chemical tools to probe the biological functions of these important enzymes.

Dermatan sulfate epimerase 1 and dermatan 4-O-sulfotransferase 1 form complexes that generate long epimerized 4-O-sulfated blocks.

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane.

BACKGROUND: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. METHODS: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. RESULTS: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. CONCLUSIONS: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. GENERAL SIGNIFICANCE: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

Gender difference in NASH susceptibility: Roles of hepatocyte Ikkß and Sult1e1.

Myeloid cell and hepatocyte IKKß may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH) are not known. Here we demonstrate myeloid IKKß deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKß deficiency (IkbkbΔhep) in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1), which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKß of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKß is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients.

Sulfatase-2: a prognostic biomarker and candidate therapeutic target in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of cancer death in the UK. Its poor prognosis is attributed to late detection and limited therapeutic options. Expression of SULF2, an endosulfatase that modulates heparan sulfate proteoglycan 6-O-sulfation and is reportedly tumourigenic in different types of cancer, was investigated. METHODS: SULF2 expression was determined immunohistochemically in archival surgical resection tissue sections from 93 patients with a confirmed histological diagnosis of PDAC between 2002 and 2008 followed for a median of 9 years. Relationships with clinico-pathological parameters and patient survival were explored. RESULTS: The majority of PDACs showed positive SULF2 staining in tumour cells and intratumoural or tumour-adjacent stroma. Greater than 25% SULF2-positive tumour cells was present in 60% of cancers and correlated with tumour stage (P=0.002) and perineural invasion (P=0.024). SULF2 intensity was scored moderate or strong in 81% of cancers and positively correlated with vascular invasion (P=0.015). High SULF2 expression, defined as >50% SULF2-positive tumour cells and strong SULF2 staining, was associated with shorter time to radiological progression (P=0.018, HR 1.98, CI 1.13-3.47). Similarly, by multivariate analysis, high SULF2 expression was independently associated with poorer survival (P=0.004, HR 2.10, CI 1.26-3.54), with a median survival of 11 months vs 21 months for lower PDAC SULF2. CONCLUSIONS: Elevated SULF2 in PDAC was associated with advanced tumour stage, vascular invasion, shorter interval to radiological progression and shorter overall survival. SULF2 may have roles as a prognostic biomarker and as a therapeutic target for patients with PDAC.

Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck.

Sulfatase 2 (SULF2), an extracellular sulfatase that alters sulfation on heparan sulfate proteoglycans, is involved in the tumorigenesis and progression of several carcinomas. SULF2 expression has not been evaluated in squamous cell carcinoma of the head and neck (HNSCC). Here we report results of IHC of SULF2 expression in HNSCC tissue. SULF2 was detected in 57% of tumors (n = 40) with a significant increase in intensity and number of stained cells compared to adjacent cancer-free tissue (p-value < 0.01), increasing with cancer stage when comparing stages 1 and 2 to stages 3 and 4 (p-value 0.01). SULF2 was not detected in epithelial cells of cancer-free controls, and expression was independent of patient demographics, tumor location and etiological factors, smoking and HPV infection by p16 IHC analysis. Sandwich ELISA was performed on serum of HNSCC patients (n = 28) and controls (n = 35), and although SULF2 was detectable, no change was observed in HNSCC. Saliva, collected by mouthwash, from HNSCC patients (n = 8) and controls (n = 8) was also tested by ELISA in a preliminary investigation and an increase in SULF2 was observed in HNSCC (p-value 0.041). Overall, this study shows that SULF2 is increased in HNSCC independent of tissue location (oral cavity, oropharynx, larynx and hypopharynx), patient demographics and etiology. Although no change in SULF2 was detected in HNSCC serum, its detection in saliva makes it worthy of further investigation as a potential HNSCC biomarker.

Dexamethasone suppresses the growth of human non-small cell lung cancer via inducing estrogen sulfotransferase and inactivating estrogen.

AIM: Dexamethasone (DEX) is a widely used synthetic glucocorticoid, which has shown anti-cancer efficacy and anti-estrogenic activity. In this study we explored the possibility that DEX might be used as an endocrine therapeutic agent to treat human non-small cell lung cancer (NSCLC). METHODS: The viability and proliferation of human NSCLC cell lines A549 and H1299 were assessed in vitro. Anti-tumor action was also evaluated in A549 xenograft nude mice treated with DEX (2 or 4 mg·kg(-1)·d(-1), ig) or the positive control tamoxifen (50 mg·kg(-1)·d(-1), ig) for 32 d. The expression of estrogen sulfotransferase (EST) in tumor cells and tissues was examined. The intratumoral estrogen levels and uterine estrogen responses were measured. RESULTS: DEX displayed mild cytotoxicity to the NSCLC cells (IC50 >500 µmol/L) compared to tamoxifen (IC50 <50 µmol/L), but it was able to inhibit the cell proliferation at low micromolar ranges. Furthermore, DEX (0.1-10 µmol/L) dose-dependently up-regulated EST expression in the cells, and inhibited the cell migration in vitro. Triclosan, a sulfation inhibitor, was able to diminish DEX-caused inhibition on the cell viability. In A549 xenograft nude mice, DEX or tamoxifen administration remarkably suppressed the tumor growth. Moreover, DEX administration dose-dependently increased EST expression in tumor tissues, and reduced intratumoral estrogen levels as well as the volumes and weights of uterine. CONCLUSION: DEX suppresses the growth of A549 xenograft tumors via inducing EST and decreasing estradiol levels in tumor tissues, suggesting that DEX may be used as anti-estrogenic agent for the treatment of NSCLC.

SULF1/SULF2 reactivation during liver damage and tumour growth.

Both SULF1 and SULF2 enzymes are undetectable in normal adult liver tissue despite their high level expression during foetal development. Most hepatocellular carcinomas unlike the normal adult liver, however, express variable levels of these enzymes with a small proportion not expressing either SULF1 or SULF2. SULF1 expression, however, is not restricted to only foetal and tumour tissues but is also abundant in liver tissues undergoing injury-induced tissue regeneration as we observed during fatty liver degeneration, chronic hepatitis and cirrhosis. Unlike SULF1, the level of SULF2 activation during injury-induced regeneration, however, is much lower when compared to foetal or tumour growth. Although a small fraction of liver tumours and some liver tumour cell lines can grow in the absence of Sulf1 and/or Sulf2, the in vitro overexpression of these genes further confirms their growth-promoting effect while transient reduction in their levels by neutralisation antibodies reduces growth. Hedgehog signalling appeared to regulate the growth of both Hep3B and PRF5 cell lines since cyclopamine demonstrated a marked inhibitory effect while sonic hedgehog (SHH) overexpression promoted growth. All Sulf isoforms promoted SHH-induced growth although the level of increase in PRF5 cell line was higher with both Sulf2 variants than Sulf1. In addition to promoting growth, the Sulf variants, particularly the shorter Sulf2 variant, markedly promoted PRF5 cell migration in a scratch assay. The SULF1/SULF2 activation thus does not only promote regulated foetal growth and injury-induced liver regeneration but also dysregulated tumour growth.

Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography-tandem mass spectrometry.

Sulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3→97.0) and cortisol (m/z 407.2→331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2pmol DHEA-S and the calibration curve was linear from 0.2 to 200pmol. The intra-day and inter-day accuracy and precision was <11.7%. DHEA-S in the quality control samples was stable at room temperature, 4°C, and -20°C. The cytosolic matrix (20-100µg cytosolic protein) did not affect DHEA-S quantification. Our UPLC-MS/MS method was applied to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. The optimal levels of MgCl2 and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) cofactor were 2.5mM and 20µM, respectively. Reducing agents, including 2-mercaptoethanol and DL-dithiothreitol, did not affect the enzyme activity. A linear relationship existed between DHEA sulfation and amount of human liver cytosol (20-200µg cytosolic protein) or incubation time (5-30min). This UPLC-MS/MS approach is safer, easier, and faster than existing radiometric-based sulfotransferase enzyme assays, and it is the first UPLC-MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.

A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase.

Metachromatic leukodystrophy (MLD) is a rare and severe genetic disease. Inhibition of cerebroside sulfotransferase (CST) has been proposed as a promising new therapeutic strategy for the treatment of MLD. CST catalyzes the transfer of a sulfate group from the coenzyme 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulfate and adenosine-3',5'-diphosphate (PAP). So far only a few weak CST inhibitors have been described. The goal of the present study was to establish a suitable assay for identifying and characterizing novel CST inhibitors. To this end, we developed and optimized a capillary electrophoresis (CE) based assay for monitoring the catalytic activity of CST by measuring the formation of PAP. A sample matrix consisting of 5mM phosphate buffer with about 0.0001% polybrene at pH 7.4 and a background electrolyte (BGE) containing 75 mM phosphate buffer with 0.002% polybrene at pH 5.6 were utilized to achieve a stacking effect for PAP by dynamic pH junction. This led to a limit of detection for the enzymatic product PAP of 66.6 nM. The CE method was sensitive, robust, and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Selected reference compounds were tested in order to validate the assay, including the substrates cerebroside and psychosine, and the inhibitor Congo Red. The newly developed CE method will be useful for the identification and development of novel CST inhibitors which are urgently needed for the treatment of MLD.

Intratumoral estrogen sulfotransferase induction contributes to the anti-breast cancer effects of the dithiocarbamate derivative TM208.

AIM: Sulfotransferase-catalyzed sulfation is the most important pathway for inactivating estrogens. Thus, activation of estrogen sulfotransferase (EST) may be an alternative approach for the treatment of estrogen-dependent breast cancer. In this study we investigated the involvement of EST in anti-breast cancer effects of the dithiocarbamate derivative TM208 in vitro and in vivo. METHODS: The viability of human breast cancer MCF-7 cells was determined using a SBB assay. Nude mice bearing MCF-7 cells were orally administered TM208 (50 and 150 mg·kg(-1)·d(-1)) for 18 days. The xenograft tumors and uteri were collected. The mRNA expression of EST was examined with real-time PCR. EST protein was detected with Western blot, ELISA or immunohistochemical staining assays. A radioactive assay was used to measure the EST activity. Uterotropic bioassay was used to examine the uterine estrogen responses. RESULTS: Treatment with TM208 (10, 15 and 20 µmol/L) concentration-dependently increased EST expression in MCF-7 cells in vitro. Co-treatment with triclosan, an inhibitor of sulfonation, abolished TM208-induced cytotoxicity in MCF-7 cells. TM208 exhibited an apparent anti-estrogenic property: it exerted more potent cytotoxicity in E2-treated MCF-7 cells. In the nude mice bearing MCF-7 cells, TM208 administration time-dependently increased the expression and activity of EST, and blocked the gradual increase of E2 concentration in the xenograft tumors. Furthermore, TM208 administration blocked the estrogens-stimulated uterine enlargement. Tamoxifen, a positive control drug, produced similar effects on the expression and activity of EST in vitro and in vivo. CONCLUSION: The induction of EST and reduction of estrogen concentration contribute to the anti-breast cancer action of TM208 and tamoxifen. TM208 may be developed as anticancer drug for the treatment of estrogen receptor-positive breast cancer.

Increased sulfatase 1 gene expression in degenerative intervertebral disc cells.

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non-degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non-degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration-specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time- and dose-dependent manner in response to TNF-α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non-degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration.