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1.
Phytomedicine ; 79: 153357, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33011631

RESUMO

BACKGROUND: Actaea racemosa L., also known as black cohosh, is a popular herb commonly used for the treatment of menopausal symptoms. Because of its purported estrogenic activity, black cohosh root extract (BCE) may trigger breast cancer growth. STUDY DESIGN/METHODS: The potential effects of standardized BCE and its main constituent actein on cellular growth rates and steroid hormone metabolism were investigated in estrogen receptor alpha positive (ERα+) MCF-7 and -negative (ERα-) MDA-MB-231 human breast cancer cells. Cell numbers were determined following incubation of both cell lines with the steroid hormone precursors dehydroepiandrosterone (DHEA) and estrone (E1) for 48 h, in the presence and absence of BCE or actein. Using a validated liquid chromatography-high resolution mass spectrometry assay, cell culture supernatants were simultaneously analyzed for the ten main steroids of the estrogen pathway. RESULTS: Inhibition of MCF-7 and MDA-MB-231 cell growth (up to 36.9%) was observed following treatment with BCE (1-25 µg/ml) or actein (1-50 µM). Incubation of MCF-7, but not of MDA-MB-231 cells, with DHEA and BCE caused a 20.9% reduction in DHEA-3-O-sulfate (DHEA-S) formation, leading to a concomitant increase in the androgens 4-androstene-3,17-dione (AD) and testosterone (T). Actein was shown to exert an even stronger inhibitory effect on DHEA-S formation in MCF-7 cells (up to 89.6%) and consequently resulted in 12- to 15-fold higher androgen levels compared with BCE. The formation of 17ß-estradiol (E2) and its glucuronidated and sulfated metabolites was not affected by BCE or actein after incubation with the estrogen precursor estrone (E1) in either cell line. CONCLUSIONS: The results of the present study demonstrated that actein and BCE do not promote breast cancer cell growth or influence estrogen levels. However, androgen formation was strongly stimulated by BCE and actein, which may contribute to their ameliorating effects on menopausal symptoms in women. Future studies monitoring the levels of AD and T upon BCE supplementation of patients are warranted to verify an association between BCE and endogenous androgen metabolism.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/metabolismo , Cimicifuga/química , Extratos Vegetais/farmacologia , Esteroides/metabolismo , Androgênios/metabolismo , Antineoplásicos Fitogênicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Extratos Vegetais/química , Raízes de Plantas/química , Saponinas/farmacologia , Sulfotransferases/metabolismo , Triterpenos/farmacologia
2.
Sci Rep ; 10(1): 12565, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724080

RESUMO

Current strategies to treat tuberculosis (TB) and co-morbidities involve multidrug combination therapies. Rifamycin antibiotics are a key component of TB therapy and a common source of drug-drug interactions (DDIs) due to induction of drug metabolizing enzymes (DMEs). Management of rifamycin DDIs are complex, particularly in patients with co-morbidities, and differences in DDI potential between rifamycin antibiotics are not well established. DME profiles induced in response to tuberculosis antibiotics (rifampin, rifabutin and rifapentine) were compared in primary human hepatocytes. We identified rifamycin induced DMEs, cytochrome P450 (CYP) 2C8/3A4/3A5, SULT2A, and UGT1A4/1A5 and predicted lower DDIs of rifapentine with 58 clinical drugs used to treat co-morbidities in TB patients. Transcriptional networks and upstream regulator analyses showed FOXA3, HNF4α, NR1I2, NR1I3, NR3C1 and RXRα as key transcriptional regulators of rifamycin induced DMEs. Our study findings are an important resource to design effective medication regimens to treat common co-conditions in TB patients.


Assuntos
Antituberculosos/efeitos adversos , Hepatócitos/efeitos dos fármacos , Rifamicinas/efeitos adversos , Antituberculosos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Hepatócitos/enzimologia , Humanos , Rifamicinas/farmacologia , Sulfotransferases/genética , Sulfotransferases/metabolismo , Tuberculose/tratamento farmacológico
3.
J Med Chem ; 63(15): 8059-8068, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32643929

RESUMO

Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis. Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been documented, even along with death cases resulting from acute liver failure. The present study aimed at the investigation of metabolic activation of pirfenidone possibly participating in the reported adverse reactions. Pirfenidone-derived glutathione/N-acetylcysteine (GSH/NAC) conjugates were detected in microsomal/primary hepatocyte incubations after exposure to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats given pirfenidone, respectively. The observation of the conjugates suggests the formation of a quinone methide intermediate derived from pirfenidone. The intermediate was possibly generated through two pathways. First, pirfenidone was directly metabolized to the quinone methide intermediate via dehydrogenation; second, pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal chemists to minimize toxicities in the development of new pharmaceutical agents.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Piridonas/metabolismo , Sulfotransferases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Sistema Enzimático do Citocromo P-450/química , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Piridonas/química , Piridonas/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sulfotransferases/química
4.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32522852

RESUMO

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.


Assuntos
Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/virologia , Sistemas CRISPR-Cas , Orthobunyavirus/genética , Orthobunyavirus/fisiologia , Animais , Bunyaviridae , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Europa (Continente) , Técnicas de Inativação de Genes , Células HEK293 , Heparitina Sulfato/metabolismo , Humanos , Gado , Glicoproteínas de Membrana/genética , Orthobunyavirus/patogenicidade , Vírus da Febre do Vale do Rift , Transportadores de Sulfato/metabolismo , Sulfotransferases/metabolismo , Células Vero , Ligação Viral
5.
Cancer Sci ; 111(8): 2907-2922, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32573871

RESUMO

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.


Assuntos
Neoplasias da Mama/patologia , Sulfotransferases/metabolismo , Antígenos CD/metabolismo , Butadienos/farmacologia , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células MCF-7 , Invasividade Neoplásica/patologia , Nitrilos/farmacologia , RNA Interferente Pequeno/metabolismo , Sulfotransferases/genética
6.
BMC Plant Biol ; 20(1): 271, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32527219

RESUMO

BACKGROUND: As a unique sulfated polysaccharide, fucoidan is an important component of cell wall in brown seaweeds. Its biochemical properties are determined by the positions and quantity of sulfate groups. Sulfotransferases (STs) catalyze the sulfation process, which transfer the sulfuryl groups to carbohydrate backbones and are crucial for fucoidan biosynthesis. Nevertheless, the structures and functions of STs in brown seaweeds are rarely investigated. RESULTS: There are a total of 44 ST genes identified from our genome and transcriptome analysis of Saccharina japonica, which were located in the 17 scaffolds and 11 contigs. The S. japonica ST genes have abundant introns and alternative splicing sites, and five tandem duplicated gene clusters were identified. Generally, the ST genes could be classified into five groups (Group I ~ V) based on phylogenetic analysis. Accordingly, the ST proteins, which were encoded by genes within the same group, contained similar conserved motifs. Members of the S. japonica ST gene family show various expression patterns in different tissues and developmental stages. Transcriptional profiles indicate that the transcriptional levels of more than half of the ST genes are higher in kelp basal blades than in distal blades. Except for ST5 and ST28, most ST genes are down-regulated with the kelp development stages. The expression levels of nine ST genes were detected by real-time quantitative PCR, which demonstrates that they responded to low salinity and drought stresses. CONCLUSIONS: Various characteristics of the STs allow the feasibilities of S. japonica to synthesize fucoidans with different sulfate groups. This enables the kelp the potential to adapt to the costal environments and meet the needs of S. japonica growth.


Assuntos
Proteínas de Algas/genética , Genoma , Feófitas/genética , Sulfotransferases/genética , Transcrição Genética , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Perfilação da Expressão Gênica , Feófitas/enzimologia , Feófitas/crescimento & desenvolvimento , Filogenia , Alinhamento de Sequência , Estresse Fisiológico/genética , Sulfotransferases/química , Sulfotransferases/metabolismo , Transcrição Genética/fisiologia
7.
PLoS One ; 15(5): e0230354, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413029

RESUMO

Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.


Assuntos
Neoplasias Ósseas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Neoplasias da Próstata/metabolismo , Sulfotransferases/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte/química , Via de Sinalização Wnt , Neoplasias Ósseas/secundário , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Hidrogéis/química , Macrófagos/metabolismo , Masculino , Neoplasias da Próstata/patologia , Células Estromais/metabolismo
8.
Plant Mol Biol ; 103(4-5): 511-525, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32279151

RESUMO

KEY MESSAGE: TPST is involved in fructose signaling to regulate the root development and expression of genes in biological processes including auxin biosynthesis and accumulation in Arabidopsis. Sulfonation of proteins by tyrosine protein sulfotransferases (TPST) has been implicated in many important biological processes in eukaryotic organisms. Arabidopsis possesses a single TPST gene and its role in auxin homeostasis and root development has been reported. Here we show that the Arabidopsis tpst mutants are hypersensitive to fructose. In contrast to sucrose and glucose, fructose represses primary root growth of various ecotypes of Arabidopsis at low concentrations. RNA-seq analysis identified 636 differentially expressed genes (DEGs) in Col-0 seedlings in response to fructose verses glucose. GO and KEGG analyses of the DEGs revealed that fructose down-regulates genes involved in photosynthesis, glucosinolate biosynthesis and IAA biosynthesis, but up-regulates genes involved in the degradation of branched amino acids, sucrose starvation response, and dark response. The fructose responsive DEGs in the tpst mutant largely overlapped with that in Col-0, and most DEGs in tpst displayed larger changes than in Col-0. Interestingly, the fructose up-regulated DEGs includes genes encoding two AtTPST substrate proteins, Phytosulfokine 2 (PSK2) and Root Meristem Growth Factor 7 (RGF7). Synthesized peptides of PSK-α and RGF7 could restore the fructose hypersensitivity of tpst mutant plants. Furthermore, auxin distribution and accumulation at the root tip were affected by fructose and the tpst mutation. Our findings suggest that fructose serves as a signal to regulate the expression of genes involved in various biological processes including auxin biosynthesis and accumulation, and that modulation of auxin accumulation and distribution in roots by fructose might be partly mediated by the TPST substrate genes PSK-α and RGF7.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Frutose/metabolismo , Raízes de Plantas/metabolismo , Sulfotransferases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Ácidos Indolacéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Meristema/metabolismo , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Proteínas de Plantas , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Plântula/crescimento & desenvolvimento , Transdução de Sinais , Sulfotransferases/genética , Transcriptoma
9.
Arch Insect Biochem Physiol ; 104(3): e21671, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32227386

RESUMO

Sulfoconjugation plays a vital role in the detoxification of xenobiotics and in the metabolism of endogenous compounds. In this study, we aimed to identify new members of the sulfotransferase (SULT) superfamily in the silkworm Bombyx mori. Based on amino acid sequence and phylogenetic analyses, two new enzymes, swSULT ST1 and swSULT ST2, were identified that appear to belong to a distinct group of SULTs including several other insect SULTs. We expressed, purified, and characterized recombinant SULTs. While swSULT ST1 sulfated xanthurenic acid and pentachlorophenol, swSULT ST2 exclusively utilized xanthurenic acid as a substrate. Based on these results, and those concerning the tissue distribution and substrate specificity toward pentachlorophenol analyses, we hypothesize that swSULT ST1 plays a role in the detoxification of xenobiotics, including insecticides, in the silkworm midgut and in the induction of gametogenesis in silkworm ovary and testis. Collectively, the data obtained herein contribute to a better understanding of SULT enzymatic functions in insects.


Assuntos
Bombyx/enzimologia , Inativação Metabólica , Sulfotransferases/química , Sequência de Aminoácidos , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Feminino , Gametogênese , Trato Gastrointestinal/enzimologia , Proteínas de Insetos , Larva/enzimologia , Masculino , Ovário , Pentaclorofenol/metabolismo , Filogenia , Sulfotransferases/metabolismo , Testículo , Xanturenatos/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(17): 9311-9317, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32277030

RESUMO

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Heparina/metabolismo , Heparitina Sulfato/biossíntese , Animais , Anticoagulantes , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica/genética , Células HeLa , Heparina/biossíntese , Heparina/genética , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mastócitos/metabolismo , Sulfotransferases/metabolismo , Suínos , Fatores de Transcrição
11.
Chem Biol Interact ; 324: 109062, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198087

RESUMO

Ginsenoside Rg1 is an active ingredient extracted from the roots of ginsenoside, and an α-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis was used to investigate the protective effect of Rg1 on cholestasis. 48 SD male rats were randomly divided into 6 groups: control group, model group, UDCA group (ursodeoxycholic acid), low-dose Rg1 group (10 mg/kg), medium-dose Rg1 group (20 mg/kg) and high-dose Rg1 group (40 mg/kg). The model group, the UDCA group and all the Rg1 group were then intragastrically administered with 80 mg/kg ANIT, and the control group were given equal volume of olive oil. Then the pathological changes in liver tissue were observed, the secretion of bile in the bile duct was measured, and the biochemical markers in serum were quantified, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transfer peptidase (GTP) and the content of total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA). The contents of inflammatory mediators in serum were quantified, including tumor necrosis factor (TNF-α), γ-interferon (IFN-γ) and interleukin-1ß (IL-1ß). The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in liver homogenate were quantified. Expression of farnesoid X receptor (FXR), transporters and metabolic enzymes in liver tissue was monitored. Rg1 treatment improved liver tissue pathological damage, promoted bile secretion and significantly reduced serum levels of the intrahepatic cholestasis markers ALT, AST, ALP, GTP, TBIL, DBIL and TBA. Rg1 increased the activity of SOD and GSH-Px in liver homogenate, while, reducing the serum levels of MDA and inflammatory mediators. Rg1 also regulated the expression of FXR, bile acid transporters and metabolic enzymes. Overall, Rg1 alleviated liver injury by improving secretion of bile and normalizing the activity of enzymes in the serum. The protective mechanism appeared to be related to the activation of FXR and regulation of liver transporters and metabolic enzymes.


Assuntos
Colestase Intra-Hepática/tratamento farmacológico , Ginsenosídeos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Substâncias Protetoras/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , 1-Naftilisotiocianato , Animais , Bile/metabolismo , Biomarcadores/metabolismo , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/patologia , Citocromo P-450 CYP3A/metabolismo , Citocinas/metabolismo , Glucuronosiltransferase/metabolismo , Glutationa Peroxidase/metabolismo , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Ratos Sprague-Dawley , Sulfotransferases/metabolismo , Superóxido Dismutase/metabolismo
12.
Nat Commun ; 11(1): 1481, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198425

RESUMO

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.


Assuntos
Heparitina Sulfato/química , Íons , Espectrometria de Massas/métodos , Oligossacarídeos/química , Epitopos , Fatores de Crescimento de Fibroblastos/metabolismo , Ácido Glucurônico/química , Heparina , Heparitina Sulfato/metabolismo , Análise de Sequência/métodos , Relação Estrutura-Atividade , Sulfotransferases/metabolismo
13.
Sci Rep ; 10(1): 5001, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193417

RESUMO

Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sulfotransferases/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Fenobarbital/metabolismo , Fosforilação , Sulfotransferases/genética
14.
Mol Biochem Parasitol ; 236: 111257, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32027942

RESUMO

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.


Assuntos
Hicantone , Oxamniquine/análogos & derivados , Esquistossomose/tratamento farmacológico , Sulfotransferases , Animais , Cristalização/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Resistência a Medicamentos , Humanos , Hicantone/efeitos adversos , Hicantone/análogos & derivados , Hicantone/química , Oxamniquine/química , Oxamniquine/uso terapêutico , Praziquantel/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Schistosoma haematobium/efeitos dos fármacos , Schistosoma haematobium/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Esquistossomicidas/uso terapêutico , Sulfotransferases/efeitos dos fármacos , Sulfotransferases/metabolismo
15.
Endocrinology ; 161(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31894239

RESUMO

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3ß-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.


Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Carcinoma/enzimologia , Neoplasias da Próstata/enzimologia , Sulfotransferases/metabolismo , Animais , Transição Epitelial-Mesenquimal , Humanos , Masculino , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Receptores Androgênicos/metabolismo
16.
Clin Sci (Lond) ; 134(2): 273-287, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31957803

RESUMO

The current main treatment for coronary artery disease (CAD) is to reduce low-density lipoprotein cholesterol (LDL-C) by statins, which could decrease the incidence of major adverse cardiovascular events (MACEs) by 30%. However, many residual risks still remain. To clarify the mechanism involved, we studied patients with acute myocardial infarction (AMI) with low LDL-C levels. Lymphocytes were isolated, and it was found that despite no difference in plasma LDL-C level, the lymphocyte cholesterol content was higher in AMI patient than those in non-CAD patients; thus, the decrease in intracellular cholesterol content was inconsistent with that in the plasma. Additionally, [3H]-cholesterol efflux rates were lower and mRNA levels of the inflammatory factors tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) higher in AMI lymphocytes. It was found that sulphotransferase 2B1b (SULT2B1b) expression was higher in AMI lymphocytes. Further research using Jurkat T lymphocytes confirmed that SULT2B1b knockdown increased cholesterol efflux capacity and decreased mRNA levels of TNF-α and IFN-γ by increasing liver X receptor (LXR)-ß levels. Furthermore, the degree of CpG island methylation in the SULT2B1b promoter was reduced in cells from AMI patients. In conclusion, SULT2B1b up-regulation due to hypomethylation of its promoter promotes cholesterol accumulation and inflammation by inhibiting LXR-ß in lymphocytes of AMI patients with low LDL-C levels. Therefore, reducing intracellular cholesterol is also important as plasma cholesterol levels. Therapeutic approaches to decrease SULT2B1b expression might be potentially beneficial for CAD prevention by decreasing intracellular cholesterol.


Assuntos
Colesterol/metabolismo , Interferon gama/metabolismo , Linfócitos/metabolismo , Sulfotransferases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transporte Biológico , Colesterol/sangue , LDL-Colesterol/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/prevenção & controle , Metilação de DNA , Humanos , Mediadores da Inflamação/metabolismo , Interferon gama/genética , Células Jurkat , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/prevenção & controle , Regiões Promotoras Genéticas/genética , Sulfotransferases/genética , Fator de Necrose Tumoral alfa/genética
17.
Nat Prod Res ; 34(6): 797-803, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30398375

RESUMO

Previous studies have revealed sulfation as a major pathway for the metabolism of hesperetin, naringenin and apigenin. The current study was designed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of sulfating these flavonoid compounds. Of the thirteen human SULTs, six (1A1, 1A2, 1A3, 1B2, 1C4, 1E1) displayed significant sulfating activity toward hesperetin, five (1A1, 1A2, 1A3, 1B2, 1C4) displayed sulfating activity towards naringenin, and four (1A1, 1A2, 1A3, 1C4) showed sulfating activity towards apigenin. Of the four human organ specimens tested, liver and intestine cytosols displayed much higher hesperetin-, naringenin- and apigenin-sulfating activity than lung and kidney cytosols. Moreover, sulfation of hesperetin, naringenin and apigenin was shown to take place in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under cultured conditions. Taken together, these results provided a biochemical basis underlying the metabolism of hesperetin, naringenin and apigenin through sulfation in humans.[Formula: see text].


Assuntos
Apigenina/metabolismo , Flavanonas/metabolismo , Hesperidina/metabolismo , Redes e Vias Metabólicas , Sulfatos/metabolismo , Sulfotransferases/metabolismo , Linhagem Celular Tumoral , Citosol/enzimologia , Humanos , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo
18.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140301, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31678194

RESUMO

Heparin is a naturally occurring glycosaminoglycan isolated from animal tissues and is medically used as an anticoagulant drug. Adulteration attempts of isolated heparin with chondroitin sulfate in the past resulted in great safety concerns. Also, increasing demands on batch-to-batch homogeneity for better evaluation and control of its pharmacodynamic and pharmacokinetic properties kindled the development of synthetic routes for the production of heparin and its derivatives. The discovery of enzymes involved in glycosaminoglycan biosynthesis and their application in chemoenzymatic synthesis makes it feasible to generate low molecular weight heparins (LMWHs) and ultra-low molecular weight heparins (ULMWHs). Understanding the scope and limitations of these enzymes currently used in the production of synthetic heparins will help to achieve more defined heparins with controlled medicative properties. Here, we summarized the recent advances in the chemoenzymatic synthesis of LMW/ULMW heparins.


Assuntos
Heparina de Baixo Peso Molecular/biossíntese , Animais , Configuração de Carboidratos , Dissacarídeos/química , Dissacarídeos/metabolismo , Glucosiltransferases/metabolismo , Heparina de Baixo Peso Molecular/química , Oligossacarídeos/metabolismo , Racemases e Epimerases/metabolismo , Sulfotransferases/metabolismo
19.
Methods Mol Biol ; 2089: 191-207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31773656

RESUMO

A variety of oxidative and conjugative enzymes are involved in the metabolism of compounds including drugs, which can be converted into toxic metabolites by Phase I drug-metabolizing enzymes (DMEs), such as the cytochromes P450 (CYP450s), and/or detoxified by Phase II DMEs, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and glutathione S-transferases (GSTs). Traditionally, primary hepatocytes containing a complete set of DMEs have been widely used as a gold standard to assess metabolism-induced compound toxicity. However, primary hepatocytes are expensive, have high donor variability in expression levels of DMEs, and rapidly lose liver-specific functions when the cells are maintained under standard in vitro cell culture conditions over time. To address this issue and rapidly profile metabolism-induced drug toxicity, we have developed a 384-pillar plate, which is complementary to conventional 384-well plates. In this chapter, we provide step-by-step procedures for three-dimensional (3D) cell printing on the 384-pillar plate coupled with DMEs and compounds in the 384-well plate for high-throughput assessment of metabolism-induced toxicity.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Inativação Metabólica/fisiologia , Preparações Farmacêuticas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Células HEK293 , Hepatócitos/metabolismo , Humanos , Desentoxicação Metabólica Fase I/fisiologia , Sulfotransferases/metabolismo
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