Formation of DNA Adducts by 1-Methoxy-3-indolylmethylalcohol, a Breakdown Product of a Glucosinolate, in the Mouse: Impact of the SULT1A1 Status-Wild-Type, Knockout or Humanised.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as "probably carcinogenic to humans". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

SULT1A1-dependent sulfonation of alkylators is a lineage-dependent vulnerability of liver cancers.

Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the second leading cause of cancer-related deaths worldwide. Most individuals are treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for selection. Here using high-throughput screens, proteomics and in vitro resistance models, we identify the small molecule YC-1 as selectively active against a defined subset of cell lines derived from both liver cancer types. We demonstrate that selectivity is determined by expression of the liver-resident cytosolic sulfotransferase enzyme SULT1A1, which sulfonates YC-1. Sulfonation stimulates covalent binding of YC-1 to lysine residues in protein targets, enriching for RNA-binding factors. Computational analysis defined a wider group of structurally related SULT1A1-activated small molecules with distinct target profiles, which together constitute an untapped small-molecule class. These studies provide a foundation for preclinical development of these agents and point to the broader potential of exploiting SULT1A1 activity for selective targeting strategies.

Sulfation of 12-hydroxy-nevirapine by human SULTs and the effects of genetic polymorphisms of SULT1A1 and SULT2A1.

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

O-Sulfation disposition of curcumin and quercetin in SULT1A3 overexpressing HEK293 cells: the role of arylsulfatase B in cellular O-sulfation regulated by transporters.

Extensive phase II metabolic reactions (i.e., glucuronidation and sulfation) have resulted in low bioavailability and decreased biological effects of curcumin and quercetin. Compared to glucuronidation, information on the sulfation disposition of curcumin and quercetin is limited. In this study, we identified that BCRP and MRP4 played a critical role in the cellular excretion of curcumin-O-sulfate (C-O-S) and quercetin-O-sulfate (Q-O-S) by integrating chemical inhibition with transporter knock-down experiments. Inhibited excretion of sulfate (C-O-S and Q-O-S) caused significant reductions in cellular O-sulfation of curcumin (a maximal 74.4% reduction) and quercetin (a maximal 76.9% reduction), revealing a strong interplay of sulfation with efflux transport. It was further identified that arylsulfatase B (ARSB) played a crucial role in the regulation of cellular O-sulfation by transporters. ARSB overexpression significantly enhanced the reduction effect of MK-571 on the cellular O-sulfation (fmet) of the model compound (38.8% reduction for curcumin and 44.2% reduction for quercetin). On the contrary, ARSB knockdown could reverse the effect of MK-571 on the O-sulfation disposition of the model compound (29.7% increase for curcumin and 47.3% increase for quercetin). Taken together, ARSB has been proven to be involved in cellular O-sulfation, accounting for transporter-dependent O-sulfation of curcumin and quercetin. A better understanding of the interplay beneath metabolism and transport will contribute to the exact prediction of in vivo drug disposition and drug-drug interactions.

Aha1 Is an Autonomous Chaperone for SULT1A1.

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Bacterial Aryl Sulfotransferases in Selective and Sustainable Sulfation of Biologically Active Compounds using Novel Sulfate Donors.

Regioselective sulfation of bioactive compounds is a vital and scarcely studied topic in enzyme-catalyzed transformations and metabolomics. The major bottleneck of enzymatic sulfation consists in finding suitable sulfate donors. In this regard, 3'-phosphoadenosine 5'-phosphosulfate (PAPS)-independent aryl sulfotransferases using aromatic sulfate donors are a favored choice due to their cost-effectiveness. This work presents a unique study of five sulfate donors differing in their leaving group pKa values with a new His-tagged construct of aryl sulfotransferase from Desulfitobacterium hafniense (DhAST-tag). DhAST-tag was purified to homogeneity and biochemically characterized. Two new donors (3-nitrophenyl sulfate and 2-nitrophenyl sulfate) were synthesized. The kinetic parameters of these and other commercial sulfates (4-nitrophenyl, 4-methylumbelliferyl, and phenyl) revealed large differences with respect to the structure of the leaving group. These donors were screened for the sulfation of selected flavonoids (myricetin, chrysin) and phenolic acids (gallate, 3,4-dihydroxyphenylacetate). The donor impact on the sulfation regioselectivity and yield was assessed. The obtained regioselectively sulfated compounds are authentic human metabolites required as standards in clinical trials.

Fluorinated Phosphoadenosine 5'-Phosphosulfate Analogues for Continuous Sulfotransferase Activity Monitoring and Inhibitor Screening by 19F NMR Spectroscopy.

Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO3) transfer. 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the universal ST cofactor, serving as the "active sulfate" source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.

Impact of Pineapple Juice on Expression of CYP3A4, NAT2, SULT1A1 and OATP1B1 mRNA in HepG2 Cells.

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i>) is a popular fruit worldwide with natural antioxidant properties. This study examined how pineapple modified the expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and SULT1A1) and a drug transporter (OATP1B1) in human hepatocarcinoma (HepG2) cells. <b>Materials and Methods:</b> HepG2 cells (2.5×10⁵ cells/well in a 24-well plate) were incubated with pineapple juice extract (125-1,000 µg mL¹) for 48 hrs in phenol red-free medium. Resazurin reduction, ROS, AST and ALT assays were performed. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple juice slightly reduced HepG2 cell viability to 80% of the control, while ROS, AST and ALT levels were not changed. Pineapple juice did not alter the expression of CYP1A2, CYP2C9 and UGT1A6 mRNA. All tested concentrations of pineapple juice suppressed CYP3A4, NAT2 and OATP1B1 expression, while SULT1A1 expression was induced. <b>Conclusion:</b> Though pineapple juice slightly decreased the viability of HepG2 cells, cell morphology and cell function remained normal. Pineapple juice disturbed the expression of phase I (CYP3A4) and phase II (NAT2 and SULT1A1) metabolizing genes and the drug transporter OATP1B1. Therefore, the consumption of excessive amounts of pineapple juice poses a risk for drug interactions.

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.

Influence of breast cancer risk factors on proliferation and DNA damage in human breast glandular tissues: role of intracellular estrogen levels, oxidative stress and estrogen biotransformation.

Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).

SULT1A1 and SULT1A2 Associated Extensive Prolapse-Type Inflammatory Polyposis in Crohn's Colitis.

Mucosal prolapse syndrome most commonly involves the rectum and presents as solitary rectal ulcer syndrome and proctitis cystica profunda. Symptoms and endoscopic appearances are nonspecific. Histologically, mucosal prolapse is characterized by fibromuscular obliteration of the lamina propria, and displacement of crypts into submucosa and muscularis mucosae. Mucosal prolapse presenting as polyposis is rare and has only been reported involving the rectosigmoid colon. In this report, we describe a case of mucosal prolapse syndrome presenting as diffuse polyposis and colitis cystica profunda involving the hepatic, splenic flexures and descending colon in a teenage boy suffering from refractory fibrostenosing Crohn's disease. This patient was found to have possibly deleterious homozygous single nucleotide polymorphisms in both SULT1A1 and SULT1A2 genes within a unique polygenic variation of altered cell adhesion.

Genetic polymorphism of Arg213His variant in the SULT1A1 gene is associated with reduced susceptibility to lung cancer in North Indian population.

Sulfotransferases (SULTs) are phase II detoxification enzymes that is involved in the biotransformation of many compounds including tobacco carcinogens. A polymorphism in the SULT1A1 (Arg213His) gene results in reduced enzyme activity.We investigated the association between the SULT1A1 (Arg213/His) genotype and lung cancer (LC). This case-control study comprised of 550 cases and controls, matched on age, gender and smoking status.The variant genotype exhibited no association with LC risk, even after stratification on basis of histological subtypes. Male LC patients carrying the variant His213 allele (p = 0.02) did not exhibit an increased risk towards LC. Smokers harbouring the Arg/His genotype did demonstrate a reduced risk towards LC (AOR = 0.70; p = 0.019). Furthermore, the LC subjects who were heavy smokers and harbouring the Arg/His genotype (AOR = 0.28; p = 0.019) did not show a genetic predisposition towards LC susceptibility. The subjects who smoked pack years of above 40 and carrying the His/His (AOR = 0.28; p = 0.036) genotype were found to have a reduced risk for LC. Furthermore, 473 subjects were analysed in regards to overall survival, wherein the His/His genotype exhibited better OS than Arg/Arg genotype (11.30 vs. 8.07 months).This study provides evidence of no genetic predisposition towards LC risk associated with SULT1A1 Arg213His polymorphism in relation to tobacco smoking.

Insights into the substrate binding mechanism of SULT1A1 through molecular dynamics with excited normal modes simulations.

Sulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein-ligand interactions studies where major rearrangements are expected.

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

Suppressed Hepatic Production of Indoxyl Sulfate Attenuates Cisplatin-Induced Acute Kidney Injury in Sulfotransferase 1a1-Deficient Mice.

Endogenous factors involved in the progression of cisplatin nephropathy remain undetermined. Here, we demonstrate the toxico-pathological roles of indoxyl sulfate (IS), a sulfate-conjugated uremic toxin, and sulfotransferase 1A1 (SULT1A1), an enzyme involved in its synthesis, in cisplatin-induced acute kidney injury using Sult1a1-deficient (Sult1a1-/- KO) mice. With cisplatin administration, severe kidney dysfunction, tissue damage, and apoptosis were attenuated in Sult1a1-/- (KO) mice. Aryl hydrocarbon receptor (AhR) expression was increased by treatment with cisplatin in mouse kidney tissue. Moreover, the downregulation of antioxidant stress enzymes in wild-type (WT) mice was not observed in Sult1a1-/- (KO) mice. To investigate the effect of IS on the reactive oxygen species (ROS) levels, HK-2 cells were treated with cisplatin and IS. The ROS levels were significantly increased compared to cisplatin or IS treatment alone. IS-induced increases in ROS were reversed by downregulation of AhR, xanthine oxidase (XO), and NADPH oxidase 4 (NOX4). These findings suggest that SULT1A1 plays toxico-pathological roles in the progression of cisplatin-induced acute kidney injury, while the IS/AhR/ROS axis brings about oxidative stress.

Small-molecule control of neurotransmitter sulfonation.

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder-a disease that afflicts ∼20% of the world's population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. To this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3)-the isoform responsible for sulfonating ∼80% of the serotonin in the extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a side chain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free-energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization - the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable with those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

1-Methylpyrene induces chromosome loss and mitotic apparatus damage in a Chinese hamster V79-derived cell line expressing both human CYP1A2 and sulfotransferase 1A1.

1-Methylpyrene (1-MP) is a ubiquitous environmental pollutant and rodent carcinogen. Its mutagenic activity depends on sequential activation by various CYP and sulfotransferase (SULT) enzymes. Previously we have observed induction of micronuclei and mitotic arrest by 1-MP in a Chinese hamster (V79)-derived cell line expressing both human CYP1A2 and SULT1A1 (V79-hCYP1A2-hSULT1A1), however, the mode of chromosome damage and the involvement of mitotic tubulin structures have not been clarified. In this study, we used immunofluorescent staining of centromere protein B (CENP-B) with the formed micronuclei, and that of ß- and γ-tubulin reflecting the structures of mitotic spindle and centrioles, respectively, in V79-hCYP1A2-hSULT1A1 cells. The results indicated that 1-MP induced micronuclei in V79-hCYP1A2-hSULT1A1 cells from 0.125 to 2 µM under a 24 h/0 h (exposure/recovery) regime, while in the parental V79-Mz cells micronuclei were induced by 1-MP only at concentrations ≥ 8 µM; in both cases, the micronuclei induced by 1-MP were predominantly CENP-B positive. Following 54 h of exposure, 1-MP induced mitotic spindle non-congression and centrosome amplification (multipolar mitosis) in V79-hCYP1A2-hSULT1A1 cells, and anaphase/telophase retardation, at concentrations ≥ 0.125 µM with concentration-dependence; while in V79-Mz cells it was inactive up to 8 µM. This study suggests that in mammalian cells proficient in activating enzymes 1-MP may induce chromosome loss and mitotic disturbance, probably by interfering with the mitotic spindle and centrioles.

Optimizing the sulfation-modification system for scale preparation of chondroitin sulfate A.

Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.