A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

Microsomal glutathione transferase 1 controls metastasis and therapeutic response in melanoma.

While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.

Anatomical pancreatic variants in intraductal papillary mucinous neoplasm patients: a cross-sectional study.

BACKGROUND: No previous studies have examined the possible relationship between intraductal papillary mucinous neoplasm (IPMN) and the developmental ductal variations of the pancreas, such as an ansa pancreatica and a meandering main pancreatic duct (MMPD). METHODS: This retrospective cross-sectional study enrolled 214 patients, 108 with IPMN disease and 106 subjects from a community at the tertiary care unit. The main pancreatic duct (MPD) was evaluated in the head of the pancreas by its course, which were non-MMPD: descending, vertical, and sigmoid, or MMPD including loop types, reverse-Z subtypes, and an N-shape, which was identified for the first time in this study. IPMN patients were also evaluated for worrisome features (WF) or high-risk stigmata (HRS), and the extent of IPMN cysts. RESULTS: Among IPMN patients, 18.4% had MMPD, which we observed in only 3.0% of the control group (P < 0.001). Patients with MMPD were more likely to belong to the IPMN group compared with non-MMPD patients [odds ratio (OR) 6.4, 95% confidence interval (CI) 2.2-24.9]. Compared with a descending shape MPD, IPMN patients with an N-shaped MPD were more likely to have a cystic mural nodule (OR 5.9, 95% CI 1.02-36.0). The presence of ansa pancreatica associated with more extent IPMN disease (OR 12.8, 95% CI 2.6-127.7). CONCLUSIONS: IPMN patients exhibited an MMPD more often than control patients. Ansa pancreatica associated with multiple cysts. Furthermore, an N-shape in IPMN patients associated with cystic mural nodules, suggesting that this shape serves as a risk factor for more severe IPMN.

Celiac disease and upper secondary school achievement in Sweden A retrospective cohort study.

BACKGROUND: Both undiagnosed celiac disease and some chronic childhood diseases are associated with lower academic achievement. However, there is little knowledge of achievements in those diagnosed with celiac disease. Our aim was to investigate school achievements in upper secondary school among Swedish adolescents with celiac disease. METHODS: We performed a retrospective cohort study using register data. We analyzed choice of upper secondary school program, completion of upper secondary school including achievements of basic eligibility for college/university, and final grade in individuals with celiac disease diagnosed before 15 years of age, born 1991-97. We compared with the Swedish population of the same birth years. Analyses were adjusted for sex, year of birth, living region at 17 years of age, and parental education as well as income. RESULTS: The cohort included 734 074 individuals, whereof 3 257 (62% females) with celiac disease. There was no significant difference in choice of upper secondary school program. No significant difference was found in completion or achieving basic eligibility for college/university in adjusted analyses. The mean final grade in the celiac disease group was 13.34 (standard deviation 4.85) compared to 12.78 (standard deviation 5.01) in the reference population (p < 0.001), out of a maximum of 20. The effect of celiac disease on final grade remained in adjusted analyses (p = 0.012). CONCLUSIONS: We found that diagnosed celiac disease does not negatively affect school achievements in upper secondary school. This finding suggests the diagnosis, treatment and follow-up programs of celiac disease could reverse potential deleterious academic processes.

Celiac Dietary Adherence Test simplifies Determining Adherence to a Gluten-free Diet in Swedish Adolescents.

OBJECTIVES: The aims of the study were to ascertain whether the Celiac Dietary Adherence Test (CDAT) could contribute in determining adherence to a gluten-free diet in patients with celiac disease and to evaluate the diet adherence and well being of a study population 5 years after a celiac disease screening known as "Exploring the Iceberg of Celiacs in Sweden." METHODS: Through the screening, 90 adolescents (born 1997) were diagnosed with biopsy-proven celiac disease at 12 years of age. Of them, 70 (78%) came to a 5-year follow-up where anti-tissue transglutaminase antibodies 2 was tested and a questionnaire was filled in, including CDAT, which consists of 7 questions related to adherence. Nonparametrical tests were used to determine associations between adherence measures. RESULTS: Among the adolescents, 86% were adherent to a gluten-free diet 5 years after screening, 38% reported their general well being as excellent, 50% very well, and 12% well. Statistically significant associations were seen between anti-tissue transglutaminase antibodies 2 and the CDAT score (P = 0.033), and the self-reported adherence question and the CDAT score (P < 0.001). CONCLUSIONS: The screening-detected adolescents reported a high level of well being and adherence to a gluten-free diet 5 years after screening. We conclude that the CDAT can be used in clinical practice as an estimation of adherence to a gluten-free diet. It would be most suitable to use in conjunction with currently used adherence measures, but can also be used as a stand-alone method when others are not accessible.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

Site-specifically 11C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death.

BACKGROUND: Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging. METHODS: AnxA5 and the control protein were recombinantly expressed with a C-terminal "Sel-tag", the tetrapeptide -Gly-Cys-Sec-Gly-COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with 11C for dynamic in vivo PET studies. RESULTS: AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The 11C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease. CONCLUSIONS: These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy. GENERAL SIGNIFICANCE: The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of 11C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.

Health care resource use, diagnostic delay and disease burden in transthyretin amyloid cardiomyopathy in Sweden.

AIMS: To estimate healthcare resource use and direct healthcare costs of Transthyretin Amyloid Cardiomyopathy (ATTR-CM) in Sweden over 12 months across severity stages as defined by the New York Heart Association (NYHA). Secondary to investigate the current diagnostic trajectory for patients with ATTR-CM in Sweden. METHODS: A stratified inclusion of patients with a confirmed diagnosis of ATTR-CM in different NYHA classes. Data was extracted from medical records in two cardiology clinics in Sweden. Healthcare resource use data were retrospectively collected for 12 months. RESULTS: 38 patients were included, of whom 7 were in NYHA class II, 20 in class III and 4 in class IV. The total cost of health care per patient increased from SEK 69,000 (€6800) in NYHA stage II, SEK 219,000 (€21,500) in NYHA stage III, to SEK 638,000 (€62,900) in stage IV, mainly due to an increase in inpatient stays. Mean time (standard deviation, SD) from any cardiac related diagnosis prior to ATTR-CM diagnosis was 3.5 (3.1) years. CONCLUSIONS: Advanced ATTR-CM stages are associated with significant healthcare costs, as patients more often require resource-intensive inpatient care. The current diagnostic trajectory of ATTR-CM in this study was characterized by a diagnostic delay of several years.

This study shows that both healthcare resource use and healthcare costs increased considerably with a higher degree of ATTR-CM severity.The diagnostic trajectory of ATTR-CM in this study was characterized by a diagnostic delay of several years.Greater disease awareness and a lower threshold for screening risk groups for TTR-amyloidosis is prompted to establish an earlier diagnosis.

A retrospective study of intraductal papillary neoplasia of the pancreas (IPMN) under surveillance.

BACKGROUND AND OBJECTIVE: The growing number of identified intraductal papillary mucinous neoplasm (IPMN) patients places greater pressure on healthcare systems. Only a minority of patients have IPMN-related symptoms. Thus, more precise surveillance is required. METHODS: In this retrospective single-center cross-sectional study, patients with an active diagnosis of branch duct IPMN (BD-IPMN) and >6 months of surveillance were classified as follows: presence/absence of worrisome features (WF) or high-risk stigmata (HRS), newly developed WF/HRS, under/over 15 mm cyst, growing/not growing <15 mm cyst, and elevated serum carbohydrate antigen 19-9 (CA 19-9). RESULTS: In all, 377 patients with BD-IPMN were followed for a median of 5.4 years, 28% with WF at diagnosis, and 14% who developed WF/HRS during surveillance. Half had a <15 mm primary cyst, 40% of which did not grow during surveillance. CA 19-9 was elevated in 12%. None of the patients with normal CA 19-9 levels developed cancer or high-grade dysplasia (HGD). CONCLUSIONS: No carcinomas or HGDs appeared with normal CA 19-9 levels. Patients with <15 mm cysts that do not grow and have no WF/HRS could undergo imaging less frequently.

MRI follow-up for pancreatic intraductal papillary mucinous neoplasm: an ultrashort versus long protocol.

PURPOSE: To evaluate whether an ultrashort-protocol (USP) MRI including only T2-weighted HASTE axial and 3D MRCP SPACE sequences adequately measures the largest diameter of the largest cyst and the main pancreatic duct (MPD) and identifies worrisome features (WF) and high-risk stigmata (HRS) when compared to longer protocols (LP, long protocol; SP, short protocol; S-LP, short or long protocol). We also calculated reductions in costs associated with USP. METHODS: This retrospective study included 183 IPMN patients. Two radiologists compared two imaging sets (USP versus S-LP) per patient, comparing the mean values of the largest cyst and MPD and agreement regarding the presence or absence of cystic or MPD mural nodules and solid pancreatic tumors. The interobserver agreement for cystic mural nodules and WF/HRS was evaluated, using the Bland-Altman plot and Cohen's Kappa. RESULTS: A total of 112 IPMN patients were evaluated. For detecting cysts or MPD nodules, WF/HRS, and solid pancreatic tumors, USP and S-LP coincided in 94.9%, 99.1%, 92.4%, and 99.1% of cases, respectively. Both USP and S-LP identified all true cystic mural nodules. The mean size of the largest cyst and MPD was 19.48/19.67 mm and 3.24/3.33 mm using USP versus S-LP, while the mean differences for USP versus S-LP were 0.19 mm and 0.08 mm. The USP cost was 39% of LP cost and 77% of SP. Interobserver agreement was moderate to strong. CONCLUSIONS: For IPMN surveillance, an ultrashort-protocol MRI provides nearly identical information to the more expensive longer protocols.

Prevalence, characteristics, and mortality of patients with transthyretin amyloid cardiomyopathy in the Nordic countries.

AIMS: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive condition caused by deposition of transthyretin amyloid fibrils in the heart and is associated with poor quality of life and a shortened lifespan. This study aimed to describe the prevalence, clinical characteristics, and mortality of patients with ATTR-CM, using multiple national health registers in Denmark, Finland, Norway, and Sweden. METHODS AND RESULTS: Transthyretin amyloid cardiomyopathy patients were identified during 2008-2018 using a combination of diagnosis codes for amyloidosis and heart disease and were matched to patients with non-ATTR heart failure (HF). An identical study design was used in each country to facilitate comparison and aggregation of results. A total of 1930 ATTR-CM patients were identified from national health registers in the four countries. In 2018, prevalence of ATTR-CM per 100 000 inhabitants ranged from 1.4 in Denmark to 5.0 in Sweden; a steep increase over time was observed in Sweden and Norway. Median survival from diagnosis was 30 months for ATTR-CM patients and 67 months for matched HF patients. Survival was significantly lower for female than for male ATTR-CM patients (median survival: 22 and 36 months), while no significant difference was observed in the HF cohort. CONCLUSIONS: This study provides the first nationwide estimates of the prevalence, clinical characteristics, and mortality of patients with ATTR-CM, using identical study design across several countries. Findings corroborate previous case series showing high mortality in ATTR-CM, two-fold higher than for other HF patients and higher in women than men, highlighting the need for more precise and early diagnosis to reduce the disease burden.

Healthcare resource use of patients with transthyretin amyloid cardiomyopathy.

AIMS: Transthyretin amyloid cardiomyopathy (ATTR-CM) is the cardiac manifestation of transthyretin amyloidosis (ATTR). The aim of this study was to estimate healthcare resource use for ATTR-CM patients compared with heart failure (HF) patients, in Denmark, Finland, Norway, and Sweden. METHODS AND RESULTS: Data from nationwide healthcare registers in the four countries were used. ATTR-CM patients were defined as individuals diagnosed with amyloidosis and cardiomyopathy or HF between 2008 and 2018. Patients in the ATTR-CM cohort were matched to patients with HF but without ATTR-CM diagnosis. Resource use included number of visits to specialty outpatient and inpatient hospital care. A total of 1831 ATTR-CM and 1831 HF patients were included in the analysis. The mean number of hospital-based healthcare contacts increased in both the ATTR-CM and HF cohort during 3 years pre-diagnosis and was consistently higher for the ATTR-CM cohort compared with the HF cohort, with 6.1 [CI: 5.9-6.3] vs. 3.2 [CI: 3.1-3.3] outpatient visits and 1.03 [CI: 0.96-1.1] vs. 0.7 [CI: 0.7-0.8] hospitalizations. In the first year following diagnosis, patients with ATTR-CM continued to visit outpatient care (10.2 [CI: 10.1, 10.4] vs. 5.7 [CI: 5.6, 5.9]) and were admitted to hospital more frequently (3.3 [CI: 3.2, 3.4] vs. 2.5 [CI: 2.5, 2.6]) than HF patients. CONCLUSIONS: Transthyretin amyloid cardiomyopathy imposes a high burden on healthcare systems with twice as many outpatient specialist visits and 50% more hospitalizations in the year after diagnosis compared with HF patients without ATTR-CM. Studies to investigate if earlier diagnosis and treatment of ATTR-CM may lower resource use are warranted.

Microsomal glutathione transferase 1: mechanism and functional roles.

Microsomal glutathione transferase 1 (MGST1) belongs to a superfamily named MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism). This family is represented in all life forms, except archae. Of the six human members, three are specialized in the synthesis of leukotrienes and prostaglandin E, whereas the others (MGST1-3) have potential roles in drug metabolism. MGST1 has a well-established role in the conjugation of electrophiles and oxidative stress protection, whereas MGST2 and 3 have been less studied. Here, we review the recent advances regarding the structure, mechanism, and functional roles of MGST1. Emerging data show that the enzyme is overexpressed in certain tumors and support a role for the enzyme in protecting cells from cytostatic drugs.

Characterization of new potential anticancer drugs designed to overcome glutathione transferase mediated resistance.

Resistance against anticancer drugs remains a serious obstacle in cancer treatment. Here we used novel strategies to target microsomal glutathione transferase 1 (MGST1) and glutathione transferase pi (GSTP) that are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, including cisplatin and doxorubicin (DOX). By synthetically combining cisplatin with a GST inhibitor, ethacrynic acid, to form ethacraplatin, it was previously shown that cytosolic GST inhibition was improved and that cells became more sensitive to cisplatin. Here we show that ethacraplatin is easily taken up by the cells and can reverse cisplatin resistance in MGST1 overexpressing MCF7 cells. A second and novel strategy to overcome GST mediated resistance involves using GST releasable cytostatic drugs. Here we synthesized two derivatives of DOX, 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) and 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) and showed that they are substrates for MGST1 and GSTP (releasing DOX). MGST1 overexpressing cells are resistant to DOX. The resistance is partially reversed by DNS-DOX. Interestingly, the less reactive MNS-DOX was more cytotoxic to cells overexpressing MGST1 than control cells. It would appear that, by controlling the reactivity of the prodrug, and thereby the DOX release rate, selective toxicity to MGST1 overexpressing cells can be achieved. In the case of V79 cells, DOX resistance proportional to GSTP expression levels was noted. In this case, not only was drug resistance eliminated by DNS-DOX but a striking GSTP-dependent increase in toxicity was observed in the clonogenic assay. In summary, MGST1 and GSTP resistance to cytostatic drugs can be overcome and cytotoxicity can be enhanced in GST overexpressing cells.

Nickel sensitization in orthodontically treated and non-treated female adolescents.

BACKGROUND: The importance of the nickel exposure from fixed orthodontic appliances is under continuous discussion. OBJECTIVES: Our aim was to investigate nickel allergy and the risk of nickel sensitization among female adolescents during orthodontic treatment with fixed appliances as compared with non-treated female adolescents. SUBJECTS AND METHODS: Female patients starting or with ongoing orthodontic treatment (n = 30) and young females without a history of orthodontic treatment (n = 140) were studied. Patch testing with 5% nickel sulfate was carried out twice on each participant with an approximately 1-year interval. The subjects completed a questionnaire before the first testing. RESULTS: None of the 7 orthodontic patients with a positive patch test reaction to nickel had any clinically visible intraoral allergic symptoms during their treatment. No significant difference was seen in the occurrence of positive nickel reactions in regard to orthodontic treatment, or between the first and second tests. In the treatment group, 2 patients changed from nickel-positive to nickel-negative during the observation period, and one patient showed an opposite change. The quantity and course of changes in the repeated nickel patch test reactions did not differ significantly between the subjects with and without orthodontic treatment experience. CONCLUSIONS: Nickel sensitization from orthodontic appliances is improbable, but nickel sensitization may develop also during orthodontic treatment.

Characterization of More Selective Central Nervous System Nrf2-Activating Novel Vinyl Sulfoximine Compounds Compared to Dimethyl Fumarate.

The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1-CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NFκB, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders.

Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1.

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k(cat)=0.075+/-0.005 s(-1) and K(m)=21+/-3 microM (k(cat)/K(m)=3.6 x 10(3)+/-5.6 x 10(2)M(-1)s(-1)), giving a rate enhancement of 10(6) compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.