Johanna Ungerstedt

Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase

Motexafin gadolinium (MGd) is a chemotherapeutic drug that selectively targets tumor cells and mediates redox reactions generating reactive oxygen species. Thioredoxin (Trx), NADPH, and thioredoxin reductase (TrxR) of the cytosol/nucleus or mitochondria are major thiol-dependent reductases with many functions in cell growth, defense against oxidative stress, and apoptosis. Mammalian TrxRs are selenocysteine-containing flavoenzymes; MGd was an NADPH-oxidizing substrate for human or rat TrxR1 with a Km value of 8.65 μm (kcat/Km of 4.86 × 104 m–1 s–1). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 μm) for rat TrxR. In contrast, direct reaction between MGd and reduced human Trx was negligible. The corresponding reaction with reduced Escherichia coli Trx was also negligible, but MGd was a better substrate (kcat/Km of 2.23 × 105 m–1 s–1) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Ribonucleotide reductase (RNR), a 1:1 complex of the non-identical R1- and R2-subunits, catalyzes the essential de novo synthesis of deoxyribonucleotides for DNA synthesis using electrons from Trx and TrxR. MGd inhibited recombinant mouse RNR activity with either 3 μm reduced human Trx (IC50 2 μm) or 4 mm dithiothreitol (IC50 6 μm) as electron donors. Our results demonstrate MGd-induced enzymatic generation of reactive oxygen species by TrxR plus a powerful inhibition of RNR. This may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair.

Clotting onset time may be a predictor of outcome in human brain injury: a pilot study.

In this study we assess the clotting onset time (COT) in samples from a population of traumatic brain injury patients. The patients were randomized to standard treatment plus high dose antithrombin (AT group) or standard treatment alone (nonAT group), during the first 16 hours after hospital admission. Our aim was to study the two patient groups during the first 5 days after injury, to assess COT as a coagulation monitoring method compared to routine parameters (thrombin-antithrombin complex (TAT), D-dimer, and soluble fibrin), and to correlate COT to clinical parameters and outcome. Clotting onset time measurements are carried out using free oscillating rheometry, where the endpoint of coagulation onset is determined by a deviation from initial viscoelastic properties of an oscillating sample. Both patient groups initially showed hypercoagulation. In the AT group, a significant increase of COT (i.e., decrease in hypercoagulation), was already seen 16 hours after hospital admission, but not until day 3 in the non AT group. Routine coagulation tests were not able to discriminate AT patients from nonAT patients. Clotting onset time correlated significantly to soluble fibrin, D-dimer, TAT, and leukocyte count. Additionally, COT levels at hospital admission correlated to outcomes measured with the Glasgow Outcome Scale (GOS) after 3 months. These results indicate that COT may be a clinically relevant variable with prognostic value, able to monitor the degree of hypercoagulation over time.

In vivo redox state of Human thioredoxin and redox shift by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

The cytosolic thioredoxin (Trx1) system is essential for maintaining a reduced intracellular environment, via reduced Trx1 acting as a general protein disulfide reductase. Trx1 is implicated in cell signaling such as proliferation, DNA synthesis, enzyme activation, cell cycle regulation, transcription, gene activation, and prevention of apoptosis. Human Trx1 contains the active-site cysteines, Cys32 and Cys35, and three additional structural cysteines, Cys62, Cys69, and Cys73, that regulate Trx1 structure and activity via a second disulfide formation, S-glutathionylation or S-nitrosylation. The present study uses an electrophoretic redox Western blot method to analyze the oxidation state of Trx1 in vivo separating the protein-changed isoform following alkylation with iodoacetic acid in 8M urea. Treatment with the histone deacetylase inhibitor SAHA increased Trx1 inhibitor thioredoxin interacting protein (Txnip) levels, decreased Trx1 activity, and switched the Trx1 oxidation state toward a more oxidized one, as a result of complex formation with Trx1, and increased reactive oxygen species (ROS). SAHA is currently in clinical trials for cancer treatment, and one possible mechanism for its anticancer effect is via effects on the Trx1 system. Determining the exact oxidation state of human cytosolic Trx1 may be useful in developing and evaluating cancer drugs and antioxidant agents.

Genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in multiple myeloma reveals the importance of Polycomb gene targeting and highlights EZH2 as a potential therapeutic target.

Multiple myeloma (MM) is a malignancy of the antibody-producing plasma cells. MM is a highly heterogeneous disease, which has hampered the identification of a common underlying mechanism for disease establishment as well as the development of targeted therapy. Here we present the first genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in MM patient samples, defining a common set of active H3K4me3-enriched genes and silent genes marked by H3K27me3 (H3K27me3 alone or bivalent) unique to primary MM cells, when compared to normal bone marrow plasma cells. Using this epigenome profile, we found increased silencing of H3K27me3 targets in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also demonstrated that pharmacological inhibition of EZH2 had anti-myeloma effects in both MM cell lines and CD138+ MM patient cells. In addition, EZH2 inhibition decreased the global H3K27 methylation and induced apoptosis. Taken together, these data suggest an important role for the Polycomb repressive complex 2 (PRC2) in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM.

Plasma glutaredoxin activity in healthy subjects and patients with abnormal glucose levels or overt type 2 diabetes

Oxidative stress induced by hyperglycemia is a key factor in the pathogenesis of diabetes complications. Glutaredoxin 1(Grx1) is a cytosolic redox protein that catalyzes GSH-dependent thiol redox reactions and reversible protein S-glutathionylation. In humans, Grx1 antigen has previously been detected in plasma; however, it has hitherto been unclear if plasma Grx1 is enzymatically active, which would indicate an extracellular function of the protein. Given that glucose overload damages cells through oxidative stress responses, we investigated whether postprandial hyperglycemia induces changes in extracellular Grx1 in patients with abnormal glucose tolerance and healthy subjects. Using a novel sensitive fluorescent substrate assay, we demonstrated that plasma Grx consists of active protein. Grx antigen, activity and total antioxidant capacity were significantly elevated in patients compared to healthy subjects. In response to oral glucose tolerance test, Grx activity and antioxidant capacity increased significantly in healthy volunteers, however, not to the high levels of the patients. In conclusion, these results indicate an extracellular function of plasma Grx in blood glucose metabolism. Thus, Grx may be a marker of increased oxidative stress during hyperglycemia in healthy subjects and may be a risk marker of progression toward diabetes onset.

KIT signaling is dispensable for human mast cell progenitor development

Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

Mutations in histone modulators are associated with prolonged survival during azacitidine therapy

Early therapeutic decision-making is crucial in patients with higher-risk MDS. We evaluated the impact of clinical parameters and mutational profiles in 134 consecutive patients treated with azacitidine using a combined cohort from Karolinska University Hospital (n=89) and from Kings College Hospital, London (n=45). While neither clinical parameters nor mutations had a significant impact on response rate, both karyotype and mutational profile were strongly associated with survival from the start of treatment. IPSS high-risk cytogenetics negatively impacted overall survival (median 20 vs 10 months; p<0.001), whereas mutations in histone modulators (ASXL1, EZH2) were associated with prolonged survival (22 vs 12 months, p=0.01). This positive association was present in both cohorts and remained highly significant in the multivariate cox model. Importantly, patients with mutations in histone modulators lacking high-risk cytogenetics showed a survival of 29 months compared to only 10 months in patients with the opposite pattern. While TP53 was negatively associated with survival, neither RUNX1-mutations nor the number of mutations appeared to influence survival in this cohort. We propose a model combining histone modulator mutational screening with cytogenetics in the clinical decision-making process for higher-risk MDS patients eligible for treatment with azacitidine.

Comprehensive mapping of the effects of azacitidine on DNA methylation, repressive/permissive histone marks and gene expression in primary cells from patients with MDS and MDS-related disease

Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median β-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.

Glutaredoxin mediated redox effects of coenzyme Q10 treatment in type 1 and type 2 diabetes patients.

The possible beneficial effects of coenzyme Q10 (CoQ10) supplementation on disease progression and oxidant status in diabetes remains debated. In the present study, patients with type 1 and type 2 diabetes were treated with oral CoQ10, 100 mg twice daily for 12 weeks. We assessed total antioxidant capacity, intra- and extracellular levels of the redox regulating protein glutaredoxin 1 (Grx1), CoQ10, oxidized LDL-cholesterol, lipid profile and HbA1c. We have previously shown that extracellular Grx1 is increased in patients with type 2 diabetes compared to healthy subjects. In the present study, CoQ10 treatment significantly decreased serum Grx1 activity as well as total antioxidant capacity independent of type of diabetes, indicating an improvement to a less oxidized extracellular environment. The effect on serum Grx1 activity was more prominent in patients not on statin treatment. Conversely, intracellular Grx1 activity as well as mRNA levels increased independent of statin treatment. There was a significant improvement in oxidized LDL-cholesterol and lipid profile, with a tendency to improved metabolic control (HbA1c). Additionally, we describe for the first time that CoQ10 is a direct substrate for glutathione, and that Grx1 catalyzes this reaction, thus presenting a novel mechanism for CoQ10 reduction which could explain our findings of an increased intracellular Grx1. In conclusion, 12 weeks CoQ10 treatment significantly improved the extracellular redox balance and lipid profile, indicating that prolonged treatment may have beneficial effects also on clinical outcome in diabetes.

Selenite and methylseleninic acid epigenetically affects distinct gene sets in myeloid leukemia: A genome wide epigenetic analysis

ABSTRACT Selenium compounds have emerged as promising chemotherapeutic agents with proposed epigenetic effects, however the mechanisms and downstream effects are yet to be studied. Here we assessed the effects of the inorganic selenium compound selenite and the organic form methylseleninic acid (MSA) in a leukemic cell line K562, on active (histone H3 lysine 9 acetylation, H3K9ac and histone H3 lysine 4 tri‐methylation, H3K4me3) and repressive (histone H3 lysine 9 tri‐methylation, H3K9me3) histone marks by Chromatin immunoprecipitation followed by DNA sequencing (ChIP‐Seq). Both selenite and MSA had major effects on histone marks but the effects of MSA were more pronounced. Gene ontology analysis revealed that selenite affected genes involved in response to oxygen and hypoxia, whereas MSA affected distinct gene sets associated with cell adhesion and glucocorticoid receptors, also apparent by global gene expression analysis using RNA sequencing. The correlation to adhesion was functionally confirmed by a significantly weakened ability of MSA treated cells to attach to fibronectin and linked to decreased expression of integrin beta 1. A striking loss of cellular adhesion was also confirmed in primary patient AML cells. Recent strategies to enhance the cytotoxicity of chemotherapeutic drugs by disrupting the interaction between leukemic and stromal cells in the bone marrow are of increasing interest; and organic selenium compounds like MSA might be promising candidates. In conclusion, these results provide new insight on the mechanism of action of selenium compounds, and will be of value for the understanding, usage, and development of new selenium compounds as anticancer agents. Graphical abstract Figure. No Caption available. HighlightsGenome wide epigenetic alterations caused by selenite and MSA were studied.These compounds exert cytotoxic effects by affecting key histone modifications.Selenite mainly affects pathways involving oxygen and hypoxia responses.MSA mainly affects adhesion and migration pathways, causing cellular detachment.MSA along with conventional therapy might overcome resistance phenotype in leukemia.