Diaryldienone derivatives with accessible β-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmarks of their activities. These compounds have been characterized as inhibitors of isopeptidases, a family of cysteine-proteases, which de-conjugate ubiquitin and ubiquitin-like proteins from their targets. However, it is unclear whether they can also react with additional proteins. In this work, we utilized the biotin-conjugated diaryldienone-derivative named 2c, as a bait to purify novel cellular targets of these small molecules. Proteomic analyses have unveiled that, in addition to isopeptidases, these inhibitors can form stable covalent adducts with different intracellular proteins, thus potentially impacting on multiple functions of the cells, from cytoskeletal organization to metabolism. These widespread activities can explain the ability of diaryldienone derivatives to efficiently trigger different cell death pathways.
Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.
Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3β was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3β is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3β with the CRISPR/Cas9 technology. In response to DMNQ GSK3β is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pronecrotic stress, GSK3β gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3β. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3β the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3β by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.
Background Soluble suppression of tumorigenesis-2 (sST2) and galectin (Gal)-3 are two biomarkers related to inflammation, metabolic disturbances and to myocardial fibrosis that characterize several cardiac pathological conditions. Increased circulating levels of these molecules have been associated with risk of cardiovascular death. Treatment with liraglutide, a glucagon-like peptide 1 analog, is associated with weight loss, improved glycemic control, and reduced cardiovascular risk. We wanted to assess (I) potential differences between subjects with prediabetes or type 2 diabetes mellitus (T2DM) and healthy controls in sST2 and Gal-3 circulating levels, and their relationship with glycemic control and markers of beta cell function and myocardial injury; (II) whether liraglutide treatment modulates these markers in subjects with prediabetes or early T2DM independently of weight loss; (III) whether baseline levels of any of these two molecules may predict the response to liraglutide treatment. Methods Forty metformin-treated obese subjects (BMI ≥ 30) with prediabetes [impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) or both (n = 23)] or newly diagnosed T2DM (n = 17), were randomized to liraglutide or lifestyle counseling until achieving a comparable weight loss (7% of initial body weight). Thirteen subjects were enrolled as healthy controls for baseline sST2 and Gal-3 levels. Results Baseline sST2 levels were comparable between controls and obese patients (p = 0.79) whereas Gal-3 levels were significantly higher in patients as compared to controls (p < 0.001). Liraglutide treatment, but not weight loss achieved by lifestyle counseling, decreased plasma sST2 levels (− 9%, beta = − 14.9, standard deviation 6.9, p = 0.037) while Gal-3 levels did not change. A reduction in serum hs-Troponin I was observed after intervention, due to a 19% (p = 0.29) increase in the lifestyle arm, and a 25% decrease (p = 0.033) in the liraglutide arm (between-group difference p = 0.083). Lower baseline Gal-3 levels predicted a better improvement in beta cell function after liraglutide treatment. Conclusions Liraglutide-induced reduction in sST2 and possibly hs-TnI suggests that in obese patients with prediabetes or early T2DM this drug may have a positive effect on (cardiac) fibrosis, whereas plasma level of Gal-3 before liraglutide initiation may predict response to the drug in terms of beta cell function improvement. Trial registration Eudract: 2013-001356-36
Cardiovascular prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet cyclooxygenase (COX)-1 activity during the 24 hours dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced platelet glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin production and higher rates of newly-formed platelets, escaping aspirin inhibition over 24 hours. Two-hundred aspirin-treated patients with high cardiovascular risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of platelet COX-1 activity recovery during the 10-24h dosing interval. Whole proteome analysis showed that platelets from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma thrombopoietin, megakaryocyte maturation and proplatelet formation, and conversely increased platelet galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin release. Treatment of HepG2 cells with recombinant glycocalicin led to a dose-dependent reduction in liver thrombopoietin mRNA, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher thrombopoietin/glycocalicin ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating thrombopoietin/glycocalicin ratio, reflecting a dysregulation of platelet lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.
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