Oximes, investigated as antidotes against organophosphates (OP) poisoning, are known to display toxic effects on a cellular level, which could be explained beyond action on acetylcholinesterase as ...their main target. To investigate this further, we performed an in vitro cell-based evaluation of effects of two structurally diverse oxime groups at concentrations of up to 800 μM, on several cell models: skeletal muscle, kidney, liver, and neural cells. As indicated by our results, compounds with an imidazolium core induced necrosis, unregulated cell death characterized by a cell burst, increased formation of reactive oxygen species, and activation of antioxidant scavenging. On the other hand, oximes with a pyridinium core activated apoptosis through specific caspases 3, 8, and/or 9. Interestingly, some of the compounds exhibited a synergistic effect. Moreover, we generated a pharmacophore model for each oxime series and identified ligands from public databases that map to generated pharmacophores. Several interesting hits were obtained including chemotherapeutics and specific inhibitors. We were able to define the possible structural features of tested oximes triggering toxic effects: chlorine atoms in combination with but-2(E)-en-1,4-diyl linker and adding a second benzene ring with substituents such as chlorine and/or methyl on the imidazolium core. Such oximes could not be used in further OP antidote development research, but could be introduced in other research studies on new specific targets. This could undoubtedly result in an overall improved wider use of unexplored oxime database created so far in OP antidotes field of research in a completely new perspective.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific ...kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the ...abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by ...hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Epidemiological studies indicate that metformin, a widely used type 2 diabetes drug, might reduce breast cancer risk and mortality in patients with type 2 diabetes. Metformin might protect against ...breast cancer indirectly by ameliorating systemic glucose homeostasis. Alternatively, it might target breast cancer cells directly. However, experiments using MDA-MB-231 cells, a standard in vitro breast cancer model, produced inconsistent results regarding effectiveness of metformin as a direct anti-cancer agent. Metformin treatments in cultured MDA-MB-231 cells are usually performed for 48-96 hours, but protocols describing renewal of cell culture medium during these prolonged treatments are rarely reported. We determined whether medium renewal protocol might alter sensitivity of MDA-MB-231 cells treated with metformin. Using the MTS assay, BrdU incorporation and Hoechst staining we found that treatment with metformin for 48-72 hours failed to suppress viability and proliferation of MDA-MB-231 cells if low-glucose (1 g/L) medium was renewed every 24 hours. Conversely, metformin suppressed their viability and proliferation if medium was not renewed. Without renewal glucose concentration in the medium was reduced to 0.1 g/L in 72 hours, which likely explains increased sensitivity to metformin under these conditions. We also examined whether 2-deoxy-D-glucose (2-DG) reduces resistance to metformin. In the presence of 2-DG metformin reduced viability and proliferation of MDA-MB-231 cells with or without medium renewal, thus demonstrating that 2-DG reduces their resistance to metformin. In sum, we show that medium renewal blocks anti-proliferative effects of metformin during prolonged treatments in low-glucose medium. Differences in medium renewal protocols during prolonged treatments might therefore lead to apparently inconsistent results as regards effectiveness of metformin as a direct anti-cancer agent. Finally, our results indicate that co-therapy with 2-DG and metformin might provide an effective strategy to overcome metformin resistance of breast cancer cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Current research has shown that several imidazolium and chlorinated bispyridinium oximes are cytotoxic and activate different mechanisms or types of cell death. To investigate this further, we ...analysed interactions between these oximes and acetylcholine receptors (AChRs) and how they affect several signalling pathways to find a relation between the observed toxicities and their effects on these specific targets. Chlorinated bispyridinium oximes caused time-dependent cytotoxicity by inhibiting the phosphorylation of STAT3 and AMPK without decreasing ATP and activated ERK1/2 and p38 MAPK signal cascades. Imidazolium oximes induced a time-independent and significant decrease in ATP and inhibition of the ERK1/2 signalling pathway along with phosphorylation of p38 MAPK, AMPK, and ACC. These pathways are usually triggered by a change in cellular energy status or by external signals, which suggests that oximes interact with some membrane receptors. Interestingly,
analysis also indicated that the highest probability of interaction for all of our oximes is with the family of G-coupled membrane receptors (GPCR). Furthermore, our experimental results showed that the tested oximes acted as acetylcholine antagonists for membrane AChRs. Even though oxime interactions with membrane receptors need further research and clarification, our findings suggest that these oximes make promising candidates for the development of specific therapies not only in the field of cholinesterase research but in other fields too, such as anticancer therapy via altering the Ca
flux involved in cancer progression.
Na
+
,K
+
-ATPase (NKA) is essential for maintenance of cellular and whole-body water and ion homeostasis. In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight ...coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor (EGFR, gefitinib). AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100–1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.
Graphical Abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and ...decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces
expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed
mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na
,K
-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed
mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed
mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.
We evaluated the potential of nine vitamin B3 scaffold-based derivatives as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors, as a starting point for the development of novel ...drugs for treating disorders with cholinergic neurotransmission-linked pathology. As the results indicate, all compounds reversibly inhibited both enzymes in the micromolar range pointing to the preference of AChE over BChE for binding the tested derivatives. Molecular docking studies revealed the importance of interactions with AChE active site residues Tyr337 and Tyr124, which dictated most of the observed differences. The most potent inhibitor of both enzymes with
of 4 μM for AChE and 8 μM for BChE was the nicotinamide derivative 1-(4'-phenylphenacyl)-3-carbamoylpyridinium bromide. Such a result places it within the range of several currently studied novel cholinesterase inhibitors. Cytotoxicity profiling did not classify this compound as highly toxic, but the induced effects on cells should not be neglected in any future detailed studies and when considering this scaffold for drug development.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Effects of low‐load blood flow restricted (LL‐BFR) training remain unexplored in patients with ACL rupture. Our hypothesis was that LL‐BFR training triggers augmented gains in knee muscle strength ...and size, which are paralleled with transcriptional responses of hypoxia‐regulated genes and myokines. Eighteen volunteers (age 37.5 ± 9 years) planned for ACL reconstruction, participated in the study. Twelve were divided between BFR group, performing 9 sessions of LL‐BFR exercise, and SHAM‐BFR group performing equal training with sham vascular occlusion. Six subjects served as a control for muscle biopsy analysis. Cross‐sectional area (CSA) and isokinetic strength of knee muscles were assessed before and after the training. Change in CSAquad was significantly (p < 0.01) larger in BFR (4.9%) compared with SHAM‐BFR (1.3%). Similarly, change in peak torque of knee extensors was significantly (p < 0.05) larger in BFR (14%) compared with SHAM‐BFR (−1%). The decrease in fatigue index of knee extensors (6%) was larger (p < 0.01) in BFR than in SHAM‐BFR (2%). mRNA expression of HIF‐1α in the vastus lateralis was reduced (p < 0.05) in SHAM‐BFR, while VEGF‐A mRNA tended to be higher in BFR. The mRNA expression of myostatin and its receptor were reduced (p < 0.05) in the semitendinosus after both types of training. Expression of IL‐6, its receptors IL‐6Rα and gp130, as well as musclin were similar in control and training groups. In conclusion, our results show augmented strength and endurance of knee extensors but less of the flexors. LL‐BFR training is especially effective for conditioning of knee extensors in this population.
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BFBNIB, FSPLJ, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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