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Thiosemicarbazones and their transition metal complexes are biologically active compounds and anticancer agents with versatile structural properties. In this contribution, the structural features and stability of four pyridoxal-thiosemicarbazone (PLTSC) complexes with Fe, Co, Ni, and Cu were investigated using the density functional theory and natural bond orbital approach. Special emphasis was placed on the analysis of the donor atom-metal interactions. The geometry of compounds and crystallographic structures were further examined by Hirshfeld surface analysis, and the main intermolecular interactions were outlined. It has been shown that the geometry and the number of PLTSC units in the structure determine the type and contribution of the specific interactions. The binding of all four complexes to bovine and human serum albumin was investigated through spectrofluorometric titration. The dependency of the thermodynamic parameters on the present metal ion and geometry was explained by the possible interactions through molecular docking simulations. The binding of complexes to DNA, as one of the possible ways the compounds could induce cell death, was examined by molecular docking. The cytotoxicity was measured towards HCT116, A375, MCF-7, A2780, and MCF5 cell lines, with Cu-PLTSC being the most active, as it had the highest affinity towards DNA and proteins.

Differentiation-inducing factor 1 (DIF-1) is a morphogen produced by Dictyostelium discoideum that inhibits the proliferation and migration of both D. discoideum and most mammalian cells. Herein, we assessed the effect of DIF-1 on mitochondria, because DIF-3, which is similar to DIF-1, reportedly localizes in the mitochondria when added exogenously, however the significance of this localization remains unclear. Cofilin is an actin depolymerization factor that is activated by dephosphorylation at Ser-3. By regulating the actin cytoskeleton, cofilin induces mitochondrial fission, the first step in mitophagy. Here, we report that DIF-1 activates cofilin and induces mitochondrial fission and mitophagy mainly using human umbilical vein endothelial cells (HUVECs). AMP-activated kinase (AMPK), a downstream molecule of DIF-1 signaling, is required for cofilin activation. Pyridoxal phosphatase (PDXP)-known to directly dephosphorylate cofilin-is also required for the effect of DIF-1 on cofilin, indicating that DIF-1 activates cofilin through AMPK and PDXP. Cofilin knockdown inhibits mitochondrial fission and decreases mitofusin 2 (Mfn2) protein levels, a hallmark of mitophagy. Taken together, these results indicate that cofilin is required for DIF-1- induced mitochondrial fission and mitophagy., Competing Interests: Conflict of interest The authors indicated no potential conflict of interest., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Tuberculosis (TB) is one of the most dangerous infectious diseases and is caused by Mycobacterium bovis (Mb) and Mycobacterium tuberculosis (Mt). Branched-chain amino acid aminotransferases (BCATs) were reported to be the key enzyme for methionine synthesis in Mycobacterium. Blocking the methionine synthesis in Mycobacterium can inhibit the growth of Mycobacterium. Therefore, in silico screening of inhibitors can be a good way to develop a potential drug for treating TB. A pyridoxal 5'-phosphate (PLP)-form of Mycobacterium bovis branched-chain amino acid aminotransferases (MbBCAT), an active form of MbBCAT, was constructed manually for docking approximately 150 000 compounds and the free energy was calculated in Autodock Vina. The 10 compounds which had the highest affinity to MbBCAT were further evaluated for their inhibitory effects against MbBCAT. Within the selected compounds, compound 4 (ZINC12359007) was found to be the best inhibitor against MbBCAT with the inhibitory constant K i of 0·45 μmol l -1 and IC 50 of 2·37 μmol l -1 . Our work provides potential candidates to develop effective drugs to prevent TB since the well-known structural information would be beneficial in the structure-based modification and design., (© 2022 Society for Applied Microbiology.)

In this work four vanadium complexes (compounds 1, 2, 3 and 4) and one molybdenum complex (compound 5) with hydrazone ligands derived from pyridoxal were synthesized and characterized. All compounds are mononuclear species, two of them (compounds 3 and 5) are dioxide complexes and the other three (compounds 1, 2 and 4) monoxide complexes. The vanadium atom of the compound 3 is five-coordinated and all the other compounds have a six coordinated environment polyhedron. The poses for the potential intercalation of the compounds 2 and 3 with DNA were obtained by using AutoDock software. Optimizations were also performed at PM6-D3H4 semi-empirical level whereas the study of the nature of the interaction was carried out by means of the Energy Decomposition Analysis and the Non-Covalent Interaction index by using in both cases Density Functional Theory computations. The cytotoxicity in lung cancer cells (A549 cell line) of all the compounds was also evaluated. After 24 h of treatment, vanadium complexes showed high values of IC 50 , between 419.93 ± 22.58 and 685.88 ± 46.55 μM. After 48 h, the results showed that the compound 3 had the lowest IC 50 value, 65.32 ± 9.95 μM, and the compound 2 the highest value, 375.28 ± 32.09 μM. The molybdenum complex showed the lowest IC 50 value at 48 h (11.22 ± 1.34 μM). The toxicity of the compounds 3, 4 and 5 was tested in vivo, using zebrafish model, and the molybdenum complex showed higher toxic effects than the studied vanadium complexes., (Copyright © 2022. Published by Elsevier Inc.)

Topoisomerase (Topo) accelerates cell growth and division, and has been a theoretical target for anti-cancer drugs for decades. A series of pyridoxal thiosemicarbazone (PLT) ligands were designed and synthesized, and the dependence of their antiproliferative activity on copper was investigated. The insertion of N-cyclohexyl-2-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methylene)-N-methylhydrazinecarbothioamide hydrochloride (compound 9) and Chlorido(N-cyclohexyl-2-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methylene)-N-methylhydrazinecarbothioamide hydrochloride-O,N,S)‑copper(II) nitrate (9-Cu complex) into Topo-I and Topo-II prevented uncoiling of DNA through hydrogen bonds and intermolecular forces. The combination of PLT derivatives and copper gluconate (CuGlu) improved their anti-tumour activity against a cell line with high expression of topoisomerase (SK-BR-3). The non-linear regression equations of the inhibitory activity and anti-tumour activity of Topo-I and Topo-IIɑ in SK-BR-3 cells had R 2 values of 0.93 and 0.94, respectively. In addition to lipophilicity, inhibition of topoisomerase also affected the activity of PLT ligands by coordinating with copper ions. At the cellular level, PLTs and CuGlu penetrate the cell membrane to form metabolites in the cell, thus selectively inhibiting the activity of Topo-I and Topo-IIɑ, and ultimately inhibiting cell division. These findings will inform the design of future anti-cancer thiosemicarbazone drugs., (Copyright © 2022. Published by Elsevier Inc.)

Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate-dependent enzymes (PLP-DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP-DEs in clinically relevant pathogens including challenging Gram-negative strains. Putative PLP-DEs with unknown function were exemplarily characterized via in-depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP-dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP-DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica. Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica. IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contributes to their functional differences in a Salmonella enterica host.

Ferroptosis and inflammation induced by cerebral hemorrhage result in an excessive inflammatory response and irreversible neuronal injury. Alleviating ferroptosis might be an effective way to prevent neuroinflammatory injury and promote neural functional recovery. Pyridoxal isonicotinoyl hydrazine (PIH), a lipophilic iron-chelating agent, has been reported to reduce excess iron-induced cytotoxicity. However, whether PIH could ameliorate the effects of hemorrhagic stroke is not completely understood. In the present study, the preventive effects of PIH in an intracerebral hemorrhage (ICH) mouse model were investigated. Neurological score, rotarod test, and immunofluorescence around the hematoma were assessed to evaluate the effects of PIH on hemorrhagic injury. The involvement of ferroptosis and inflammation was also examined in vitro to explore the underlying mechanism. Results showed that administration of PIH prevented neuronal cell death and reduced lipid peroxidation in Erastin-treated PC-12 cells. In vivo, mice treated with PIH after ICH attenuated neurological deficit scores. Additionally, we found PIH reduced ROS production, iron accumulation, and lipid peroxidation around the hematoma peripheral tissue. Meanwhile, ICH mice treated with PIH showed an upregulation of the key ferroptosis enzyme, glutathione peroxidase 4, and downregulation of cyclooxygenase-2. Moreover, PIH administration inhibited proinflammatory polarization and reduced interleukin-1 beta and tumor necrosis factor alpha in ICH mice. Collectively, these results demonstrated that PIH protects mice against hemorrhage stroke, which was associated with mitigation of inflammation and ferroptosis., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Hengli Zhang et al.)

Proteomic analysis of foodborne pathogen Listeria monocytogenes after treatment with three disinfectants based on ammonium salts of pyridoxal oxime (POD) reveal perturbation of cellular processes. These inhibitors caused disturbance in the synthesis of plasma membrane proteins and cell wall proteoglycans. Some of key proteins and proteoglycans from these two groups that are important for bacterial growth are down-regulated. Additionally, we demonstrated that the main bacterial toxin Listeriolysin O (LLO) is significantly down-regulated after treatment with each of three investigated inhibitors. These investigations confirm already postulated mechanism of action of POD-based inhibitors that results in disturbance of key cell surface proteins and proteoglycans in Gram-positive bacteria. Additionally, the use of some proteins such as LLO, as potential biomarker candidates of food poisoning with this bacterium is discussed., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest, (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the efficacy and safety of pyridoxal for treating West syndrome., Methods: We retrospectively investigated pyridoxal's efficacy and safety in 117 patients with West syndrome at Saitama Children's Medical Center from July 1993 to May 2016. Pyridoxal was administered at doses of 10-50 mg/kg/day. We evaluated seizure outcomes and electroencephalographic findings at 4 weeks after pyridoxal therapy. The responders were those with complete cessation of spasms for more than 4 weeks and those with resolution of hypsarrhythmia on EEG at 1-4 weeks after pyridoxal therapy., Results: Five of the 117 patients (4.3%) were responders. The median duration between pyridoxal therapy to spasm cessation was 6 (5-13) days. Among the responders, four had hypsarrhythmia resolution, no spasm relapse, and no other seizure types more than 2 years after pyridoxal therapy. One responder had partial seizures and spasm relapse. No serious adverse effects occurred. There were no significant differences in sex, etiologies, complication, other seizure types preceding the spasms, onset age of spasms, age of pyridoxal therapy, treatment lag, initial and maintenance doses of pyridoxal, and adverse effects between pyridoxal responders and non-responders., Conclusions: The efficacy rate of pyridoxal monotherapy as first-line treatment for West syndrome was low. However, pyridoxal therapy showed a rapid response within 1 week and was safe. We consider pyridoxal therapy as a kind of challenge therapy during the evaluation period concerning differential diagnosis and etiologies of West syndrome and immunological risks before adrenocorticotrophic hormone therapy or vigabatrin therapy., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

We found that a large inoculum of Cryptococcus gattii cells, when plated on Dulbecco's modified eagle's medium (DMEM) incorporated into agar, died within a few hours provided that DMEM agar plates had been stored in darkness for approximately 3 days after preparation. Standard conditions were developed for quantification of killing. The medium lost its fungicidal activity when exposed to visible light of wave length ∼400 nm. The amount of energy required was estimated at 5.8 × 104 joules @ 550 nm. Liquid DMEM conditioned by incubation over DMEM agar plates stored in darkness was fungicidal. We found that fungicidal activity was heat-stable (100°C). Dialysis tubing with MWC0 < 100 Daltons retained fungicidal activity. Neutral pH was required. Strains of Cryptococcus were uniformly sensitive, but some Candida species were resistant. Components of DMEM required for killing were pyridoxal and cystine. Micromolar amounts of iron shortened the time required for DMEM agar plates to become fungicidal when stored in the dark. Organic and inorganic compounds bearing reduced sulfur atoms at millimolar concentrations inhibited fungicidal activity. Our results point to a light-sensitive antifungal compound formed by reaction of pyridoxal with cystine possibly by Schiff base formation., (Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology 2018.)

In a mouse model, rifampicin and isoniazid combination treatment results in cholestatic liver injury that is associated with an increase in protoporphyrin IX, the penultimate heme precursor. Both ferrochelatase (FECH/Fech) and aminolevulinic acid synthase 1 (ALAS1/Alas1) are crucial enzymes in regulating heme biosynthesis. Isoniazid has recently been reported to upregulate Alas1 but downregulate Fech protein levels in mice; however, the mechanism by which isoniazid mediates disruption of heme synthesis has been unclear. Two metabolites of isoniazid, pyridoxal isonicotinoyl hydrazone (PIH, the isoniazid-vitamin B6 conjugate) and hydrazine, have been detected in the urine of humans treated with isoniazid. Here we show that, in primary human hepatocytes and the human hepatocellular carcinoma cell line HepG2/C3A, (1) isoniazid treatment increases Alas1 protein levels but decreases Fech levels; (2) hydrazine treatment upregulates Alas1 protein and Alas1 mRNA levels; (3) PIH treatment decreases Fech protein levels, but not Fech mRNA levels; and (4) PIH is detected after isoniazid treatment, with levels increasing further when exogenous vitamin B6 analogs are coadministered. In addition, the PIH-mediated downregulation of human FECH is associated with iron chelation. Together, these data demonstrate that hydrazine upregulates ALAS1, whereas PIH downregulates FECH, suggesting that the metabolites of isoniazid mediate its disruption of heme biosynthesis by contributing to protoporphyrin IX accumulation., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The breast cancer is the most common type of cancer in women. In this project, the breast cancer was transplanted in vivo with the TUBO cells. Then, the cancerous mice were treated by radiation of low frequency electromagnetic fields and injection of the Mn(II) complex of the N,N'-dipyridoxyl(1,2-diaminobenzene) Schiff base. Three different concentrations of the Mn(II) complex were used. Cytotoxicity and morphological alterations caused by the Mn(II) complex in the TUBO breast cancer cell line have been evaluated. Apoptotic properties of the Mn(II) complex was studied using the flow cytometry. The Mn(II) complex has a cytotoxic effect on cancer cells. Also, both of the Mn(II) complex and low frequency electromagnetic field induced apoptosis, which was confirmed by flow cytometry. Both of them result in considerable changes in the treated tissues such as decrease of the tumor mass, induction of apoptosis and decrease in number of the blood vessels., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the current study, we investigated the inhibitory activity of pyridoxine, pyridoxal, and pyridoxamine, against various digestive enzymes such as α-glucosidases, sucrase, maltase, and glucoamylase. Inhibition of these enzymes involved in the absorption of disaccharide can improve post-prandial hyperglycemia due to a carbohydrate-based diet. Pyridoxal (4.14 mg/mL of IC 50 ) had the highest rat intestinal α-glucosidase inhibitory activity, followed by pyridoxamine and pyridoxine (4.85 and 5.02 mg/mL of IC 50 , respectively). Pyridoxal demonstrated superior inhibition against maltase (0.38 mg/mL IC 50 ) and glucoamylase (0.27 mg/mLIC 50 ). In addition, pyridoxal showed significant higher α-amylase inhibitory activity (10.87 mg/mL of IC 50 ) than that of pyridoxine (23.18 mg/mL of IC 50 ). This indicates that pyridoxal can also inhibit starch hydrolyzing by pancreatic α-amylase in small intestine. Based on these in vitro results, the deeper evaluation of the anti-hyperglycemic potential of pyridoxine and its derivatives using Sprague-Dawley (SD) rat models, was initiated. The post-prandial blood glucose levels were tested two hours after sucrose/starch administration, with and without pyridoxine and its derivatives. In the animal trial, pyridoxal ( p < 0.05) had a significantly reduction to the postprandial glucose levels, when compared to the control. The maximum blood glucose levels (C max ) of pyridoxal administration group were decreased by about 18% (from 199.52 ± 22.93 to 164.10 ± 10.27, p < 0.05) and 19% (from 216.92 ± 12.46 to 175.36 ± 10.84, p < 0.05) in sucrose and starch loading tests, respectively, when compared to the control in pharmacodynamics study. The pyridoxal administration significantly decreased the minimum, maximum, and mean level of post-prandial blood glucose at 0.5 h after meals. These results indicate that water-soluble vitamin pyridoxine and its derivatives can decrease blood glucose level via the inhibition of carbohydrate-hydrolyzing and absorption-linked enzymes. Therefore, pyridoxal may have the potential to be used as a food ingredient for the prevention of prediabetes progression to type 2 diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The purinergic system consists of two large receptor families - P2X and P2Y. Both are activated by adenosine triphosphate (ATP), although presenting different functions. These receptors are present in several brain regions, including those involved in emotion and stress-related behaviors. Hence, they seem to participate in fear- and anxiety-related responses. However, few studies have investigated the purinergic system in threatening situations, as observed in contextual fear conditioning (CFC). Therefore, this study investigated the involvement of purinergic receptors in the expression and extinction of aversive memories. C57Bl/6 background mice were submitted to the CFC protocol. Wildtype (WT) mice received i.p. injection of either a nonselective P2 receptor (P2R) antagonist, P178 (10 or 30 mg/kg); a selective P2X7 receptor (P2X7R) antagonist, A438079 (10 mg/kg); a selective P2Y1 receptor (P2Y1R) antagonist, MRS2179 (10 mg/kg); or vehicle 10 min prior to or immediately after the extinction session. Additionally, P2X7R KO mice were tested in the CFC protocol. After P2R antagonist treatment, contextual fear recall increased, while acquisition of extinction was impaired. Similar results were observed with the selective P2X7R antagonist, but not with the selective P2Y1R antagonist. Interestingly, P2X7R KO mice showed increased contextual fear recall, associated with impaired acquisition of extinction, in accordance with pharmacologic P2X7R antagonism. Our results suggest that specific pharmacological or genetic blockade of P2X7R promotes anxiogenic-like effects, along with deficits in extinction learning. Thus, these receptors could present an alternative treatment of stress-related psychiatric disorders., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A comprehensive proteomic analysis of food borne pathogens after treatment with disinfectants based on ammonium salts of pyridinium oxime was performed. Changes in proteomes of the Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli, were evaluated. Up and down-regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanisms of action of these disinfectants. In the Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and cause significant alterations of membrane and cell surface proteins. On the other hand, intracellular proteins were more affected in the Gram-negative pathogen Escherichia coli. This research is a contribution to the investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

UVA-driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3-hydroxypyridine-derived chromophores including B 6 -vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA-induced photooxidative stress in human skin cells. Here, we report that the B 6 -vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA-induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ-H2AX (Ser139)], comet analysis indicated the formation of Fpg-sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA-driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B 6 -vitamers., (© 2017 The American Society of Photobiology.)

In a search for the safe vitamin carrier the PAMAM G3 dendrimer covalently substituted with 9 and 10 molecules of vitamin B7 (biotin) and B6 (pyridoxal), respectively (BC-PAMAM) was investigated. Dendrimer substitution with B-group vitamins significantly alters its biological properties as compared to native form. Observed effects on investigated cell parameters including morphology, adhesion, migration and ATP level were different for normal human fibroblasts (BJ) and squamous cell carcinoma (SCC-15) cell lines. BC-PAMAM revealed significantly less pronounced effects on investigated parameters, particularly at higher concentrations (5-50μM), which is relevant with its lower positive surface charge, as compared with native form. The bioconjugate, up to 50μM concentration, appeared to be a safe vitamin carrier to normal fibroblasts, without significant effect on their adhesion, shape and migration as well as on intracellular ATP level. In SCC-15 cells BC-PAMAM, at low concentrations (0.1-0.5μM), altered the cell shape and increase adhesion, whereas at higher concentrations opposite effects were seen. Measurements of cellular level of ATP showed that higher resistance of cancer cells to toxic effects of native PAMAM dendrimers may be due to higher energy supply of cancer cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Organophosphorus (OP) nerve agents (sarin, tabun VX and soman) inhibit the enzyme acetylcholinesterase (AChE, EC 3.1.1.7) by binding to its active site while preventing neurotransmission in the cholinergic synapses. The protection and treatment of this kind of poisoning are still a challenge as we are yet to discover an antidote that would be effective in all cases of poisoning. To aid the search for more efficient antidotes, we evaluated the ability of nine pyridoxal oxime derivatives, prepared by a novel synthetic pathway, to reactivate recombinant human AChE and the related purified human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) inhibited by VX, tabun and paraoxon. Oximes are derivatives of vitamin B6 bearing a phenacyl moiety attached to the quaternary nitrogen atom and having various substituents on the phenyl ring. As the results have shown, the tested oximes were in general more efficient in the reactivation of OP-inhibited BChE than AChE. The highest observed rate was in the case of VX-inhibited BChE reactivation, where k obs was 0.0087min -1 and the reactivation maximum of 90% was achieved within 5h. The cholinesterases displayed a binding affinity for these derivatives in a μmolar range no matter the substituent on their rings which was in accordance with the molecular modelling results showing a similar binding pattern for all oximes within the active site of both AChE and BChE. Such a positioning reveals also that hydroxy and a metoxy substituents at the vicinity of the oxime moiety present a possible steric hindrance explaining the reactivation results., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

As commented by the Nobelist James Black that "The most fruitful basis of the discovery of a new drug is to start with an old drug", drug repurposing represents an attractive drug discovery strategy. Despite the success of several repurposed drugs on the market, the ultimate therapeutic potential of a large number of non-cancer drugs is hindered during their repositioning due to various issues including the limited efficacy and intellectual property. With the increasing knowledge about the pharmacological properties and newly identified targets, the scaffolds of the old drugs emerge as a great treasure-trove towards new cancer drug discovery. In this review, we summarize the recent advances in the development of novel small molecules for cancer therapy by scaffold repurposing with highlighted examples. The relevant strategies, advantages, challenges and future research directions associated with this approach are also discussed.

H2S is produced mainly by two enzymes:cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), using L-cysteine (L-Cys) as the substrate. In this study, we investigated the role of H2S in gastric accommodation using CBS(+/-) mice, immunohistochemistry, immunoblot, methylene blue assay, intragastric pressure (IGP) recording and electrical field stimulation (EFS). Mouse gastric fundus expressed H2S-generating enzymes (CBS and CSE) and generated detectable amounts of H2S. The H2S donor, NaHS or L-Cys, caused a relaxation in either gastric fundus or body. The gastric compliance was significantly increased in the presence of L-Cys (1 mM). On the contrary, AOAA, an inhibitor for CBS, largely inhibited gastric compliance. Consistently, CBS(+/-) mice shows a lower gastric compliance. However, PAG, a CSE inhibitor, had no effect on gastric compliances. L-Cys enhances the non-adrenergic, non-cholinergic (NANC) relaxation of fundus strips, but AOAA reduces the magnitude of relaxations to EFS. Notably, the expression level of CBS but not CSE protein was elevated after feeding. Consistently, the production of H2S was also increased after feeding in mice gastric fundus. In addition, AOAA largely reduced food intake and body weight in mice. Furthermore, a metabolic aberration of H2S was found in patients with functional dyspepsia (FD). In conclusion, endogenous H2S, a novel gasotransmitter, involves in gastric accommodation.

Vitamin B6 in the form of pyridoxine (PN) is one of the most widespread pharmacological therapies for inherited diseases involving pyridoxal phosphate (PLP)-dependent enzymes, including primary hyperoxaluria type I (PH1). PH1 is caused by a deficiency of liver-peroxisomal alanine: glyoxylate aminotransferase (AGT), which allows glyoxylate oxidation to oxalate leading to the deposition of insoluble calcium oxalate in the kidney. Only a minority of PH1 patients, mostly bearing the F152I and G170R mutations, respond to PN, the only pharmacological treatment currently available. Moreover, excessive doses of PN reduce the specific activity of AGT in a PH1 cellular model. Nevertheless, the possible effect(s) of other B6 vitamers has not been investigated previously. Here, we compared the ability of PN in rescuing the effects of the F152I and G170R mutations with that of pyridoxamine (PM) and PL. We found that supplementation with PN raises the intracellular concentration of PN phosphate (PNP), which competes with PLP for apoenzyme binding leading to the formation of an inactive AGT-PNP complex. In contrast, PNP does not accumulate in the cell upon PM or PL supplementation, but higher levels of PLP and PM phosphate (PMP), the two active forms of the AGT coenzyme, are found. This leads to an increased ability of PM and PL to rescue the effects of the F152I and G170R mutations compared with PN. A similar effect was also observed for other folding-defective AGT variants. Thus, PM and PL should be investigated as matter of importance as therapeutics for PH1 patients bearing folding mutations., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin-pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander's coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%-35% of the total nuclei area at all investigated concentrations, with lower level (15%-25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%-20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, [Fe(B)(L)](NO3) (1-5), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L(1) is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridine (1-3) and H2L(2) is 2-[(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 μM with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Vitamin B6 compound, pyridoxine (PN), has shown antitumor action. Our previous experiments showed that PN induces expression of insulin-like growth factor binding protein-3 to arrest proliferation and induce cell death. This induction is inhibited by the p53-specific inhibitor pifithrin-α. Here, we report that another B6 compound, pyridoxal (PL), strongly inhibited MCF-7 cell growth compared to PN. PL induced the G0/G1 arrest and the accumulation of subG1 population. Although p53 mRNA was not changed by PL, 0.5 mM PL increased the protein level in MCF-7 cells. The cell growth suppression by 0.5 mM PL did not occur when p53 expression was knocked down using siRNA. Together, these data suggest that PL accumulate p53 and PL-induced cell growth suppression is dependent on p53 in MCF-7 breast cancer cells.

Increasing evidence indicates vitamin B6 acts as a protective factor against colon cancer. However, the mechanisms of the effect of vitamin B6 are poorly understood. The present preliminary study using DNA microarray and real-time PCR indicates p21 mRNA is upregulated in human colon carcinoma (HT29) cells exposed to pyridoxal (PL, 500 µM). A similar effect was observed in human epithelial colorectal adenocarcinoma (Caco2) cells, human colon adenocarcinoma (LoVo) cells, human embryonic kidney (HEK293T) cells, and human hepatoma (HepG2) cells. Adding other B6-vitamers such as pyridoxal 5'-phosphate (PLP), pyridoxine (PN), and pyridoxamine (PM) caused no such effect. In order to understand the mechanism of higher mRNA expression of p21 by PL, effect of PL on the p53 activation was examined (the upstream factor for p21 mRNA transcription) in HT29 cells, LoVo cells, and HepG2 cells. PL increased the phosphorylated p53 protein levels (active form) in whole-cell lysates and the nuclei of the cells. Noteworthy, the consumption of a vitamin B6-deficient diet for 5 weeks significantly reduced p21 mRNA levels and tended to reduce phosphorylated p53 protein levels (P=0.053) in the colons of mice compared to a diet with adequate vitamin B6. Thus, these results suggest vitamin B6 plays a role in increasing p21 gene expression via p53 activation in several cancer cells and the mouse colon.

We have selectively synthesized by Pictet-Spengler condensation of tryptophan and pyridoxal the four stereoisomers of a pyridoxal β-carboline derivative that was designed to inhibit the proliferation of Plasmodium falciparum. Biological investigation of the four compounds revealed that they all inhibit the growth of P. falciparum. With an IC50 value of 8 ± 1 μM, the highest inhibitory effect on the proliferation of the parasite was found for the 1,3-trans-substituted tetrahydro-β-carboline that was obtained from d-tryptophan. Lower activity was found for its enantiomer, while the two diastereomeric cis-products were markedly less effective. Apparently a distinct spacial orientation of the carboxyl group of the substituted tetrahydropyridine unit of the compounds is needed for high activity, while the absolute configuration of the molecules is of lesser importance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

The rise in non-heme iron (NHI) concentration observed in skeletal muscle of aging rodents is thought to contribute to the development of sarcopenia. The source of the NHI has not been identified, nor have the physiological ramifications of elevated iron status in aged muscle been directly examined. Therefore, we assessed plantaris NHI and heme iron (HI) levels in addition to expression of proteins involved in iron uptake (transferrin receptor-1; TfR1), storage (ferritin), export (ferroportin; FPN), and regulation (iron regulatory protein-1 (IRP1) and -2 (IRP2)) of male F344xBN F1 rats (n=10/group) of various ages (8, 18, 28, 32, and 36 months) to further understand iron regulation in aging muscle. In a separate experiment, iron chelator (pyridoxal isonicotinoyl hydrazone; PIH) or vehicle was administered to male F344xBN F1 rats (n=8/group) beginning at 30 months of age to assess the impact on plantaris muscle mass and function at ~36 months of age. Principle findings revealed the increased NHI concentration in old age was consistent with concentrating effects of muscle atrophy and reduction in HI levels, with no change in the total iron content of the muscle. The greatest increase in muscle iron content occurred during the period of animal growth and was associated with downregulation of TfR1 and IRP2 expression. Ferritin upregulation did not occur until senescence and the protein remained undetectable during the period of muscle iron content elevation. Lastly, administration of PIH did not significantly (p>0.05) impact NHI or measures of muscle atrophy or contractile function. In summary, this study confirms that the elevated NHI concentration in old age is largely due to the loss in muscle mass. The increased muscle iron content during aging does not appear to associate with cytosolic ferritin storage, but the functional consequences of elevated iron status in old age remains to be determined., (© 2013.)

Hydrogen sulfide (H2S), mainly produced by cystathionine γ-lyase (CSE) in vascular system, emerges as a novel gasotransmitter exerting anti-inflammatory and anti-atherosclerotic effects. Alterations of CSE/H2S pathway may thus be involved in atherosclerosis pathogenesis. However, the underlying mechanisms are poorly understood. The present study showed that the levels of CSE mRNA and protein expression, as well as H2S production were decreased in ox-LDL-treated macrophage. CSE overexpression reduced the ox-LDL-stimulated tumor necrosis factor-α (TNF-α) generation in Raw264.7 and primary macrophage while CSE knockdown enhanced it. Exogenous supplementation of H2S with NaHS and Na2S also decreased the production of TNF-α and intercellular adhesion molecule-1 (ICAM-1) in ox-LDL-stimulated macrophage, and alleviated the adhesion of macrophage to endothelial monolayer. Cysteine, a CSE preferential substrate for H2S biosynthesis, produced similar effects on the pro-inflammatory cytokine generation, which were reversed by CSE inhibitors PAG and BCA, respectively. Moreover, NaHS and Na2S attenuated the phosphorylation and degradation of IκBα and p65 nuclear translocation, as well as JNK activation caused by ox-LDL. The JNK inhibitor suppressed the NF-κB transcription activity in ox-LDL-treated cells. Furthermore, inhibitors of NF-κB (PDTC), ERK (U0126 and PD98059) and JNK (SP600125) partially blocked the suppression by ox-LDL on the CSE mRNA levels. Taken together, the findings demonstrate that ox-LDL may down-regulate the CSE/H2S pathway, which plays an anti-inflammatory role in ox-LDL-stimulated macrophage by suppressing JNK/NF-κB signaling. The study reveals new therapeutic strategies for atherosclerosis, based on modulating CSE/H2S pathway., (© 2013.)

Vanadium compounds can exert anticancer effects, partly due to inhibition of tyrosine phosphatases. Here, we report the effect of N,N'-ethylenebis (pyridoxylideneiminato) vanadium (IV) complex (Pyr2 enV(IV)), that induced 93% and 57% of cell mortality in A375 (human melanoma) and A549 (human lung carcinoma) cells, respectively; the mortality was <24% in other cancer cell lines and in human normal epidermal keratinocytes, lung cells and peripheral blood mononuclear cells. The mechanism of Pyr2 enV(IV) effect relied on apoptosis induction; this was triggered by ROS increase, followed by mitochondrial membrane depolarization. Indeed, the addition of N-acetyl cysteine to cell cultures abated Pyr2 enV(IV)-induced apoptosis. These results disclose the pro-apoptotic activity of Pyr2 enV(IV) and its mechanism, relying on intracellular ROS increase., (Copyright © 2013 Wiley Periodicals, Inc.)

Increasing evidence suggests that dietary vitamin B6 is linked to the prevention of cancer and cardiovascular disease. However, the molecular mechanisms involved in this process are not yet understood. Preliminary results in the current study indicated, following DNA microarray analysis and quantitative PCR, that insulin‑like growth factor‑binding protein 1 (IGFBP1) mRNA is upregulated in HT29 colon carcinoma cells exposed to pyridoxal (PL, 500 µM). IGFBP1 is secreted from the liver and is hypothesized to exert a protective role in the development of cancer and cardiovascular disease. Thus, further experiments were performed to investigate the effect of PL on the expression of IGFBP1 in HepG2 hepatocellular carcinoma cells. The addition of PL (500 µM) markedly increased the expression of IGFBP1 mRNA in HepG2 cells at 6, 12 and 24 h (P<0.01), whereas other vitamers (500 µM), including pyridoxal 5'‑phosphate (PLP), pyridoxine (PN) and pyridoxamine (PM), caused no such effect. The expression of the IGFBP1 protein in the cell lysate and culture medium was elevated in the presence of PL. PL elevated expression of the active form of ERK1 protein, p‑ERK1, and the p‑c‑Jun protein, a downstream factor of ERK. Furthermore, IGFBP1 expression, elevated by PL, was suppressed by PD98059, an ERK inhibitor. Higher expression of IGFBP1 protein by PL was suppressed by cycloheximide. These results suggest that PL may induce the expression of IGFBP1 in hepatoma cells via a mechanism involving the ERK/c‑Jun pathway.

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B-vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed., (© 2013 American Institute of Chemical Engineers.)

Carboxylic acid derivatives of pyridoxal were developed as potent P2X(1) and P2X(3) receptor antagonists with modifications of a lead compound, pyridoxal-5'-phosphate-6-azophenyl-2',5'-disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5'-phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24-28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X(3) receptors with 100 nM range of IC(50) values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X(1) and human P2X(3) receptors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A third-generation polyamidoamine dendrimer (PAMAM G3) was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G3(9B)), and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G3(9B10P)), were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using (1)H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM) was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15) cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet) and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 μM (NR) and 10 μM (XTT), and BC-PAMAM was not cytotoxic up to 50 μM (both assays) for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 μM and BC-PAMAM at 10 μM in both cell lines) corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules significantly decreases that effect, confirming that PAMAM G3 is a useful candidate as a carrier for active biocompound delivery.

Purpose: Corneal cross-linking (CXL) is a treatment for keratoconus that eliminates the need for keratoplasty in most patients. However, its molecular mechanisms remain under study. Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis., Methods: Corneas of young rabbits and sharks were pretreated with pyridoxal hydrochloride and copper ions before CXL. Two known inhibitors of AGE formation, aminoguanidine and rifampicin, were applied during CXL in the treatment solution. Tensile strength tests were conducted after these experiments to detect diminished or accentuated corneal stiffening after CXL. SDS-PAGE was performed on type I collagen cross-linked in the absence and presence of AGE inhibitors., Results: Pretreatment with pyridoxal hydrochloride resulted in significantly higher corneal stiffening after CXL. AGE inhibitors significantly diminished cross-linking as detected by both tensile strength measurements using whole corneas and gel electrophoresis of in vitro cross-linking of type I collagen in solution, in the presence and absence of the inhibitors. Rifampicin inhibited CXL more significantly than aminoguanidine in gel electrophoresis and tensile strength tests, confirming recent findings on its efficacy as an AGE inhibitor., Conclusions: Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.

Background: H(2)S synthesis inhibitors (HSSI) have been shown to impact respiratory control. For instance, the HSSI hydroxylamine (HA) decreases the respiratory discharge rate from isolated medullary sections, and HA in addition to other HSSIs propargylglycine and amino-oxyacetic acid (AOAA) have been found to reduce hypoxic responsiveness., Objectives: The aim of this study was to determine if administration of HSSIs could improve respiratory stability in an intact organism prone to recurrent central apneas., Methods: Saline and HSSI compounds were administered to C57BL/6J mice (n = 24), a strain predisposed to recurrent central apneas, prior to measurement of hypoxic and posthypoxic ventilatory behavior., Results: Administration of HA and AOAA resulted in a significantly smaller percentage of animals expressing one or more apneas during reoxygenation compared to saline control, and animals given AOAA demonstrated a smaller coefficient of variation for frequency during reoxygenation, a marker suggesting greater respiratory stability. This occurred despite varying effects of the three HSSI compounds on hypoxic ventilatory response., Conclusions: Instability and pause expression are improved by targeting H(2)S synthesis, an effect not predicted by effects on hypoxic responsiveness., (Copyright © 2011 S. Karger AG, Basel.)

Oxidative damage is considered to play an important role in the pathogenesis of several diseases, such as diabetes mellitus (DM), atherosclerosis, cardiovascular complications and chronic renal failure. DM is associated with the oxidative stress and formation of advanced glycation end products (AGEs). Different drugs inhibit oxidative stress and formation of advanced glycation end products. Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of DM. Recent reports show a pro-oxidant activity of AG. Therefore we examined the effect of structural analogue of AG, its Schiff base with pyridoxal-pyridoxylidene aminoguanidine (PAG) on the level of selected markers of oxidative stress. We found that PAG decreased total damage to DNA in controls as well as in diabetic group of rats. However, we also found that PAG supplementation increases susceptibility of lipoproteins to oxidation and formation of conjugated dienes in both, diabetic as well as control animals. Its administration to diabetic rats decreases antioxidant capacity of plasma. Therefore, it is necessary to search for other structural modifications of AG that would combine its higher anti-diabetic activity with less toxicity.

Taking into account the ability of vitamin B6 vitamers (i.e., pyridoxal, pyridoxine, pyridoxamine) to stimulate adrenomedullary catecholamine outflow and biochemical and cardiovascular changes consistent with adrenoceptor stimulation, this study was undertaken in mice to verify the possibility that pyridoxal (PL), the most potent of the B6 vitamers, could enter into an adverse vitamin-drug interaction when coadministered with a cardioactive drug, and that compounds like taurine (TAU) and labetalol (LAB) could be protective. To this end, mice were treated with PL (200 mg/kg) and isoproterenol (ISO 200 mg/kg), with and without a pretreatment with TAU (300 mg/kg), LAB (30 mg/kg), or TAU plus LAB, and monitored for heart rate, electrocardiographic (ECG) and myocardial electrolyte changes. PL plus ISO lowered the LD50 of ISO and magnified the changes in heart rate, ECG and myocardial electrolytes seen with ISO alone. Pretreating rats with TAU or TAU plus LAB attenuated the cardiovascular changes induced by PL plus ISO to a greater extent than LAB alone. The attenuating actions of TAU and LAB probably reflect independent and complementary mechanisms of action.

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.

The aim of this study was to analyze the ECG time intervals in the course of the development of chronic anthracycline cardiomyopathy in rabbits. Furthermore, this approach was employed to study the effects of a model cardioprotective drug (dexrazoxane) and two novel iron chelating compounds--salicylaldehyde isonicotinoyl hydrazone (SIH) and pyridoxal 2-chlorobenzoyl hydrazone (o-108). Repeated daunorubicin administration induced a significant and progressive prolongation of the QRS complex commencing with the eighth week of administration. At the end of the study, we identified a significant correlation between QRS duration and the contractility index dP/dt(max) (r = -0.81; P<0.001) as well as with the plasma concentrations of cardiac troponin T (r = 0.78; P<0.001). In contrast, no alterations in ECG time intervals were revealed in the groups co-treated with either dexrazoxane or both novel cardioprotective drugs (SIH, o-108). Hence, in this study, the QRS duration is for the first time shown as a parameter suitable for the non-invasive evaluation of the anthracycline cardiotoxicity and cardioprotective effects of both well established and investigated drugs. Moreover, our results strongly suggest that novel iron chelators (SIH and o-108) merit further study as promising cardioprotective drugs against anthracycline cardiotoxicity.

Background and Purpose: The anticancer drugs doxorubicin and bleomycin are well-known for their oxidative stress-mediated side effects in heart and lung, respectively. It is frequently suggested that iron is involved in doxorubicin and bleomycin toxicity. We set out to elucidate whether iron chelation prevents the oxidative stress-mediated toxicity of doxorubicin and bleomycin and whether it affects their antiproliferative/proapoptotic effects., Experimental Approach: Cell culture experiments were performed in A549 cells. Formation of hydroxyl radicals was measured in vitro by electron paramagnetic resonance (EPR). We investigated interactions between five iron chelators and the oxidative stress-inducing agents (doxorubicin, bleomycin and H(2)O(2)) by quantifying oxidative stress and cellular damage as TBARS formation, glutathione (GSH) consumption and lactic dehydrogenase (LDH) leakage. The antitumour/proapoptotic effects of doxorubicin and bleomycin were assessed by cell proliferation and caspase-3 activity assay., Key Results: All the tested chelators, except for monohydroxyethylrutoside (monoHER), prevented hydroxyl radical formation induced by H(2)O(2)/Fe(2+) in EPR studies. However, only salicylaldehyde isonicotinoyl hydrazone and deferoxamine protected intact A549 cells against H(2)O(2)/Fe(2+). Conversely, the chelators that decreased doxorubicin and bleomycin-induced oxidative stress and cellular damage (dexrazoxane, monoHER) were not able to protect against H(2)O(2)/Fe(2+)., Conclusions and Implications: We have shown that the ability to chelate iron as such is not the sole determinant of a compound protecting against doxorubicin or bleomycin-induced cytotoxicity. Our data challenge the putative role of iron and hydroxyl radicals in the oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin and have implications for the development of new compounds to protects against this toxicity.

Iron chelation is the only pharmacological intervention against anthracycline cardiotoxicity whose effectiveness has been well documented both experimentally and clinically. In this study, we aimed to assess whether pyridoxal 2-chlorobenzoyl hydrazone (o-108, a strong iron chelator) can provide effective protection against daunorubicin (DAU)-induced chronic cardiotoxicity in rabbits. First, using the HL-60 leukemic cell line, it was shown that o-108 has no potential to blunt the antiproliferative efficacy of DAU. Instead, o-108 itself moderately inhibited cell proliferation. In vivo, chronic DAU treatment (3 mg/kg weekly for 10 weeks) induced mortality (33%), left ventricular (LV) dysfunction, a troponin T rise, and typical morphological LV damage. In contrast, all animals treated with 10 mg/kg o-108 before DAU survived without a significant drop in the LV ejection fraction (63.2 +/- 0.5 versus 59.2 +/- 1.0%, beginning versus end, not significant), and their cardiac contractility (dP/dt(max)) was significantly higher than in the DAU-only group (1131 +/- 125 versus 783 +/- 53 kPa/s, p < 0.05), which corresponded with histologically assessed lower extent and intensity of myocardial damage. Although higher o-108 dose (25 mg/kg) was well tolerated when administered alone, in combination with DAU it led to rather paradoxical and mostly negative results regarding both cardioprotection and overall mortality. In conclusion, we show that shielding of free intracellular iron using a potent lipophilic iron chelator is able to offer a meaningful protection against chronic anthracycline cardiotoxicity. However, this approach lost its potential with the higher chelator dose, which suggests that iron might play more complex role in the pathogenesis of this disease than previously assumed.

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.

Taurine (2-aminoethanesulfonic acid) is known to attenuate the release of adrenomedullary catecholamines and ensuing hepatic glycogenolysis and hyperglycemia induced by pyridoxal in the rat. Using this animal model, the present study was undertaken to assess the impact that simple structural modifications of the taurine molecule might have on its antagonistic actions against PL. Removal of the amino group (ethanesulfonic acid) or shortening the carbon chain by one methylene (2-aminomethanesulfonic acid) raised the protective actions of taurine. While N-alkylation (N-methyltaurine) or replacement of the amino group by hydroxyl (isethionic acid) had a lowering effect, substituting a sulfhydryl group (2-mercaptoethanesulfonic acid) for the 2-amino group abolished all antagonistic properties associated with taurine. The sulfinic acid analog (hypotaurine) was equipotent with taurine, but the carboxylate isostere (beta-alanine) was inactive. Propranolol, a nonspecific beta-adrenoceptor antagonist, enhanced the antiglycogenolytic effect of taurine and of all structurally related compounds capable of attenuating the outflow of adrenal catecholamines elicited by pyridoxal.

Although vitamin B6 has been supposed to have anti-inflammatory effects, the molecular mechanism is not fully understood. To explore the mechanism of anti-inflammatory effects of vitamin B6, we have examined the effect of vitamin B6 on lipopolysaccharide (LPS)-stimulated inflammatory response in RAW 264.7 macrophages. This study demonstrated that vitamin B6 (pyridoxal) pretreatment of RAW cells inhibited LPS-induced expression of iNOS and COX-2 at the mRNA and protein levels. Vitamin B6 inhibited LPS-induced nuclear translocation of the NF-kappaB, the proinflammatory transcription factor, with reduction of the extent of LPS-induced IkappaBalpha degradation in RAW cells. Although vitamin B6 did not affect cellular proteasome activity, in vitro phosphorylation analysis with glutathione S-transferase-fused IkappaBalpha has shown that vitamin B6 suppressed LPS-induced IkappaB kinase activation. Furthermore, we demonstrated that elevating dietary vitamin B6 suppressed NO production in vivo in response to LPS administration. These observations suggest that the anti-inflammatory effect of vitamin B6 is mediated by suppression of NF-kappaB activation.

Risk of cardiotoxicity is the most serious drawback of the clinical usefulness of anthracycline antineoplastic antibiotics, which however, remain among the most powerful and widely employed anticancer drugs. In this study we have used daunorubicin-induced cardiomyopathy in rabbits as a model to investigate possible cardioprotective effects of pyridoxal isonicotinoyl hydrazone (PIH)-a principal representative of a novel group of aroylhydrazone iron chelators. Three groups of animals were used: a control group (n=11; i.v. saline), daunorubicin-treated animals (n=11; 3mg/kg, i.v.), and animals pretreated with PIH (n=9, 25 mg/kg, i.p.) 60 min before daunorubicin administration. All substances were administered once weekly for 10 weeks. Repeated administration of daunorubicin caused premature death in four animals and induced conspicuous histopathological changes in the myocardium, progressive and significant impairment of systolic heart function (a decrease in left ventricular dP/dt(max), ejection fraction, an increase in the pre-ejection period/left ventricular ejection time index), and a gradual increase in cardiac troponin T plasma concentrations. On the contrary, all the PIH-treated animals have survived all daunorubicin applications. Furthermore, in this group, the daunorubicin-induced cardiac changes were in most functional, biochemical as well as morphological parameters less pronounced than in the group receiving daunorubicin alone. Hence, PIH and other aroylhydrazones merit further investigation as potentially protective agents against anthracycline-induced cardiotoxicity.

We previously demonstrated anti-angiogenic activity of pyridoxal 5'-phosphate (PLP) and pyridoxal (PL) using in vitro and ex vivo angiogenesis models and anti-colon tumor effect of vitamin B(6) in mice. There is growing evidence that endothelial progenitor cells (EPCs) in circulation contribute to tumor-induced angiogenesis. It is of importance to clarify whether EPC differentiation is involved in the mechanism of anti-angiogenic effect of vitamin B(6). In this study, we investigated the effect of vitamin B(6) on angiogenesis in embryonic stem cell-derived embryoid bodies (EBs). PLP suppressed angiogenesis in EBs at >or=100 microM. Among the vitamin B(6) compounds at the concentration of 200 microM, PL, as well as PLP, was the most effective suppressor of vasculogenesis, while pyridoxine was inactive. These results were consistent with their effects in rat aorta and endothelial cell assays. Interestingly, pyridoxamine (PM), which had no effect in rat aorta and endothelial cell assays, also exerted a significant suppressive effect in this model. This study demonstrated an inhibitory effect of PM on vasculogenesis in this EB model as well as PLP and PL, and suggests that suppression of EPC differentiation is at least in part responsible for the mechanism of the anti-angiogenesis effect of vitamin B(6).