Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

doi:10.1016/0304-4165(88)90197-3

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 967, Issue 1, 13 October 1988, Pages 122-129

https://doi.org/10.1016/0304-4165(88)90197-3 Get rights and content

Abstract

A model consisting of ⁵⁹Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of ⁵⁹Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent ⁵⁹Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides perpared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate ⁵⁹Fe release from ⁵⁹Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme ⁵⁹Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrzaones. Several compounds proved to be very potent in mobilizing ⁵⁹Fe. These included hydrazones derived from 2-hydroxyl-1-naphthylaldehyde and benzoic acid hydrazide, p-hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m-fluorobenzyol hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.

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Citation Excerpt :
However, the utilization of cisplatin and its derivatives as successful anticancer drugs is often hindered by severe side effects, lack in resistance, and narrow activity range, leading to further findings for other metal based anticancer compounds [3–9]. Vanadium, being naturally present in the human body, has effective role in therapeutic purposes; depending on the type of complex, nature of the ligands, and route of administration, it received increasing attention in many biological applications [10–21]. Among all, the oxidovanadium complexes of VVO3+ and VIVO2+ ions have the utmost attention because their wide distribution in all organisms and role in many biological functions such as haloperoxidation [22], nitrogen fixation [23], phosphorylation [24], glycogen metabolism [25–27].
Herein we report the synthesis of five new mononuclear mixed ligand oxidovanadium(IV) complexes [V^IVO(L¹⁻³)(L^NN)] (1–5) with tridentate O,N,O-donor aroylhydrazones as main ligand (H₂L^1–3) and N,N-chelating 2,2′-bipyridine (bipy) and 1,10-phenanthroline (phen) as co-ligand (L^NN). The complexes were characterized by elemental and thermogravimetric analysis (TGA), IR, UV–vis, and electron paramagnetic resonance (EPR) spectroscopy, electrospray ionization-mass spectrometry (ESI–MS) and cyclic voltammetry (CV). The structure of 1–5 was confirmed by single crystal X-ray analysis and also optimized by density functional theory (DFT) methods. At physiological pH an equilibrium [V^IVO(L^1–3)(L^NN)] + H₂O ⇄ [V^IVO(L^1–3)(H₂O)] + L^NN, shifted towards left, is established, with water molecule that could be replaced by the biomolecules of the organism. The studies on the interaction with two proteins, lysozyme (Lyz) chosen as a representative model of a small protein, and human serum albumin (HSA) show that two types of binding are possible: a non-covalent binding through the accessible residues on protein surface with [V^IVO(L^1–3)(L^NN)] keeping its octahedral structure, and a covalent binding upon the replacement of water in [V^IVO(L^1–3)(H₂O)] with His-N donors to form V^IVO(L^1–3)(HSA). In vitro cytotoxicity of ligands and complexes were screened against human cervical cancer (HeLa) (IC₅₀ = 7.39–15.13 μM), colon cancer (HT-29) (IC₅₀ = 11.04–28.20 μM) and mouse embryonic fibroblast (NIH-3T3) cell lines (IC₅₀ = 62.22–87.75 μM) by MTT assay. Particularly, 5 showed higher cytotoxicity than cisplatin and cyclophosphamide, with an IC₅₀ of 7.39 ± 1.21 μM and 11.04 ± 0.29 μM against HeLa and HT-29.
Bone morphogenetic protein 6-mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro
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Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.
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A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The “gold standard” histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca⁺² channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.
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Higher levels of copper, reduced glutathione (GSH) and reactive oxygen species (ROS) observed in cancer cells than in normal cells, favor the idea of developing copper ionophores as prooxidative anticancer agents (PAAs) to hit the altered redox homeostasis (redox Achilles heel) of cancer cells. In this work, we used salicylaldehyde isonicotinoyl hydrazone (SIH-1) as a basic scaffold to design Cu(II) ionophores with tunable chelation and release of Cu(II) by introducing electron-withdrawing nitro and electron-donating methoxyl groups in the para position to phenolic hydroxyl, or by blocking the phenolic hydroxyl site using methyl. These molecules were used to probe how chelation and release of copper influence their ionophoric role and ability to target redox Achilles heel of cancer cells. Among these molecules, SIH-1 was identified as the most potent Cu(II) ionophore to kill preferentially HepG2 cells over HUVEC cells, and also superior to clioquinol, a copper ionophore evaluated in clinical trials, in terms of its relatively higher cytotoxicity and better selectivity. Higher oxidative potential, despite of lower stability constant, of the Cu(II) complex formed by SIH-1 than by the other molecules, is responsible for its stronger ability in releasing copper by GSH, inducing redox imbalance and triggering mitochondria-mediated apoptosis of HepG2 cells. This work gives useful information on how to design copper ionophores as PAAs for selective killing of cancer cells.
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Mycobacterium tuberculosis (Mtb) and the Human Immunodeficiency Virus (HIV) pose a major public health threat. The 2015 World Health Organization (WHO) report estimates that one in three HIV deaths is due to Mtb, the causative agent of Tuberculosis (TB). The lethal synergy between these two pathogens leads to a decline in the immune function of infected individuals as well as a rise in morbidity and mortality rates. The deadly interaction between TB and HIV, along with the heightened emergence of drug resistance, drug-drug interactions, reduced drug efficacy and increased drug toxicity, has made the therapeutic management of co-infected individuals a major challenge. Hence, the development of new drug targets and/or new drug leads are imperative for the effective therapeutic management of co-infected patients. Here, we report the characterization of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (311), a known inhibitor of HIV-1 replication and transcription as a new inhibitor of methionine aminopeptidases (MetAPs) from Mycobacterium tuberculosis: MtMetAP1a and MtMetAP1c. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. The essential role of MetAP in microbes makes it a promising chemotherapeutic target. We demonstrated that 311 is a potent and selective inhibitor of MtMetAP1a and MtMetAP1c. Furthermore, we found that 311 is active against replicating and aged non-growing Mtb at low micromolar concentrations. These results suggest that 311 is a promising lead for the development of novel class of therapeutic agents with dual inhibition of TB and HIV for the treatment of TB-HIV co-infection.
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2014, Free Radical Biology and Medicine
Citation Excerpt :
It should be noted that the mobilization of iron through MRP1 occurs only in the event of an NO insult, and not under other conditions. Although iron efflux from cells has been described for many years in the presence and absence of pharmacological iron chelators [16,134,135], and ferroportin-1 has been shown to be responsible for physiological iron export [104,136,137], NO-mediated iron release through MRP1 is a distinct process that can be differentiated in three major ways. First, ferroportin-1 is expressed in high levels in macrophages, placenta, and hepatocytes [104,136,137], whereas MRP1 is ubiquitously expressed in tissues [125].
Nitrogen monoxide (NO) is vital for many essential biological processes as a messenger and effector molecule. The physiological importance of NO is the result of its high affinity for iron in the active sites of proteins such as guanylate cyclase. Indeed, NO possesses a rich coordination chemistry with iron and the formation of dinitrosyl–dithiolato iron complexes (DNICs) is well documented. In mammals, NO generated by cytotoxic activated macrophages has been reported to play a role as a cytotoxic effector against tumor cells by binding and releasing intracellular iron. Studies from our laboratory have shown that two proteins traditionally involved in drug resistance, namely multidrug-resistance protein 1 and glutathione S-transferase, play critical roles in intracellular NO transport and storage through their interaction with DNICs (R.N. Watts et al., Proc. Natl. Acad. Sci. USA 103:7670–7675, 2006; H. Lok et al., J. Biol. Chem. 287:607–618, 2012). Notably, DNICs are present at high concentrations in cells and are biologically available. These complexes have a markedly longer half-life than free NO, making them an ideal “common currency” for this messenger molecule. Considering the many critical roles NO plays in health and disease, a better understanding of its intracellular trafficking mechanisms will be vital for the development of new therapeutics.

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Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

Abstract

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. Pharmacol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Inorg. Chim. Acta

Ann. Intern. Med.

Semin. Hematol.

N. Engl. J. Med.

Clin. Sci. Mol. Med.

Clin. Physiol. Biochem.