Isoniazid induces a monocytic-like phenotype in HL-60 cells.

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

An evaluation of myeloperoxidase-mediated bio-activation of NSAIDs in promyelocytic leukemia (HL-60) cells for potential cytotoxic selectivity.

Several lines of evidence have pointed towards the potential therapeutic benefit of NSAIDs in cancer therapy. In this study, we have investigated the acute bio-activation of NSAIDs and their metabolites via myeloperoxidase (MPO), a highly-expressed peroxidase enzyme in acute myeloid leukemia. As bio-activation involves the formation of reactive metabolites, we hypothesized that NSAIDs which produced reactive metabolites would be correlated with leukemia cell toxicity. We tested the enzymatic peroxidation of three NSAIDs, namely diclofenac, indomethacin, and naproxen in comparison with their hepatic metabolites, 4'- hydroxydiclofenac (4'-OHD), 5-hydroxydiclofenac (5-OHD), O-desmethyl-N-deschlorobenzoylindomethacin (DMBI), O-desmethylindomethacin (DMI) and O-desmethylnaproxen (ODN). Firstly, we used purified peroxidases in kinetic UV-vis kinetic spectrophotometry, and electron paramagnetic resonance (EPR) experiments to determine oxidation of ascorbic acid and glutathione (GSH), respectively. We then used HL-60 cells, as a model of acute myelogenous leukemia to carry out trypan blue exclusion, cellular ATP analysis, mitochondrial membrane potential (MMP) and cytofluorometric GSH assays. Our results present evidence that diclofenac, 4'-OHD, 5-OHD, DMBI and DMI demonstrated significant cytotoxic effect in the leukemic cells through oxidation by intracellular MPO. In the same vein, only diclofenac and its two metabolites caused a significant drop in the MMP and cellular ATP level; however, the cell death induced by indomethacin metabolites reflected a subtle effect on MMP or GSH content. Interestingly, only diclofenac and 4'-OHD (and not 5- OHD) caused a significant drop in HL-60 cells' GSH content. Among diclofenac compounds, only 4'-OHD also generated GS radical and caused a significant increase in ascorbate co-oxidation rate. Lastly, even though ODN also generated GS radical and potently cooxidized ascorbate, it showed no significant cytotoxicity. These results provide evidence of a correlation between acute cytotoxicity and MPO-bioactivated NSAIDs, though this was not correlated for all compounds (e.g., ODN). Further studies are required to determine both the MPO-dependent and MPO-independent mechanisms of cytotoxicity.

Cytoprotective effect of isoniazid against H2O2 derived injury in HL-60 cells.

To combat tuberculosis (TB), host phagocytic cells need to survive against self-generating oxidative stress-induced necrosis. However, the effect of isoniazid (INH) in protecting cells from oxidative stress-induced necrosis has not been previously investigated. In this in vitro study, the cytotoxic effect of H2O2 generation using glucose oxidase (a model of oxidative stress) was found to be abrogated by INH in a concentration-dependent manner in HL-60 cells (a human promyelocytic leukemia cell). In cells treated with glucose oxidase, both ATP and mitochondrial membrane potential were found to be decreased. However, treatment with INH demonstrated small but significant attenuation in decreasing ATP levels, and complete reversal for the decrease in mitochondrial membrane potential. Quantitative proteomics analysis identified up-regulation of 15 proteins and down-regulation of 14 proteins which all together suggest that these proteomic changes signal for increasing cellular replication, structural integrity, ATP synthesis, and inhibiting cell death. In addition, studies demonstrated that myeloperoxidase (MPO) was involved in catalyzing INH-protein adduct formation. Unexpectedly, these covalent protein adducts were correlated with INH-induced cytoprotection in HL-60 cells. Further studies are needed to determine whether the INH-protein adducts were causative in the mechanism of cytoprotection.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.