Antioxidants

Research

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19 pages, 4526 KiB

Open AccessArticle

Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy

by Sankaranarayan Kannan, Mary E. Irwin, Shelley M. Herbrich, Tiewei Cheng, LaNisha L. Patterson, Marisa J. L. Aitken, Kapil Bhalla, M. James You, Marina Konopleva, Patrick A. Zweidler-McKay and Joya Chandra

Antioxidants 2022, 11(4), 717; https://doi.org/10.3390/antiox11040717 - 05 Apr 2022

Cited by 14 | Viewed by 2523

Abstract

Acute myeloid leukemia (AML) is a molecularly heterogenous hematological malignancy, with one of the most common mutations being internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (FLT3). Despite the development of FLT3-directed tyrosine kinase inhibitors (TKI), relapse [...] Read more.

Acute myeloid leukemia (AML) is a molecularly heterogenous hematological malignancy, with one of the most common mutations being internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (FLT3). Despite the development of FLT3-directed tyrosine kinase inhibitors (TKI), relapse and resistance are problematic, requiring improved strategies. In both patient samples and cell lines, FLT3-ITD raises levels of reactive oxygen species (ROS) and elicits an antioxidant response which is linked to chemoresistance broadly in AML. NF-E2–related factor 2 (NRF2) is a transcription factor regulating the antioxidant response including heme oxygenase -1 (HO-1), a heat shock protein implicated in AML resistance. Here, we demonstrate that HO-1 is elevated in FLT3-ITD-bearing cells compared to FLT3-wild type (WT). Transient knockdown or inhibitor-based suppression of HO-1 enhances vulnerability to the TKI, quizartinib, in both TKI-resistant and sensitive primary AML and cell line models. NRF2 suppression (genetically or pharmacologically using brusatol) results in decreased HO-1, suggesting that TKI-resistance is dependent on an active NRF2-driven pathway. In AML-patient derived xenograft (PDX) models, brusatol, in combination with daunorubicin, reduces leukemia burden and prolongs survival. Cumulatively, these data encourage further development of brusatol and NRF2 inhibition as components of combination therapy for refractory AML. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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23 pages, 3279 KiB

Open AccessArticle

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells

by James Hillier, Gemma J. Allcott, Laura A. Guest, Wayne Heaselgrave, Alex Tonks, Myra E. Conway, Amy L. Cherry and Steven J. Coles

Antioxidants 2022, 11(4), 683; https://doi.org/10.3390/antiox11040683 - 31 Mar 2022

Cited by 6 | Viewed by 3985

Abstract

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC [...] Read more.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a ‘redox-switch’ in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H₂O₂ in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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15 pages, 1778 KiB

Open AccessArticle

Effect of 5-Azacitidine Treatment on Redox Status and Inflammatory Condition in MDS Patients

by Paola Montes, Ana Guerra-Librero, Paloma García, María Elena Cornejo-Calvo, María del Señor López, Tomás de Haro, Laura Martínez-Ruiz, Germaine Escames and Darío Acuña-Castroviejo

Antioxidants 2022, 11(1), 139; https://doi.org/10.3390/antiox11010139 - 09 Jan 2022

Cited by 2 | Viewed by 1947

Abstract

This study focused on the impact of the treatment with the hypomethylating agent 5-azacitidine on the redox status and inflammation in 24 MDS patients. Clinical and genetic features of MDS patients were recorded, and peripheral blood samples were used to determine the activity [...] Read more.

This study focused on the impact of the treatment with the hypomethylating agent 5-azacitidine on the redox status and inflammation in 24 MDS patients. Clinical and genetic features of MDS patients were recorded, and peripheral blood samples were used to determine the activity of the endogenous antioxidant defense system (superoxide dismutase, SOD; catalase, CAT; glutathion peroxidase, GPx; and reductase, GRd, activities), markers of oxidative damage (lipid peroxidation, LPO, and advanced oxidation protein products, AOPP). Moreover, pro-inflammatory cytokines and plasma nitrite plus nitrate levels as markers of inflammation, as well as CoQ10 plasma levels, were also measured. Globally, MDS patients showed less redox status in terms of a reduction in the GSSG/GSH ratio and in the LPO levels, as well as increased CAT activity compared with healthy subjects, with no changes in SOD, GPx, and GRd activities, or AOPP levels. When analyzing the evolution from early to advanced stages of the disease, we found that the GPx activity, GSSG/GSH ratio, LPO, and AOPP increased, with a reduction in CAT. GPx changes were related to the presence of risk factors such as high-risk IPSS-R or mutational score. Moreover, there was an increase in IL-2, IL-6, IL-8, and TNF-α plasma levels, with a further increase of IL-2 and IL-10 from early to advanced stages of the disease. However, we did not observe any association between inflammation and oxidative stress. Finally, 5-azacitidine treatment generated oxidative stress in MDS patients, without affecting inflammation levels, suggesting that oxidative status and inflammation are two independent processes. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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21 pages, 2754 KiB

Open AccessArticle

Oxidative Stress and X-ray Exposure Levels-Dependent Survival and Metabolic Changes in Murine HSPCs

by Melis Karabulutoglu, Rosemary Finnon, Lourdes Cruz-Garcia, Mark A. Hill and Christophe Badie

Antioxidants 2022, 11(1), 11; https://doi.org/10.3390/antiox11010011 - 22 Dec 2021

Cited by 3 | Viewed by 2965

Abstract

Haematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are [...] Read more.

Haematopoietic bone marrow cells are amongst the most sensitive to ionizing radiation (IR), initially resulting in cell death or genotoxicity that may later lead to leukaemia development, most frequently Acute Myeloid Leukaemia (AML). The target cells for radiation-induced Acute Myeloid Leukaemia (rAML) are believed to lie in the haematopoietic stem and progenitor cell (HSPC) compartment. Using the inbred strain CBA/Ca as a murine model of rAML, progress has been made in understanding the underlying mechanisms, characterisation of target cell population and responses to IR. Complex regulatory systems maintain haematopoietic homeostasis which may act to modulate the risk of rAML. However, little is currently known about the role of metabolic factors and diet in these regulatory systems and modification of the risk of AML development. This study characterises cellular proliferative and clonogenic potential as well as metabolic changes within murine HSPCs under oxidative stress and X-ray exposure. Ambient oxygen (normoxia; 20.8% O₂) levels were found to increase irradiated HSPC-stress, stimulating proliferative activity compared to low oxygen (3% O₂) levels. IR exposure has a negative influence on the proliferative capability of HSPCs in a dose-dependent manner (0–2 Gy) and this is more pronounced under a normoxic state. One Gy x-irradiated HSPCs cultured under normoxic conditions displayed a significant increase in oxygen consumption compared to those cultured under low O₂ conditions and to unirradiated HSPCs. Furthermore, mitochondrial analyses revealed a significant increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and membrane potential in a dose-dependent manner under normoxic conditions. Our results demonstrate that both IR and normoxia act as stressors for HSPCs, leading to significant metabolic deregulation and mitochondrial dysfunctionality which may affect long term risks such as leukaemia. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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22 pages, 22274 KiB

Open AccessArticle

Exploring the Leukemogenic Potential of GATA-1_S, the Shorter Isoform of GATA-1: Novel Insights into Mechanisms Hampering Respiratory Chain Complex II Activity and Limiting Oxidative Phosphorylation Efficiency

by Silvia Trombetti, Raffaele Sessa, Rosa Catapano, Laura Rinaldi, Alessandra Lo Bianco, Antonio Feliciello, Paola Izzo and Michela Grosso

Antioxidants 2021, 10(10), 1603; https://doi.org/10.3390/antiox10101603 - 12 Oct 2021

Cited by 12 | Viewed by 2942

Abstract

GATA-1 is a key regulator of hematopoiesis. A balanced ratio of its two isoforms, GATA-1_FL and GATA-1_S, contributes to normal hematopoiesis, whereas aberrant expression of GATA-1_S alters the differentiation/proliferation potential of hematopoietic precursors and represents a poor prognostic factor [...] Read more.

GATA-1 is a key regulator of hematopoiesis. A balanced ratio of its two isoforms, GATA-1_FL and GATA-1_S, contributes to normal hematopoiesis, whereas aberrant expression of GATA-1_S alters the differentiation/proliferation potential of hematopoietic precursors and represents a poor prognostic factor in myeloid leukemia. We previously reported that GATA-1_S over-expression correlates with high levels of the succinate dehydrogenase subunit C (SDHC). Alternative splicing variants of the SDHC transcript are over-expressed in several tumors and act as potent dominant negative inhibitors of SDH activity. With this in mind, we investigated the levels of SDHC variants and the oxidative mitochondrial metabolism in myeloid leukemia K562 cells over-expressing GATA-1 isoforms. Over-expression of SDHC variants accompanied by decreased SDH complex II activity and oxidative phosphorylation (OXPHOS) efficiency was found associated only with GATA-1_S. Given the tumor suppressor role of SDH and the effects of OXPHOS limitations in leukemogenesis, identification of a link between GATA-1_S and impaired complex II activity unveils novel pro-leukemic mechanisms triggered by GATA-1_S. Abnormal levels of GATA-1_S and SDHC variants were also found in an acute myeloid leukemia patient, thus supporting in vitro results. A better understanding of these mechanisms can contribute to identify novel promising therapeutic targets in myeloid leukemia. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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23 pages, 3968 KiB

Open AccessArticle

Betulinic Acid–Doxorubicin-Drug Combination Induced Apoptotic Death via ROS Stimulation in a Relapsed AML MOLM-13 Cell Model

by Milan Vu, Nick Kassouf and Sandra Appiah

Antioxidants 2021, 10(9), 1456; https://doi.org/10.3390/antiox10091456 - 14 Sep 2021

Cited by 3 | Viewed by 2203

Abstract

In this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with doxorubin (Dox) and betulinic acid (BetA), singly and in combination. CyQUANT Direct^® and Annexin V/propidium iodide double staining were used [...] Read more.

In this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with doxorubin (Dox) and betulinic acid (BetA), singly and in combination. CyQUANT Direct^® and Annexin V/propidium iodide double staining were used to measure the cytotoxic and cell death induction effects of the compounds, respectively. Reactive oxygen species (ROS) generation was measured using 2′,7′-dichlorofluorescin diacetate staining. Expressions of proteins and genes were examined by Western blot and reverse transcription polymerase chain reaction analysis, respectively. BetA (20 μM) and Dox (1 μM) indicated a synergistic growth inhibitory effect on MOLM-13 cells. The combined drug caused more cells to reside in irreversible late apoptotic stage compared to the single treatments (p < 0.05). Elevation in ROS may be the synergistic mechanism involved in MOLM-13 cell death since ROS can directly disrupt mitochondrial activity. In contrast, in leukaemic U-937 cells, the combination treatments attenuated Dox-induced cell death. Dox and the drug combination selectively reduced (p < 0.05) a recently reported anti-apoptotic Bcl-2 protein isoform p15-20-Bcl-2 in MOLM-13 by our group, without affecting the usually reported p26-Bcl-2-α. Further studies using known inhibitors of apoptosis are required to confirm the potential of Dox-BetA combination to modulate these pathways. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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15 pages, 13984 KiB

Open AccessArticle

Induction of AML Preleukemic Fusion Genes in HSPCs and DNA Damage Response in Preleukemic Fusion Gene Positive Samples

by Pavol Kosik, Matus Durdik, Milan Skorvaga, Daniela Klimova, Dominika Kochanova, Zlatica Cerna, Miroslav Kubes, Marek Holop and Igor Belyaev

Antioxidants 2021, 10(3), 481; https://doi.org/10.3390/antiox10030481 - 18 Mar 2021

Cited by 5 | Viewed by 2438

Abstract

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by [...] Read more.

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by several research groups and estimated to be 1–5% in umbilical cord blood (UCB) of healthy newborns, PFGs that are relevant to acute myeloid leukemia (AML) were poorly investigated. Therefore, this study is focused on the estimation of the incidence of the most frequent AML PFGs in newborns. For the first time, this study considered the inducibility of AML PFGs in different subsets of UCB HSPCs by low-dose γ-rays and also compared endogenous DNA damage, apoptosis, and reactive oxygen species (ROS) level between UCB samples containing or lacking AML PFGs. We found that: (i) the incidence of AML PFGs in UCB was 3.19% for RUNX1-RUNX1T1, 3.19% for PML-RARα, and 1.17% for KMT2A-MLLT3, (ii) 50 cGy of γ-rays did not induce RUNX1-RUNX1T1, PML-RARα, or KMT2A-MLLT3 PFGs in different subsets of sorted and expanded HSPCs, and (iii) the AML PFG⁺ samples accumulated the same level of endogenous DNA damage, as measured by the γH2AX/53BP1 focus formation, and also the same ROS level, and apoptosis as compared to PFG⁻ controls. Our study provides critical insights into the prevalence of AML PFGs in UCB of newborns, without the evidence of a specific HSPC population more susceptible for PFG formation after irradiation to low-dose γ-rays or increased amount of ROS, apoptosis and DNA damage. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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Review

Jump to: Research

31 pages, 2900 KiB

Open AccessReview

Reactive Oxygen Species in Acute Lymphoblastic Leukaemia: Reducing Radicals to Refine Responses

by Abdul Mannan, Zacary P. Germon, Janis Chamberlain, Jonathan R. Sillar, Brett Nixon and Matthew D. Dun

Antioxidants 2021, 10(10), 1616; https://doi.org/10.3390/antiox10101616 - 14 Oct 2021

Cited by 9 | Viewed by 3982

Abstract

Acute lymphoblastic leukaemia (ALL) is the most common cancer diagnosed in children and adolescents. Approximately 70% of patients survive >5-years following diagnosis, however, for those that fail upfront therapies, survival is poor. Reactive oxygen species (ROS) are elevated in a range of cancers [...] Read more.

Acute lymphoblastic leukaemia (ALL) is the most common cancer diagnosed in children and adolescents. Approximately 70% of patients survive >5-years following diagnosis, however, for those that fail upfront therapies, survival is poor. Reactive oxygen species (ROS) are elevated in a range of cancers and are emerging as significant contributors to the leukaemogenesis of ALL. ROS modulate the function of signalling proteins through oxidation of cysteine residues, as well as promote genomic instability by damaging DNA, to promote chemotherapy resistance. Current therapeutic approaches exploit the pro-oxidant intracellular environment of malignant B and T lymphoblasts to cause irreversible DNA damage and cell death, however these strategies impact normal haematopoiesis and lead to long lasting side-effects. Therapies suppressing ROS production, especially those targeting ROS producing enzymes such as the NADPH oxidases (NOXs), are emerging alternatives to treat cancers and may be exploited to improve the ALL treatment. Here, we discuss the roles that ROS play in normal haematopoiesis and in ALL. We explore the molecular mechanisms underpinning overproduction of ROS in ALL, and their roles in disease progression and drug resistance. Finally, we examine strategies to target ROS production, with a specific focus on the NOX enzymes, to improve the treatment of ALL. Full article

(This article belongs to the Special Issue Reactive Oxygen Species (ROS), Haematopoiesis and Leukaemia)

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