Nuclear H2O2 Research Articles

Mitochondrial H2O2 release does not directly cause damage to chromosomal DNA

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging.

Histone H3.1 is a chromatin-embedded redox sensor triggered by tumor cells developing adaptive phenotypic plasticity and multidrug resistance

Chromatin structure is regulated through posttranslational modifications of histone variants that modulate transcription. Although highly homologous, histone variants display unique amino acid sequences associated with specific functions. Abnormal incorporation of histone variants contributes to cancer initiation, therapy resistance, and metastasis. This study reports that, among its biologic functions, histone H3.1 serves as a chromatin redox sensor that is engaged by mitochondrial H2O2. In breast cancer cells, the oxidation of H3.1Cys96 promotes its eviction and replacement by H3.3 in specific promoters. We also report that this process facilitates the opening of silenced chromatin domains and transcriptional activation of epithelial-to-mesenchymal genes associated with cell plasticity. Scavenging nuclear H2O2 or amino acid substitution of H3.1(C96S) suppresses plasticity, restores sensitivity to chemotherapy, and induces remission of metastatic lesions. Hence, it appears that increased levels of H2O2 produced by mitochondria of breast cancer cells directly promote redox-regulated H3.1-dependent chromatin remodeling involved in chemoresistance and metastasis.

Radio-resistance of hypoxic tumors: exploring the effects of oxygen and X-ray radiation on non-small lung cancer cell lines

BackgroundSolid tumors are often riddled with hypoxic areas, which develops as a result of high proliferation. Cancer cells willingly adapt and thrive in hypoxia by activating complex changes which contributes to survival and enhanced resistance to treatments, such as photon radiation. Photon radiation primarily relies on oxygen for the production of reactive oxygen species to induce DNA damage. The present in-vitro study aimed at investigating the biochemical responses of hypoxic non-small cell lung cancer (NSCLC) cells, particularly the effects on the DNA damage repair systems contributing to more radioresistant phenotypes and their pro- and anti-oxidant potential, within the first 24 h post-IR.MethodsNSCLC cell lines (H460, A549, Calu-1) were irradiated using varying X-ray doses under normoxia (21% O2) and hypoxia (0.1% O2). The overall cell survival was assessed by clonogenic assays. The extent of irradiation (IR)-induced DNA damage was evaluated by analyzing γ-H2AX foci induction and the altered expression of repair genes involved in non-homologous end joining and homologous recombination pathways. Moreover, cell-altered responses were investigated, including the nuclear and cytosolic hydrogen peroxide (H2O2) production, as well as the associated anti-oxidant potential, in particular some components related to the glutathione system.ResultsAnalysis of clonogenic survival revealed an enhanced radioresistance of the hypoxic NSCLC cells associated with reduced DNA damage and a downregulation of DNA repair genes. Moreover, nuclear H2O2 levels were IR-induced in a dose-dependent manner only under normoxia, and directly correlated with the DNA double-strand breaks. However, the observed nuclear H2O2 reduction in hypoxia appeared to be unaffected by IR, thus highlighting a possible reason for the enhanced radioresistance of the hypoxic NSCLC cells. The cellular antioxidant capacity was upregulated by IR in both oxygen conditions most likely helping to counteract the radiation effect on the cytosolic H2O2.ConclusionsIn conclusion, our data provide insight into the adaptive behavior of radiation-resistant hypoxic NSCLC cells, in particular their DNA repair and oxidative stress responses, which could contribute to lower DNA damage and higher cell survival rates following X-ray exposure. These findings may therefore help to identify potential targets for improving cancer treatment outcomes.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Elevated branched-chain amino acid promotes atherosclerosis progression by enhancing mitochondrial-to-nuclear H2O2-disulfide HMGB1 in macrophages

As the essential amino acids, branched-chain amino acid (BCAA) from diets is indispensable for health. BCAA supplementation is often recommended for patients with consumptive diseases or healthy people who exercise regularly. Latest studies and ours reported that elevated BCAA level was positively correlated with metabolic syndrome, diabetes, thrombosis and heart failure. However, the adverse effect of BCAA in atherosclerosis (AS) and its underlying mechanism remain unknown. Here, we found elevated plasma BCAA level was an independent risk factor for CHD patients by a human cohort study. By employing the HCD-fed ApoE−/− mice of AS model, ingestion of BCAA significantly increased plaque volume, instability and inflammation in AS. Elevated BCAA due to high dietary BCAA intake or BCAA catabolic defects promoted AS progression. Furthermore, BCAA catabolic defects were found in the monocytes of patients with CHD and abdominal macrophages in AS mice. Improvement of BCAA catabolism in macrophages alleviated AS burden in mice. The protein screening assay revealed HMGB1 as a potential molecular target of BCAA in activating proinflammatory macrophages. Excessive BCAA induced the formation and secretion of disulfide HMGB1 as well as subsequent inflammatory cascade of macrophages in a mitochondrial-nuclear H2O2 dependent manner. Scavenging nuclear H2O2 by overexpression of nucleus-targeting catalase (nCAT) effectively inhibited BCAA-induced inflammation in macrophages. All of the results above illustrate that elevated BCAA promotes AS progression by inducing redox-regulated HMGB1 translocation and further proinflammatory macrophage activation. Our findings provide novel insights into the role of animo acids as the daily dietary nutrients in AS development, and also suggest that restricting excessive dietary BCAA consuming and promoting BCAA catabolism may serve as promising strategies to alleviate and prevent AS and its subsequent CHD.

Miro1-mediated mitochondrial positioning supports subcellular redox status

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.

A glycine spacer improved peptidyl-nuclear-localized efficiency for fluorescent imaging nuclear H2O2

H2O2 generated aberrantly can attack DNA in nuclei and result in oxidative DNA damage, which has been associated with many serious diseases. Therefore, it is crucial to monitor nuclear H2O2 levels. However, the vast majority of fluorescence sensors can not localize in the nucleus. In the present study, we empolyed glycine (the simplest amino acids) as a spacer to lengthen distance between N50 and NP1. Cu2+-free click reaction (through inserting dibenzocyclooctyne-maleimide) was used to connect the two parts in situ. Three spacer sequences were tested: C, CGGGGG, and CGGGGGGGGGG, and three new NLS peptides (connection spacer sequence to N50) were generated: pep5, pep6 and pep7. The fluorescence imaging results showed the nucleus-targeting efficancy of the three peptides, especially pep6, were imporved. The calculated binding energies by molecular docking simulations with importin α5 (a member of importin) were pep6 < pep5 < pep7, consistent with the fluorescence imaging results. Importantly, the probe system consisting of pep6 and NP1 was used for imaging the increase of nuclear H2O2 level upon stimulation.

Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.

MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell

To improve the treatment of advanced prostate cancer, the development of effective and innovative antitumor agents is needed. Our previous work demonstrated that the ROS (reactive oxygen species) scavenger, MnTE-2-PyP, inhibited human prostate cancer growth and also inhibited prostate cancer migration and invasion. We showed that MnTE-2-PyP treatment altered the affinity of the histone acetyltransferase enzyme, p300, to bind to DNA. We speculate that this may be one mechanism by which MnTE-2-PyP inhibits prostate cancer progression. Specifically, MnTE-2-PyP decreased p300/HIF-1/CREB complex (p300/hypoxia-inducible factor-1/cAMP response element-binding protein) binding to a specific hypoxia-response element (HRE) motif within the plasminogen activator inhibitor-1 (PAI-1) gene promoter region, and consequently, repressed PAI-1 expression. However, it remains unclear how MnTE-2-PyP reduces p300 complex binding affinity to the promoter region of specific genes. In this study, we found that overexpression of Cu/ZnSOD (superoxide dismutase 1, SOD1) significantly suppressed PAI-1 gene expression and p300 complex binding to the promoter region of PAI-1 gene, just as was observed in cells treated with MnTE-2-PyP. Furthermore, catalase (CAT) overexpression rescued the inhibition of PAI-1 expression and p300 binding by MnTE-2-PyP. Taken together, the above findings suggest that hydrogen peroxide (H2O2) is likely the mediator through which MnTE-2-PyP inhibits the PAI-1 expression and p300 complex binding in PC3 cells. To confirm this, we measured the production of H2O2 following overexpression of SOD1 or catalase with MnTE-2-PyP treatment in the presence or absence of radiation. We found that MnTE-2-PyP increased the intracellular steady-state levels of H2O2 and increased nuclear H2O2 levels. As expected, catalase overexpression significantly decreased the levels of intracellular H2O2 induced by MnTE-2-PyP. We then determined if this increased H2O2 production could result in oxidized protein thiol groups. In the presence of MnTE-2-PyP, there was a significant increase in oxidized thiols in PC3 cell lysates and this was reversed with catalase overexpression. Specifically, we showed that p300 was oxidized after MnTE-2-PyP treatment, indicating that MnTE-2-PyP is creating a more oxidizing environment and this is altering the oxidation state of p300 thiol residues. Our data provide an in depth mechanism by which MnTE-2-PyP regulates gene transcription through induced H2O2 mediated oxidation of particular proteins, supporting an important role for MnTE-2-PyP as an effective and innovative antitumor agent to enhance treatment outcomes in prostate cancer radiotherapy.

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.

A highly sensitive ratiometric fluorescent probe for the detection of cytoplasmic and nuclear hydrogen peroxide.

As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H2O2) plays an important role in living systems. It is thus critical to monitor the changes in H2O2 in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H2O2 fluorescent probe (NP1) based on 1,8-naphthalimide and boric acid ester. In response to H2O2, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of NP1 toward H2O2 is estimated as 0.17 μM. It was capable of imaging endogenous H2O2 generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H2O2 produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications.