Reactive Oxygen Species-dependent Manner Research Articles

Reconstitution of autophagy ameliorates vascular function and arterial stiffening in spontaneously hypertensive rats.

Insufficient autophagy has been proposed as a mechanism of cellular aging, as this leads to the accumulation of dysfunctional macromolecules and organelles. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in clinical and experimental hypertension. Previously, we have reported decreased autophagy in arteries from spontaneously hypertensive rats (SHRs); however, the effects of restoring autophagic activity on blood pressure and vascular function are currently unknown. We hypothesized that reconstitution of arterial autophagy in SHRs would decrease blood pressure and improve endothelium-dependent relaxation. We treated 14- to 18-wk-old Wistar rats (n = 7 vehicle and n = 8 trehalose) and SHRs (n = 7/group) with autophagy activator trehalose (2% in drinking water) for 28 days. Blood pressure was measured by radiotelemetry, and vascular function and structure were measured in isolated mesenteric resistance arteries (MRAs) using wire and pressure myographs, respectively. Treatment with trehalose had no effect on blood pressure in SHRs; however, isolated MRAs presented enhanced relaxation to acetylcholine, in a cyclooxygenase- and reactive oxygen species-dependent manner. Similarly, trehalose treatment shifted the relaxation to the Rho kinase (ROCK) inhibitor Y-27632 to the right, indicating reduced ROCK activity. Finally, trehalose treatment decreased arterial stiffness as indicated by the slope of the stress-strain curve. Overall these data indicate that reconstitution of arterial autophagy in SHRs improves endothelial and vascular smooth muscle function, which could synergize to prevent stiffening. As a result, restoration of autophagic activity could be a novel therapeutic for premature vascular aging in hypertension.NEW & NOTEWORTHY This work supports the concept that diminished arterial autophagy contributes to premature vascular aging in hypertension and that therapeutic reconstitution of autophagic activity can ameliorate this phenotype. As vascular age is a new clinically used index for cardiovascular risk, understanding this mechanism may assist in the development of new drugs to prevent premature vascular aging in hypertension.

HDAC5 catalytic activity suppresses cardiomyocyte oxidative stress and NRF2 target gene expression

Histone deacetylase 5 (HDAC5) and HDAC9 are class IIa HDACs that function as signal-responsive repressors of the epigenetic program for pathological cardiomyocyte hypertrophy. The conserved deacetylase domains of HDAC5 and HDAC9 are not required for inhibition of cardiac hypertrophy. Thus, the biological function of class IIa HDAC catalytic activity in the heart remains unknown. Here we demonstrate that catalytic activity of HDAC5, but not HDAC9, suppresses mitochondrial reactive oxygen species generation and subsequent induction of NF-E2-related factor 2 (NRF2)-dependent antioxidant gene expression in cardiomyocytes. Treatment of cardiomyocytes with TMP195 or TMP269, which are selective class IIa HDAC inhibitors, or shRNA-mediated knockdown of HDAC5 but not HDAC9 leads to stimulation of NRF2-mediated transcription in a reactive oxygen species-dependent manner. Conversely, ectopic expression of catalytically active HDAC5 decreases cardiomyocyte oxidative stress and represses NRF2 activation. These findings establish a role of the catalytic domain of HDAC5 in the control of cardiomyocyte redox homeostasis and define TMP195 and TMP269 as a novel class of NRF2 activators that function by suppressing the enzymatic activity of an epigenetic regulator.

Tamoxifen restores extracellular trap formation in neutrophils from patients with chronic granulomatous disease in a reactive oxygen species–independent manner

Tamoxifen restores extracellular trap formation in neutrophils from patients with chronic granulomatous disease in a reactive oxygen species–independent manner

Annexin A2 Regulates AKT Upon H₂O₂-Dependent Signaling Activation in Cancer Cells.

Hydrogen peroxide (H2O2) is a main second messenger in oncogenic signaling networks including the Ras and the growth factor receptor pathways. This is achieved predominantly through the oxidation of redox-sensitive cysteine (Cys) residues in proteins resulting in changes to their structure and function. We previously identified annexin A2 (ANXA2) as a redox regulatory protein that plays an important cellular role during oxidative stress and also promoting tumorigenesis. Here we investigated the role of ANXA2 in the regulation of H2O2-dependent signaling that drives tumor progression. We show that depletion of ANXA2 leads to the enhanced activation of AKT following either EGF/EGFR stimulation or oncogenic Ras transformation. The phosphatase and tensin homologue (PTEN) protein negatively regulates the PI3K/AKT pathway. We demonstrate that ANXA2 via its reactive Cys-8 residue, binds to PTEN and that the co-expression of PTEN and ANXA2, but not ANXA2 Cys-8-Ala mutant, inhibits AKT phosphorylation on Ser 473. These results indicate that ANXA2 is important for PTEN regulation within the PI3K/AKT signaling cascade. Furthermore, we also reveal that ANXA2 inversely regulates the expression of the peroxidase, peroxiredoxin 2, in a reactive oxygen species dependent manner.

Influence of Tea Consumption on the Development of Second Esophageal Neoplasm in Patients with Head and Neck Cancer.

Alcohol is an important risk factor for the development of second esophageal squamous-cell carcinoma (ESCC) in head and neck squamous-cell carcinoma (HNSCC) patients. However, the influence of tea consumption is uncertain. We prospectively performed endoscopic screening in incident HNSCC patients to identify synchronous esophageal neoplasm. In total, 987 patients enrolled between October 2008 and December 2017 and were analyzed. In vitro studies were conducted to investigate the effect of epigallocatechin gallate (EGCG) on the betel alkaloid, arecoline-stimulated carcinogenesis in two ESCC cell lines. There were 151 patients (15.3%) diagnosed to have synchronous esophageal neoplasm, including 88 low-grade dysplasia, 30 high-grade dysplasia and 33 squamous-cell carcinoma (SCC). Tea consumption was associated with a significantly lower risk of having esophageal high-grade dysplasia or SCC in HNSCC patients, especially those who were betel nut chewers, alcohol drinkers or cigarette smokers (all adjusted odds ratio were 0.5; p-values: 0.045, 0.045 and 0.049 respectively). In vitro studies indicated that EGCG suppressed arecoline-induced ESCC cell proliferation and colony formation through the inhibition of the Akt and ERK1/2 pathway in a reactive oxygen species-independent manner. In conclusion, tea consumption may protect against the development of second esophageal neoplasms among HNSCC patients, especially those who regularly consume betel nuts, alcohol and cigarettes.

Isoliquiritigenin Induces Mitochondrial Dysfunction and Apoptosis by Inhibiting mitoNEET in a Reactive Oxygen Species-Dependent Manner in A375 Human Melanoma Cells.

The mitochondrial protein mitoNEET is a type of iron-sulfur protein localized to the outer membrane of mitochondria and is involved in a variety of human pathologies including cystic fibrosis, diabetes, muscle atrophy, and neurodegeneration. In the current study, we found that isoliquiritigenin (ISL), one of the components of the root of Glycyrrhiza glabra L., could decrease the expression of mitoNEET in A375 melanoma cells. We also demonstrated that mitoNEET could regulate the content of reactive oxygen species (ROS), by showing that the ISL-mediated increase in the cellular ROS content could be mitigated by the mitoNEET overexpression. We also confirmed the important role of ROS in ISL-treated A375 cells. The increased apoptosis rate and the decreased mitochondrial membrane potential were mitigated by the overexpression of mitoNEET in A375 cells. These findings indicated that ISL could decrease the expression of mitoNEET, which regulated ROS content and subsequently induced mitochondrial dysfunction and apoptosis in A375 cells. Our findings also highlight mitoNEET as a promising mitochondrial target for cancer therapy.

Peripheral and central oxidative stress in chemotherapy-induced neuropathic pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is an adverse side effect of many anti-cancer chemotherapeutic treatments. CIPN often causes neuropathic pain in extremities, and oxidative stress has been shown to be a major contributing factor to this pain. In this study, we determined the site of oxidative stress associated with pain (specifically, mechanical hypersensitivity) in cisplatin- and paclitaxel-treated mouse models of CIPN and investigated the neurophysiological mechanisms accounting for the pain. C57BL/6N mice that received either cisplatin or paclitaxel (2 mg/kg, once daily on four alternate days) developed mechanical hypersensitivity to von Frey filament stimulations of their hindpaws. Cisplatin-induced mechanical hypersensitivity was inhibited by silencing of Transient Receptor Potential channels V1 (TRPV1)- or TRPA1-expressing afferents, whereas paclitaxel-induced mechanical hypersensitivity was attenuated by silencing of Aβ fibers. Although systemic delivery of phenyl N-tert-butylnitrone, a reactive oxygen species scavenger, alleviated mechanical hypersensitivity in both cisplatin- and paclitaxel-treated mice, intraplantar phenyl N-tert-butylnitrone was effective only in cisplatin-treated mice, and intrathecal phenyl N-tert-butylnitrone, only in paclitaxel-treated mice. In a reactive oxygen species-dependent manner, the mechanosensitivity of Aδ/C fiber endings in the hindpaw skin was increased in cisplatin-treated mice, and the excitatory synaptic strength in the spinal dorsal horn was potentiated in paclitaxel-treated mice. Collectively, these results suggest that cisplatin-induced mechanical hypersensitivity is attributed to peripheral oxidative stress sensitizing mechanical nociceptors, whereas paclitaxel-induced mechanical hypersensitivity is due to central (spinal) oxidative stress maintaining central sensitization that abnormally produces pain in response to Aβ fiber inputs.

Streptococcus Suis Serotype 2 Stimulates Neutrophil Extracellular Traps Formation via Activation of p38 MAPK and ERK1/2.

Streptococcus suis serotype 2 is a major pathogen of swine streptococcicosis, which result in serious economic loss worldwide. SS2 is an important zoonosis causing meningitis and even death in humans. Neutrophil extracellular traps (NETs) constitute a significant bactericidal strategy of innate immune. The battle between SS2 and NETs may account for the pathogenicity of SS2. However, the molecular mechanism underlying release of SS2-induced NETs remains unclear. In this study, SS2 was found to induce NETs within 2–4 h, and was dependent on reactive oxygen species (ROS) from NADPH oxidase. Moreover, SS2 could activate neutrophil p38 MAPK and ERK1/2. Blockage of p38 MAPK or ERK1/2 activation decreased SS2-induced NETs formation by 65 and 85%, respectively. In addition, NADPH oxidase derived ROS inhibition negatively affected phosphorylation of p38 MAPK and ERK1/2 in SS2 induced neutrophils. Both TLR2 and TLR4 were significantly up-regulated by SS2 infection in blood cells in vivo and neutrophils in vitro, which indicates these two receptors are involved in SS2 recognition. Blocking TLR4 signaling could further inhibit the activation of ERK1/2, but not p38 MAPK; however, TLR4 signaling inhibition reduced NETs formation induced by SS2. In conclusion, SS2 could be recognized by TLR2 and/or TLR4, initiating NETs formation signaling pathways in a NADPH oxidase derived ROS dependent manner. ROS will activate p38 MAPK and ERK1/2, which ultimately induces NETs formation.

Inhibition of euchromatin histone-lysine N-methyltransferase 2 sensitizes breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through reactive oxygen species-mediated activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been characterized as an anti-cancer therapeutic agent with prominent cancer cell selectivity over normal cells. However, breast cancer cells are generally resistant to TRAIL, thus limiting its therapeutic potential. In this study, we found that BIX-01294, a selective inhibitor of euchromatin histone methyltransferase 2/G9a, is a strong TRAIL sensitizer in breast cancer cells. The combination of BIX-01294 and TRAIL decreased cell viability and led to an increase in the annexin V/propidium iodide-positive cell population, DNA fragmentation, and caspase activation. BIX-01294 markedly increased death receptor 5 (DR5) expression, while silencing of DR5 using small interfering RNAs abolished the TRAIL-sensitizing effect of BIX-01294. Specifically, BIX-01294 induced C/EBP homologous protein (CHOP)-mediated DR5 gene transcriptional activation and DR5 promoter activation was induced by upregulation of the protein kinase R-like endoplasmic reticulum kinase-mediated activating transcription factor 4 (ATF4). Moreover, inhibition of reactive oxygen species by N-acetyl-L-cysteine efficiently blocked BIX-01294-induced DR5 upregulation by inhibiting ATF4/CHOP expression, leading to diminished sensitization to TRAIL. These findings suggest that BIX-01294 sensitizes breast cancer cells to TRAIL by upregulating ATF4/CHOP-dependent DR5 expression with a reactive oxygen species-dependent manner. Furthermore, combination treatment with BIX-01294 and TRAIL suppressed tumor growth and induced apoptosis in vivo. In conclusion, we found that epigenetic regulation can contribute to the development of resistance to cancer therapeutics such as TRAIL, and further studies of unfolded protein responses and the associated epigenetic regulatory mechanisms may lead to the discovery of new molecular targets for effective cancer therapy.

Abstract 2835: Temporal sequencing of anticancer drugs, ex vivo, optimizes therapeutic effect by targeting drug-induced glucose-6-phosphate dehydrogenase

Abstract Background: Phenotypic cell state transitions are emerging as novel drivers of transient resistance to cancer chemotherapy. We recently demonstrated that non-cancer stem cells are able to undergo a phenotypic cell state transition that enables acquire a ‘reversible' drug tolerant state. Consequently, we made two key discoveries, drug tolerance 1) results in a cross-resistance to other classes of anticancer drugs 2) coincidental to a switch in the metabolic behavior. Further, evidences prompted to investigate if drug-induced metabolic reprogramming contributes to combination therapy resistance in a population of cancer cells that have gained tolerance to a primary therapy. Methods: We investigated the metabolic phenotype of drug tolerant cells using 3-D in-vitro models of breast cancer. A systems biology approach was used to identify key, interconnecting proteins involved in metabolic dysregulation, drawing inferences among signaling networks and events in a temporal context. Using an in-silico simulation we perturbed glucose metabolism, and tested how timing, combination and drug order impact the therapeutic effect of combination therapy and validated our findings using in-vivo experiments. Finally, to provide a direct clinical translation, we tested temporally-sequenced 3-drug combinations using CANscriptTM, a human explant tumor assay that captures the entire tumor ecosystem. Results: We report that conventional chemotherapies used to treat breast cancer, results in an adaptive cross-tolerance against an unrelated chemotherapeutic agent via induction of both glycolytic and oxidative pathways. These drug-tolerant cells switch to a CD44Hi phenotypic cell state, and rely on both the Akt pathway and HIF1α-Glut1 axis in a reactive oxygen species-dependent manner, which temporally cooperate to remodel a glucose shunt towards the pentose phosphate pathway. Mathematically modeling these pathways, we demonstrate how a sequentially-applied, 3-drug combination that includes G6PD metabolic inhibitors and cytotoxic agents can improve therapeutic effect. The use of CANscriptTM demonstrated that pharmacodynamics, biomarkers of resistance, and temporal ordering of drugs can influence the phenotypic response to therapy, reflecting in-vitro and in-vivo evidence, in a patient-specific manner. Conclusions: Timing, sequence and order of drugs is emerging as a critical component of combination therapy for cancer. Our results demonstrate that timing the order of G6PD inhibitors in exquisitely sequenced combination with chemotherapy can emerge as a new paradigm in the treatment of cancer. Ex-vivo, human tumor models that fully capture the tumor microenvironment can contribute to and potentially uncover the mechanisms of action, phenotypic effect, and pharmacodynamics of anticancer drug combinations in distinct temporal sequences. Citation Format: Baraneedharan Ulaganathan, Andrew Dhawan, Biswanath Majumder, Munisha Smalley, Saravanan Thiyagarajan, Gopinath S. Kodaganur, S Krishnamurthy, Mohammed Mamunur Rahman, Elizaveta Freinkman, Pradip Majumder, Mohammad Kohandel, Aaron Goldman. Temporal sequencing of anticancer drugs, ex vivo, optimizes therapeutic effect by targeting drug-induced glucose-6-phosphate dehydrogenase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2835.

Cold PSM, but not TRAIL, triggers autophagic cell death: A therapeutic advantage of PSM over TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) are promising novel anticancer tools due to their strong anticancer activities and high tumor-selectivity. The present study demonstrated that PSM and TRAIL may trigger autophagy in human malignant melanoma and osteosarcoma cells. Live-cell imaging revealed that even under nutritional and stress-free conditions, these cells possessed a substantial level of autophagosomes, which were localized in the cytoplasm separately from tubular mitochondria. In response to cytotoxic levels of PSM, the mitochondria became highly fragmented, and aggregated and colocalized with the autophagosomes. The cytotoxic effects of PSM were suppressed in response to various pharmacological autophagy inhibitors, including 3-methyladenine (3-MA) and bafilomycin A1, thus indicating the induction of autophagic cell death (ACD). Lethal levels of PSM also resulted in non-apoptotic, non-autophagic cell death in a reactive oxygen species-dependent manner under certain circumstances. Furthermore, TRAIL exhibited only a modest cytotoxicity toward these tumor cells, and did not induce ACD and mitochondrial aberration. The combined use of TRAIL and subtoxic concentrations of 3-MA resulted in decreased basal autophagy, increased mitochondrial aberration, colocalization with autophagosomes and apoptosis. These results indicated that PSM may induce ACD, whereas TRAIL may trigger cytoprotective autophagy that compromises apoptosis. To the best of our knowledge, the present study is the first to demonstrate that PSM can induce ACD in human cancer cells. These findings provide a rationale for the advantage of PSM over TRAIL in the destruction of apoptosis-resistant melanoma and osteosarcoma cells.

Pan-Cancer Analysis Reveals Synergistic Effects of CDK4/6i and PARPi Combination Treatment in RB-Proficient and RB-Deficient Breast Cancer Cells

DNA damage results in mutations and plays critical roles in cancer development, progression, and treatment. Targeting DNA damage response in cancers by inhibiting poly-(ADP-ribose) polymerases (PARPs) offers an important therapeutic strategy. However, the failure of PARP inhibitors to markedly benefit patients suggests the necessity for developing new strategies to improve their efficacy. Here, we show that the expression of cyclin dependent kinase 4/6 (CDK4/6) complex members significantly correlates with mutations (as proxies of DNA damages), and that the combination of CDK4/6 and PARP inhibitors shows synergy in both RB-proficient and RB-deficient breast cancer cells. As PARPs constitute sensors of DNA damage and are broadly involved in multiple DNA repair pathways, we hypothesized that the combined inhibition of PARPs and DNA repair (or repair-related) pathways critical for cancer (DRPCC) should show synergy. To identify druggable candidate DRPCC(s), we analyzed the correlation between the genome-wide expression of individual genes and the mutations for 27 different cancer types, assessing 7146 exomes and over 1,500,000 somatic mutations. Pathway enrichment analyses of the top ranked genes correlated with mutations indicated cell cycle pathway as the top candidate DRPCC. Additionally, among functional cell-cycle complexes, the CDK4/6 complex showed the most significant negative correlation with mutations, also suggesting that combined CDK4/6 and PARP inhibition might exhibit synergy. Furthermore, combination treatment showed synergy in not only RB-proficient but also RB-deficient breast cancer cells in a reactive oxygen species-dependent manner. These findings suggest a potential therapeutic strategy to improve the efficacy of PARP and CDK4/6 inhibitors in cancer treatment. Funding: This study was supported by grants from the National Natural Science Foundation of China (NSFC Nos. 31471328 and 81622040 to Q. Yang; 81502622 to L. Wang). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The animal-handling procedures were approved by the Animal Care and Use Committee of Dalian Medical University.

Mouse lung fibroblasts are highly susceptible to necroptosis in a reactive oxygen species-dependent manner

Mouse embryonic fibroblasts (MEFs) have extensively been used to study necroptosis, a recently identified form of programmed cell death. However, very little is yet known about the role of necroptosis and its regulation by reactive oxygen species (ROS) in cell types naturally exposed to high oxygen levels such as mouse lung fibroblasts (MLFs). Here, we discover that MLFs are highly susceptible to undergo necroptosis in a ROS-dependent manner upon exposure to a prototypic death receptor-mediated necroptotic stimulus, i.e. cotreatment with tumor necrosis factor (TNF)α, Smac mimetic and the caspase inhibitor zVAD.fmk (TSZ). Kinetic analysis revealed that TSZ rapidly induces cell death in MLFs. Pharmacological inhibition of receptor-interacting protein kinase (RIPK)1 by necrostatin-1 (Nec-1) or RIPK3 by GSK’872 significantly rescues TSZ-stimulated cell death. Also, genetic silencing of RIPK3 or mixed lineage kinase domain-like pseudokinase (MLKL) significantly protects MLFs from TSZ-mediated cell death. Prior to cell death, TSZ significantly increases production of ROS. Importantly, addition of radical scavengers such as butylated hydroxyanisole (BHA) or α-Tocopherol (α-Toc) significantly suppresses TSZ-induced cell death in parallel with a significant reduction of ROS generation. Consistently, BHA prevented TSZ-triggered phosphorylation of MLKL similar to the addition of GSK’872. Thus, our study demonstrates for the first time that MLFs are prone to undergo necroptosis in response to a prototypic necroptotic stimulus and identifies ROS as important mediators of TSZ-triggered necroptosis.

Autophagy Inhibition Enhances SPCA-1 Cell Proliferation Inhibition Induced by By-1 from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes)

Taiwanofungus camphoratus has been reported to have antitumor effects against various cancer cells. The aim of this study was to investigate the direct inhibitory effect of By-1 (3-isobutyl-l-methoxy-4-[4'-(3-methylbut-2-enyloxy)phenyl]-1H-pyrrole-2,5-dione), a compound from spent broth from submerged cultures of T. camphoratus, on human lung adenocarcinoma cells and to determine the molecular mechanism underlying this effect. The growth-inhibitory assay and colony formation assay showed that cell viability was significantly decreased. A By-1 concentration of 300 μmol/L caused 73.55% cell death and at a concentration of 240 μmol/L led to a 58% reduction in the number of colonies. The wound-healing assay showed that the distance of migration was 0.3 times shorter than that of untreated cells. Flow cytometry revealed that By-1 could suppress DNA synthesis, cause cell cycle arrest at the S phase, and induce apoptosis in a reactive oxygen species-dependent manner. Furthermore, the expression of caspase-3 and P53 was 4 times higher than that in untreated cells, and the antiapoptotic protein Bcl-2 was decreased 2 times compared with the protein in untreated cells. It is interesting to note that apoptosis and autophagy were both induced during treatment with By-1, and autophagy inhibition decreased cell proliferation. By-1 potently inhibited the growth of SPCA-1 cells by inducing cell cycle arrest and apoptosis. The combination of proapoptosis agents and antiautophagy agents could effectively enhance anticancer efficacy, which may be a new strategy in treating non-small cell lung cancer.

Lead Transiently Promotes Granulocyte-Macrophage Progenitor Differentiation and Subsequently Suppresses Common Myeloid Progenitor Differentiation.

Lead (Pb) is a toxic heavy metal affecting human health; it is known to be harmful to various organs or systems, yet the mechanisms by which Pb influences immune cell development remain to be defined. In this study, we show that Pb exposure (1250 ppm via drinking water) selectively impacted the development of myeloid cells (myelopoiesis). After Pb treatment of adult C57BL/6 mice, the numbers of granulocyte-macrophage progenitors (GMP) were consistently reduced, whereas the numbers of myeloid cells were increased at week (wk) 1 and decreased at wk8 after initiating the Pb exposure. Functional assays indicate that Pb accelerated GMP differentiation in a reactive oxygen species-dependent manner after treatment for 1 week and inhibited common myeloid progenitor differentiation by upregulating interferon regulatory factor 8 (IRF8) expression after treatment for 8 weeks. Consistent with the distinct Pb influences on myeloid cells observed at wk1 and wk8, Pb caused an inflammatory environment in vivo at wk8, but not at wk1. Furthermore, like the observations in mice during the Pb exposure, bloods from humans occupationally exposed to Pb had their numbers of monocytes, neutrophils and GMP negatively associated with the Pb concentration, whereas IRF8 expression in common myeloid progenitor, but not GMP, was positively correlated with the Pb concentration. These data suggest an occupationally relevant level of Pb exposure preferentially influences myelopoiesis involving reactive oxygen species and IRF8, which may contribute to the current understanding of the hematopoietic toxicology of Pb.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2

Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

429 - Hyperglycemia Primes Cells for Programmed Cell Death Shift in a Glycolysis-Dependent Manner

Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which are influenced by glycolysis, suggesting that increased cellular glucose may prime necroptosis. Here, we establish that exposure to hyperglycemic levels of glucose enhances necroptosis in U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemia induces a shift from apoptotic death to necroptosis in response to the extrinsic apoptotic stimulus TNFα. Increased membrane permeability and decreased phosphatidylserine exposure were seen in the hyperglycemic condition as compared to euglycemia. Additionally, cell death was shown to be caspase dependent under euglycemic conditions and shifted to RIP1 dependency in hyperglycemic conditions. Total levels of RIP1, RIP3, and MLKL increased following treatment with high levels of glucose suggesting a potential positive feedback mechanism. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Furthermore, the hyperglycemic state resulted in dysregulated calcium flux in a ROS dependent manner. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by inhibition of RIP1 with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD.

Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells

ABSTRACTRecently, we showed that the metal chelator TPEN targets colon cancer cells through redox cycling of copper. Here, we studied the DNA damage potential of TPEN and deciphered the role of Chk1, ATM and DNA-PK in TPEN-induced toxicity in 3 human colon cancer cell lines, HCT116, SW480 and HT29. We also investigated the role of reactive oxygen species (ROS) in TPEN-induced DNA damage. TPEN reduced cell viability in a dose- and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of γ-H2AX protein and activation of ATM/ATR signaling pathway. Cell death by TPEN was dependent on ROS generation as evidenced by the reversal of cell viability, and DNA damage and the abrogation of γ-H2AX levels in the presence of antioxidants. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10µM). TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression. Cell death by low TPEN concentrations, involved ATM/ATR signaling in all 3 cell lines, since pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells from TPEN-induced DNA damage. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of γ-H2AX and reversed cell death, suggesting that Chk1 and DNA-PK mediate TPEN-induced cytotoxicity in colon cancer cells. This study shows for the first time the involvement of Chk1, DNA-PK and ATM in TPEN-induced DNA damage and confirms our previous findings that ROS generation and the redox cycling of copper in response to TPEN are the main mechanisms by which this compound induces cell death in human colon cancer cells. Inhibition of ATM or DNA-PK did not reverse cytotoxicity at high TPEN concentrations that cause excessive levels of ROS and irreversible cellular damage.

Cigarette smoke reversibly activates hypoxia-inducible factor 1 in a reactive oxygen species-dependent manner.

Cigarette smoke (CS) is a major contributor to the development of a large number of fatal and debilitating disorders. However, the precise molecular mechanisms underlying the effects of CS in lung disease are largely unknown. To elucidate these pathophysiological processes, we examined the in vitro and in vivo effects of CS extract (CSE) and CS on the transcription factor, hypoxia-inducible factor 1 (HIF-1). CSE induced concentration- and time-dependent accumulation of HIF-1α protein in human lung epithelial-like cells under non-hypoxic conditions. Genes upregulated by HIF-1, including vascular endothelial growth factor and regulated in development and DNA damage response 1, both of which are involved in smoking-induced emphysematous changes, were increased by CSE treatment under non-hypoxic conditions in vitro and in vivo. Further investigation revealed that reactive oxygen species were generated in cells exposed to CSE and were required for CSE-mediated induction of HIF-1α protein, as was activation of phosphoinositide 3-kinase and mitogen-activated protein kinase pathways. In conclusion, we demonstrated that CSE and CS induced HIF-1 activation in vitro and in vivo, respectively. The evidence warrants further investigation to indicate that HIF-1 plays an important role in CS-induced gene expression, which is deeply involved in pulmonary cellular stress and small airway remodelling.

Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death.

Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H2O2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN-mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis.