Enhanced Reactive Oxygen Species Production Research Articles

T63, a new 4-arylidene curcumin analogue, induces cell cycle arrest and apoptosis through activation of the reactive oxygen species–FOXO3a pathway in lung cancer cells

Curcumin (diferuloylmethane) is a natural polyphenol product of the plant Curcuma longa and has a diversity of antitumor activities. T63, a new 4-arylidene curcumin analogue, was reported to inhibit proliferation of lung cancer cells. However, its precise molecular antitumor mechanisms have not been well elucidated. Here, we showed that T63 could significantly inhibit the proliferation of A549 and H460 human lung cell lines via induction of G0/G1 cell cycle arrest and apoptosis. We found that the reactive oxygen species (ROS)-activated FOXO3a cascade plays a central role in T63-induced cell proliferation inhibition. Mechanistically, enhancement of ROS production by T63 induced FOXO3a expression and nuclear translocation through activation of p38MAPK and inhibition of AKT, subsequently elevating the expression of FOXO3a target genes, including p21, p27, and Bim, and then increased the levels of activated caspase-3 and decreased the levels of cyclin D1. Moreover, the antioxidant N-acetylcysteine markedly blocked the above effects, and small interfering RNA-mediated knockdown of FOXO3a also significantly decreased T63-induced cell cycle arrest and apoptosis. In vivo experiments showed that T63 significantly suppressed the growth of A549 lung cancer xenograft tumors, associated with proliferation suppression and apoptosis induction in tumor tissues, without inducing any notable major organ-related toxicity. These data indicated that the novel curcumin analogue T63 is a potent antitumor agent that induces cell cycle arrest and apoptosis and has significant therapeutic potential for lung cancer.

Superoxide production during ischemia–reperfusion in the perfused rat heart: A comparison of two methods of measurement

Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia–reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223–38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.

Radioprotective role of H2S/CSE pathway in Chang liver cells

Radiation-induced liver cell damage may be life-threatening. Here, we investigated whether hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway could serve the protective role toward radiation in normal human liver cells. Our data showed that pretreatment of cells with H2S donor, sodium hydrosulfide (NaHS) significantly attenuated radiation induced micronuclei formation and improved cell viability. However, the use of dl-propargylglycine (PPG), a potent inhibitor of CSE, markedly enhanced the cell-killing effect induced by radiation. Exposure of cells to 2Gy γ-radiation led to significant increases of the endogenous H2S content. The mRNA and protein expressions of CSE also increased after radiation in a time-dependent manner, while the expression of cystathionine β-synthase (CBS), another endogenous H2S synthetase, did not change significantly. Notably, radiation induced production of reactive oxygen species (ROS) was significantly reversed by the pretreatment of NaHS, while blockage of CSE activity resulted in an enhanced ROS production in irradiated cells. Moreover, NaHS markedly suppressed radiation-induced phosphorylation of P53, decrease of Bcl-2/Bax, and activity of nuclear factor kappaB (NF-κB). In conclusion, our finding demonstrates that H2S/CSE pathway plays a radioprotection role by inhibiting radiation-induced ROS production, P53 phosphorylation, NF-κB activation and decrease of Bcl-2/Bax, indicating that modulation of H2S may be a novel protection strategy for liver radiation injury in radiotherapy.

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H2O2). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H2O2 and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H2O2 and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

Reactive Oxygen Species, Inflammation, and Lung Diseases

Reactive oxygen species (ROS) are well recognized for playing a dual role as both deleterious and beneficial species. ROS are products of normal cellular metabolism and under physiological conditions, participate in maintenance of cellular 'redox homeostasis. Overproduction of ROS, results in oxidative stress. Oxidative stress is a deleterious process that leads to lung damage and consequently to various disease states. The lung is a highly specialized organ that facilitates uptake of oxygen and release of carbon dioxide. Persistent inhalation of the invading pathogens or toxic agents may result in overwhelming production of ROS. Oxidants initiate a number of pathologic processes, including inflammation of the airways, which may contribute to the pathogenesis and/or exacerbation of airways disease. During inflammation, enhanced ROS production may induce recurring DNA damage, inhibition of apoptosis, and activation of protooncogenes by initiating signal transduction pathways. Therefore, it is conceivable that chronic inflammation-induced production of ROS in the lung may predispose individuals to lung diseases. In this review, we discuss mechanisms of oxidant stress in the lung, the role of oxidants in lung disease pathogenesis and exacerbation.

Tissue protection and endothelial cell signaling by 20-HETE analogs in intact ex vivo lung slices

The capacity to follow cell type-specific signaling in intact lung remains limited. 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous fatty acid that mediates signaling for a number of key physiologic endpoints in the pulmonary vasculature, including cell survival and altered vascular tone. We used confocal microscopy to identify enhanced reactive oxygen species (ROS) production in endothelial cell (EC)s in intact lung evoked by two stable analogs of 20-HETE, 20-5,14-HEDE (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid) and 20-5,14-HEDGE (N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine). These analogs generated increased ROS in cultured pulmonary artery endothelial cells as well. 20-HETE analog treatment decreased apoptosis of pulmonary tissue exposed to hypoxia-reoxygenation (HR) ex vivo. Enhanced ROS production and apoptosis were confirmed by biochemical assays. Our studies identify physiologically critical, graded ROS from ECs in live lung tissue ex vivo treated with 20-HETE analogs and protection from HR-induced apoptosis. These methodologies create exciting possibilities for studying signaling by stable 20-HETE analogs and other factors in pulmonary endothelial and other lung cell types in their native milieu.

Abstract 3094: Interferon-α induced TLR4 is indispensable for the synergistic induction of Duox2/DuoxA2 by lipopolysacharide (LPS) and IFN-γ in human pancreatic cancer cells

Abstract Several bacterial cell wall components, including lipopolysaccharide (LPS), muramyldipeptide (MDP), and flagellin, have been shown to engage their corresponding receptors and mediate NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) generation that may be involved in wound healing, host defense, and the inflammatory response in airway and gastrointestinal epithelial cells. Previously, we reported that two human pancreatic cancer cell lines, BxPC-3 and AsPC-1, respond to the pro-inflammatory cytokine IFN-γ by upregulating the expression of both Dual oxidase 2 and its cognate cofactor DuoxA2 resulting in enhanced ROS production. It has also been reported that human pancreatic cancer cells express TLR4/MyD88, and that signaling downstream of the TLR receptor may be responsible for LPS-induced invasiveness of pancreatic cancer cells. In our studies, we have demonstrated that IFN-γ up-regulates TLR4 and its adaptor protein MyD88 expression in BxPc-3 cells and augments the response of these cells to LPS, resulting in the activation of NFkB signaling, enhancing p65 subunit accumulation in the nucleus. Moreover, we found that p65 binds to the Duox2 promoter, and cooperates with the IFN-γ- induced Stat1 expression to synergistically enhance Duox2/DuoxA2 expression, leading to persistent intra- and extracellular ROS accumulation. TLR4 silencing by RNAi and two independent NF-kB inhibitors (PDTC and BAY11-70829) specifically attenuate LPS- and IFN-γ-mediated Duox2 expression in BxPc-3 cells, underscoring the importance of TLR4 engagement and the activation of NF-kB signaling in regulating Duox2 expression and subsequent ROS generation by LPS and IFN-γ. Our data suggest that sustained extracellular H2O2 accumulation, mediated by LPS- and IFN-γ- induced Duox2/DuoxA2 expression, may contribute to the deleterious effects of cytokine-mediated pancreatic inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3094. doi:1538-7445.AM2012-3094

Sub‐contractile sphingosylphosphorylcholine enhances vasoreactivity via reactive oxygen species

Sphingosylphosphorylcholine (SPC), at concentrations that do not cause contraction (<1uM), causes potentiation of vasoconstriction to other agonists or depolarisation (Snetkov et al, Hypertension 2008;51:239–245). Our objective was to determine whether this was related to activation of NADPH oxidase and production of reactive oxygen species (ROS).METHODSSmall pulmonary or mesenteric arteries were mounted on a myograph, challenged with depolarising solutions containing ~23 mM KCl to elicit a small contraction, and incubated with SPC (1uM) before being re‐challenged.RESULTS0.2 to 1uM SPC caused substantial potentiation of depolarisation‐induced force to between 400 and 800% of control in both rat and mouse arteries. Tempol (3mM) suppressed potentiation (e.g. PA: 526 +/− 220%; +tempol: 200+/−60%, n=5). SPC enhanced ROS production estimated by lucigenin and DCF. Potentiation was abolished in p47phox −/− mice (n=6). 0.2 uM SPC enhanced voltage activated calcium current in whole cell patch by ~30% (p<0.01), and this was mimicked by exogenous ROS. We conclude that low concentrations of SPC may cause hyperreactivity via NADPH oxidase and elevation of ROS, which enhances calcium entry via L‐type channels. Funded by the Wellcome Trust

Berberine improves endothelial function by reducing endothelial microparticles-mediated oxidative stress in humans

BackgroundCirculating endothelial microparticles (EMPs) lead to endothelial dysfunction by increasing oxidative stress. Berberine has a beneficial effect on endothelial function, but no data are available on the EMP-mediated oxidative stress. The present study tests the hypothesis that berberine contributes to the improvement of endothelial function in humans via inhibiting EMP-mediated oxidative stress in vascular endothelium. MethodsTwelve healthy subjects received a 1-month berberine therapy and eleven healthy subjects served as control. Endothelium-dependent and -independent function in the brachial artery was assessed by flow-mediated vasodilation (FMD) and sublingual nitroglyceride-mediated vasodilation (NMD). Circulating EMPs and serum malondialdehyde (MDA) were measured before and after therapy. Furthermore, in vitro human umbilical vein endothelial cells (HUVECs) were stimulated by EMPs with or without presence of anti-oxidant compound apocynin or berberine. Intracellular reactive oxygen species (ROS), nitric oxide (NO) production and NADPH oxidase 4 (Nox4) protein expressions were examined, respectively. ResultsThe levels of serum MDA and circulating CD31+/CD42− MPs were significantly reduced in the berberine group compared with the control group, which were associated with improvement of FMD. The EMPs in vitro facilitated ROS production and Nox4 protein expression and reduced NO synthesis in HUVECs. These alterations can be reversed by the presence of apocynin or berberine, respectively. ConclusionThe present study demonstrated for the first time that EMP-induced upregulation of Nox4 expression may enhance ROS production in HUVECs. Berberine treatment contributes to the amelioration of endothelial function through a partially reducing oxidative stress of vascular endothelium induced by circulating CD31+/CD42− microparticles in humans.

Uncoupling Protein-2 Protects Endothelial Function in Diet-Induced Obese Mice

Previous studies indicate uncoupling protein-2 (UCP2) as an antioxidant defense against endothelial dysfunction in hypertension. UCP2 also regulates insulin secretion and action. However, the role of UCP2 in endothelial dysfunction associated with diabetes and obesity is unclear. UCP2 protects against endothelial dysfunction induced by high-fat diet through inhibition of reactive oxygen species (ROS) production, and subsequent increase of nitric oxide bioavailability. Endothelium-dependent relaxation (EDR) in aortae and mesenteric arteries in response to acetylcholine was measured in wire myograph. Flow-mediated vasodilatation in 2(nd)-order mesenteric arteries was measured in pressure myograph. ROS production is measured by CM-H(2)DCFDA and DHE fluorescence. High-glucose exposure reduced EDR in mouse aortae, which was exaggerated in UCP2 knockout (KO) mice, whereas UCP2 overexpression by adenoviral infection (AdUCP2) restored the impaired EDR. Impairment of EDR and flow-mediated vasodilatation in aortae and mesenteric arteries from high-fat diet-induced obese mice (DIO) was exaggerated in UCP2KO DIO mice compared with wild-type DIO littermates, whereas AdUCP2 i.v. injection restored both EDR and flow-mediated vasodilatation in DIO mice. Improved EDR in mesenteric arteries was inhibited by nitric oxide synthase inhibitor. UCP2 overexpression also inhibited intracellular ROS production in the en face endothelium of aorta and mesenteric artery of DIO mice, whereas UCP2 deficiency enhanced ROS production. UCP2 preserves endothelial function through increasing nitric oxide bioavailability secondary to the inhibition of ROS production in the endothelium of obese diabetic mice.

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

Cytochrome c as an amplifier of ROS release in mitochondria

The influence of exogenous cytochrome c on reactive oxygen species (ROS) formation and its dependence on mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. Fluorescent probe dichlorofluorescein (DCF) was used. It was shown that MPTP activation by increasing concentrations of Ca2+ in the medium results in the increase in mitochondrial ROS production and oxygen consumption, but the decrease in matrix calcium retention, dependent on the amount of added Ca2+. Cytochrome c in the incubation medium does not much influence ROS formation when MPTP opening is blocked by cyclosporine A. However, in the presence of cytochrome c MPTP opening is accompanied by dramatic increase in ROS production. Steep rise in DCF fluorescence because of matrix ROS formation is sensitive to MPTP opening and is not resulted from the direct interaction between the probe and cytochrome c outside the mitochondria. To explain obtained data the hypothesis is put forward that MPTP could serve for ROS exchange between the matrix and the medium where heme iron of cytochrome c would act as a catalytic center to enhance ROS production. We suppose that apart of its conventional function, cytochrome c which is not involved in electron transport, could serve in such way as the amplifier of ROS production which in turn would provide a background for the development of apoptosis due to MPTP opening.

Astroglial Pentose Phosphate Pathway Rates in Response to High-Glucose Environments

ROS (reactive oxygen species) play an essential role in the pathophysiology of diabetes, stroke and neurodegenerative disorders. Hyperglycaemia associated with diabetes enhances ROS production and causes oxidative stress in vascular endothelial cells, but adverse effects of either acute or chronic high-glucose environments on brain parenchymal cells remain unclear. The PPP (pentose phosphate pathway) and GSH participate in a major defence mechanism against ROS in brain, and we explored the role and regulation of the astroglial PPP in response to acute and chronic high-glucose environments. PPP activity was measured in cultured neurons and astroglia by determining the difference in rate of 14CO2 production from [1-14C]glucose and [6-14C]glucose. ROS production, mainly H2O2, and GSH were also assessed. Acutely elevated glucose concentrations in the culture media increased PPP activity and GSH level in astroglia, decreasing ROS production. Chronically elevated glucose environments also induced PPP activation. Immunohistochemical analyses revealed that chronic high-glucose environments induced ER (endoplasmic reticulum) stress (presumably through increased hexosamine biosynthetic pathway flux). Nuclear translocation of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), which regulates G6PDH (glyceraldehyde-6-phosphate dehydrogenase) by enhancing transcription, was also observed in association with BiP (immunoglobulin heavy-chain-binding protein) expression. Acute and chronic high-glucose environments activated the PPP in astroglia, preventing ROS elevation. Therefore a rapid decrease in glucose level seems to enhance ROS toxicity, perhaps contributing to neural damage when insulin levels given to diabetic patients are not properly calibrated and plasma glucose levels are not adequately maintained. These findings may also explain the lack of evidence for clinical benefits from strict glycaemic control during the acute phase of stroke.

Differential expression of Arabidopsis defense-related genes in response to sulfur dioxide

Sulfur dioxide (SO2) is one of the most common and harmful air pollutants. To analyze cellular responses to SO2, we investigated the transcript alterations, antioxidant enzyme activities and reactive oxygen species (ROS) levels in Arabidopsisthaliana (Col-0) exposed to SO2. Transcriptional profiling using Affymetrix GeneChip technology identified 494 genes differentially expressed (⩾2-fold change) in plants exposed to 30mgm−3 SO2 for 72h, including up-regulation of some defense-related genes encoding antioxidant enzymes and heat shock proteins. Moreover, numerous genes encoding pathogenesis-related proteins and enzymes required for the phenylpropanoid pathway and for cell wall modification were highly activated upon SO2 exposure. We selected eight of the significantly differentially expressed defense-related genes for analysis using semi-quantitative RT-PCR and confirmed that their expression was up-regulated under SO2 stress. In addition, SO2 exposure caused the enhancement of ROS production, and also increased activities of antioxidant enzymes (superoxide dismutase, peroxidase, glutathione peroxidase and glutathione S-transferase) in Arabidopsis plants. Our results indicated that increased ROS may act as a signal to induce defense responses, which provide enhanced defense capacity to protect plants against SO2 and other environmental stress. This is the first transcriptional profiling that identifies novel genes and pathways involved in SO2 stress responses in plant cells. These data will be helpful for better understanding the molecular basis for plant adaptation to SO2 stress.

Astrogliopathy as a loss of astroglial protective function against glycoxidative stress under hyperglycemia

Reactive oxygen species (ROS) derived from mitochondria play an essential role in stroke as well as in neurodegenerative disorders. Although hyperglycemia associated with diabetes mellitus is well known to enhance ROS production in vascular endothelial cells, the effects of either acute or chronic high glucose environments on neurons and glial cells remain unclear. Astroglia play a pivotal role in glucose metabolism. Thus, the astroglial metabolic response to high glucose environments is an interesting subject. In particular, the glutathione/pentose phosphate pathway (PPP) system, which is a major defense mechanism against ROS in the brain, contributes to glucose metabolism and is more active in astroglia. We propose that high glucose environments activate PPP through an increased flux to the hexosamine biosynthetic pathway (HBP). HBP is known to induce endoplasmic reticulum (ER) stress under hyperglycemia, resulting in the nuclear translocation of nuclear factor-erythroid-2-related factor 2 (Nrf2), a master regulator of phase 2 detoxifying enzymes including glucose-6-phosphate dehydrogenase that regulates PPP activity, as Nrf2 is reported to be a direct substrate of protein kinase RNA (PKR)-like ER kinase (PERK), a transducer of ER stress. Therefore, the phosphorylation of Nrf2 by hyperglycemia-induced ER stress facilitates Nrf2 translocation through PERK, thus activating the PPP. If acute or chronic hyperglycemia induces PPP activation in astroglia to reduce ROS, reducing the glucose concentration may be accompanied by a risk, which may explain the lack of evidence that strict glycemic control during the acute phase of stroke conveys no beneficial effect.

MAP17, a ROS-dependent oncogene

MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas. Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition, decreased apoptotic sensitivity, and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis. MAP17 is overexpressed in a great variety of human carcinomas, including breast tumors. Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression. Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and, especially, for malignant progression.

Hirsutanol A Induces Apoptosis and Autophagy via Reactive Oxygen Species Accumulation in Breast Cancer MCF-7 Cells

Hirsutanol A is a novel sesquiterpene compound purified from the marine fungus Chondrostereum sp in the coral Sarcophyton tortuosum. Our previous studies had demonstrated that hirsutanol A exerted potent cytotoxic effect in many kinds of cancer cell lines. Here, the anticancer molecular mechanisms of hirsutanol A were investigated in breast cancer MCF-7 cells. The results showed that hirsutanol A could inhibit cell proliferation, elevate reactive oxygen species (ROS) level, and induce apoptosis and autophagy. Co-treatment with the potent antioxidant agent N-acetyl-l-cysteine could effectively reverse the effect of enhanced ROS production, which in turn, reduces growth inhibition, apoptosis, and autophagy mediated by hirsutanol A. In addition, blocking autophagy by bafilomycin A1 or Atg7-siRNA could synergistically enhance the antiproliferative effect and apoptosis induced by hirsutanol A. These data suggested that hirsutanol A could induce apoptosis and autophagy via accumulation of ROS and co-treatment with an autophagy inhibitor could sensitize MCF-7 cells to hirsutanol A.

Loss of Stem Cell Quiescence and Impaired Stem Cell Function of CD34+/CD38low Cells One Year Following Autologous Stem Cell Transplantation

Abstract 1893Autologous stem cell transplantation (ASCT) allows the application of high-dose chemotherapy and is included in the standard treatment regimens for multiple myeloma and relapsing lymphoma. The application of ASCT has considerably improved treatment outcome but in 30–50% of the patients the underlying malignant disorder relapses. In that case treatment options are limited, in part due to a diminished capacity of the transplanted bone marrow to tolerate chemotherapy. The transplanted bone marrow seems to be more vulnerable to chemotoxic stress. This is supported by our recent observations that progenitor cells from post-ASCT bone marrow demonstrate altered phenotypic and functional properties (Woolthuis et al. BMT 2011:110-115). In the present study, a more detailed examination of the hematopoietic stem cell (HSC) compartment post-ASCT was performed. The examined bone marrow cells were obtained from patients with normal peripheral blood cell counts one year after ASCT. First it was studied whether stem cell quiescence had been changed due to the ASCT procedure. Therefore the percentage of cells in the G0 phase was measured by staining the cells with Hoechst and Pyronin Y followed by flowcytometric analysis. Both the stem cell (CD34+/CD38low) and progenitor (CD34+/CD38+) fractions were analyzed. Post-ASCT bone marrow cells (n=6) were compared with normal bone marrow cells (n=9) and mobilized peripheral blood stem cells (PBSC) (n=7). Interestingly, post-ASCT bone marrow contained a significantly lower percentage of quiescent cells in the CD34+/CD38low fraction compared to normal bone marrow (mean percentage 23.6% (95%CI: 6.5–40.8) vs. 48.6% (95%CI: 31.4–65.9), p=0.045). In contrast no differences were observed in the CD34+/CD38+ fraction. To examine whether the loss of stem cell quiescence is associated with loss of stem cell function, stem cell frequency was analyzed in vitro by the long-term colony-initiating cell assay. These analyses revealed a strongly decreased stem cell frequency in post-ASCT CD34+ cells compared to normal CD34+ bone marrow cells (mean frequency 0.0016 (95%CI: 0.0003–0.0028) vs. 0.0206 (95%CI: 0.0162–0.0250), p=0.002). Importantly, CD34+ PBSC cells demonstrated a stem cell frequency comparable with the frequency observed in normal bone marrow, suggesting that the decrease in stem cell frequency observed in post-ASCT bone marrow can not simply be explained by the previous chemotherapy and the mobilization procedure. To obtain more insight into the mechanisms explaining our observations, levels of reactive oxygen species (ROS) were measured by flowcytometry. Comparable ROS levels were observed in post-ASCT and normal bone marrow CD34+/CD38low cells while the CD34+/CD38+ post-ASCT cells demonstrated significantly higher ROS levels compared to normal bone marrow (p=0.01). In addition significant higher ROS levels were observed in CD34+/CD38low PBSC compared to normal bone marrow (p=0.043). To define pathways associated with altered quiescence and/or ROS production, gene expression analysis was performed comparing CD34+ cells from post-ASCT bone marrow (n=6), normal bone marrow (n=31) and PBSC (n=5). Preliminary analysis reveals an upregulation in post-ASCT CD34+ cells of genes involved in cell cycle compared to normal bone marrow. In contrast, cell cycle genes appeared to be downregulated in CD34+ PBSC cells. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow post-ASCT is likely related to a loss of stem cell quiescence and enhanced ROS production by progenitor cells which will make the bone marrow cells more vulnerable for the effects of cytotoxic stress. Disclosures:No relevant conflicts of interest to declare.

Genomic Instability Originates From Leukemia Stem Cells in a Mouse Model of CML-CP

Abstract 445For decades, chronic myeloid leukemia (CML) has served not only as a paradigm for understanding the evolution and multi-step process of carcinogenesis but also for studying cancer stem and progenitor cells responsible for the initiation and/or maintenance of the disease. CML is initiated by BCR-ABL1 tyrosine kinase transformation of hematopoietic stem cells into leukemia stem cells (LSCs) to induce CML-chronic phase (CML-CP). The deregulated growth of LSC-derived leukemia progenitor cells (LPCs) leads to manifestation of the disease. It is unclear if LSCs and/or LPCs are able to acquire additional genetic changes that confer resistance to tyrosine kinase inhibitors (TKIs) and induce more aggressive CML blast phase (CML-BP). In addition, the mechanisms and consequences of genomic instability may differ substantially among these cells. For example, the effects of genetic aberrations acquired in quiescent LSCs may be dormant, but if the aberrations induce proliferation or appear in LSCs that are already cycling, they may generate TKI-resistant and/or more malignant clones. Alternatively, genomic instability in LPCs must be accompanied by the acquisition of LSC-like properties to prevent mutations from disappearing before they undergo terminal maturation.Previously, we reported that BCR-ABL1–transformed cell lines accumulate reactive oxygen species (ROS)-induced oxidative DNA damage [8-oxoguanine (8oxoG), double strand breaks (DSBs)] resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). To determine which populations of CML-CP cells, LSCs and/or LPCs, accumulate genomic instability we employed the SCLtTA/BCR-ABL1 tetracycline-inducible (tet-off) transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood. BCR-ABL1 mRNA expression was higher in the Lin−c-Kit+Sca1+ murine leukemia stem cell–enriched population (muLSCs) than in the Lin−c-Kit+Sca1− murine leukemia progenitor cell–enriched population (muLPCs), thus reminiscent of human CML-CP (Lin−CD34+CD38− LSCs > Lin−CD34+CD38+ LPCs). BCR-ABL1 induction increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA damage (8-oxoG, DSBs) in muLSCs, but not in muLPCs. In addition, CFSEmax/eFluor670max quiescent muLSCs displayed more ROS (superoxide, hydrogen peroxide) and oxidative DNA damage (8oxoG, DSBs) than non-induced counterparts. Currently, we are examining genomic instability in the most primitive long-term muLSCs (Lin−c-Kit+Sca1+CD34−Flt3−). Lastly, single nucleotide polymorphism (SNP) arrays detected a variety of genetic aberrations (addition, deletions) in BCR-ABL1–induced Lin− BM cells. Individual mice displayed a great degree of diversity in the intensity of genetic instability accumulating between 31 to 826 aberrations, which recapitulate heterogeneity of sporadic aberrations detected in CML-CP patients. These aberrations include deletions in Trp53 and Ikzf1, and additions in Zfp423 and Idh1 genes, which have been linked to progression from CML-CP to CML-BP.In summary, by using the SCLtTA/BCR-ABL1 inducible transgenic mouse model of CML-CP we showed that muLSCs, but not muLPCs, displayed elevated levels of ROS-induced oxidative DNA damage likely resulting in the accumulation of extensive genetic aberrations. This observation supports the hypothesis that genomic instability in CML-CP originates in LSCs. Current analysis of microarrays may shed some light on the mechanisms leading to enhanced ROS production and accumulation of oxidative DNA damage in muLSCs. Disclosures:Koschmieder:Novartis, Bristol-Myers Squibb: Consultancy.

Oxidative Stress, Mitochondrial Dysfunction, and Aging

Aging is an intricate phenomenon characterized by progressive decline in physiological functions and increase in mortality that is often accompanied by many pathological diseases. Although aging is almost universally conserved among all organisms, the underlying molecular mechanisms of aging remain largely elusive. Many theories of aging have been proposed, including the free-radical and mitochondrial theories of aging. Both theories speculate that cumulative damage to mitochondria and mitochondrial DNA (mtDNA) caused by reactive oxygen species (ROS) is one of the causes of aging. Oxidative damage affects replication and transcription of mtDNA and results in a decline in mitochondrial function which in turn leads to enhanced ROS production and further damage to mtDNA. In this paper, we will present the current understanding of the interplay between ROS and mitochondria and will discuss their potential impact on aging and age-related diseases.