PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Khz (Fusion of Ganoderma lucidum and Polyporus umbellatus Mycelia) Induces Apoptosis by Increasing Intracellular Calcium Levels and Activating JNK and NADPH Oxidase-Dependent Generation of Reactive Oxygen Species

Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47^phox and p67^phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca²⁺]_i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47^phox and p67^phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca²⁺]_i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Panaxydol induces apoptosis through an increased intracellular calcium level,activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species

Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce apoptosis through an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), activation of JNK and p38 MAPK, and generation of reactive oxygen species (ROS) initially by NADPH oxidase and then by mitochondria. Panaxydol-induced apoptosis was caspase-dependent and occurred through a mitochondrial pathway. ROS generation by NADPH oxidase was critical for panaxydol-induced apoptosis. Mitochondrial ROS production was also required, however, it appeared to be secondary to the ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the membrane translocation of regulatory p47^phox and p67^phox subunits and shown to be necessary for ROS generation by panaxydol treatment. Panaxydol triggered a rapid and sustained increase of [Ca²⁺]_i, which resulted in activation of JNK and p38 MAPK. JNK and p38 MAPK play a key role in activation of NADPH oxidase, since inhibition of their expression or activity abrogated membrane translocation of p47^phox and p67^phox subunits and ROS generation. In summary, these data indicate that panaxydol induces apoptosis preferentially in cancer cells, and the signaling mechanisms involve a [Ca²⁺]_i increase, JNK and p38 MAPK activation, and ROS generation through NADPH oxidase and mitochondria.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

ROS-challenged keratinocytes as a new model for oxidative stress-mediated skin diseases

In the current study, the effects of the reactive oxygen species (ROS) generator 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water-soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose-dependent increase in NADPH oxidase activity in intact keratinocytes. The long-term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH-treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O ₂ ⁻), while AAPH-treated keratinocytes overproduce hydrogen peroxide (H ₂O ₂) in extracellular medium. H ₂O ₂ is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost-effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes-melanocytes cross-talk studies is also suggested. The model of AAPH-stressed human keratinocytes described here can represent a useful tool for redox cross-talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.     

PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT₁) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [³H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [¹²⁵I]AngII to AT₁ receptors was blocked by Ib in a concentration-dependent manner with IC₅₀ value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.     

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.     

Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT₁R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC.     

A critical role for Romo1-derived ROS in cell proliferation

Low levels of endogenous reactive oxygen species (ROS) originating from NADPH oxidase have been implicated in various signaling pathways induced by growth factors and mediated by cytokines. However, the main source of ROS is known to be the mitochondria, and increased levels of ROS from the mitochondria have been observed in many cancer cells. Thus far, the mechanism of ROS production in cancer cell proliferation in the mitochondria is not well-understood. We recently identified a novel protein, ROS modulator 1 (Romo1), and reported that increased expression of Romo1-triggered ROS production in the mitochondria. The experiments conducted in the present study showed that Romo1-derived ROS were indispensable for the proliferation of both normal and cancer cells. Furthermore, whilst cell growth was inhibited by blocking the ERK pathway in cells transfected with siRNA directed against Romo1, the cell growth was recovered by addition of exogenous hydrogen peroxide. The results of this study suggest that Romo1-induced ROS may play an important role in redox signaling in cancer cells.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Surfactin-Induced Apoptosis Through ROS–ERS–Ca2+-ERK Pathways in HepG2 Cells

Although surfactin is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism responsible for this process remain elusive. In this study, the signaling network underlying the apoptosis of human hepatoma (HepG2) cells induced by surfactin was investigated. It is found that the reaction oxygen species (ROS) production and intracellular calcium ([Ca²⁺]_i) accumulation are both induced HepG2 cells apoptosis. The [Ca²⁺]_i exaltation was partly depended on the Ca²⁺ release from inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry) receptors channels, which both triggered endoplasmic reticulum stress (ERS). The results showed that surfactin induced the ROS production and ROS production led to ERS. The occurrence of ERS increased the [Ca²⁺]_i level and the processes associated with blocking extracellular signal-regulated kinase (ERK) pathway. According to a comprehensive review of all the evidence, it is concluded that surfactin induces apoptosis of HepG2 cells through a ROS–ERS–Ca²⁺ mediated ERK pathway.     

RIP3-Dependent Accumulation of Mitochondrial Superoxide Anions in TNF-α-Induced Necroptosis

Excessive production of reactive oxygen species (ROS) is a key phenomenon in tumor necrosis factor (TNF)-α-induced cell death. However, the role of ROS in necroptosis remains mostly elusive. In this study, we show that TNF-α induces the mitochondrial accumulation of superoxide anions, not H₂O₂, in cancer cells undergoing necroptosis. TNF-α-induced mitochondrial superoxide anions production is strictly RIP3 expression-dependent. Unexpectedly, TNF-α stimulates NADPH oxidase (NOX), not mitochondrial energy metabolism, to activate superoxide production in the RIP3-positive cancer cells. In parallel, mitochondrial superoxide-metabolizing enzymes, such as manganese-superoxide dismutase (SOD2) and peroxiredoxin III, are not involved in the superoxide accumulation. Mitochondrial-targeted superoxide scavengers and a NOX inhibitor eliminate the accumulated superoxide without affecting TNF-α-induced necroptosis. Therefore, our study provides the first evidence that mitochondrial superoxide accumulation is a consequence of necroptosis.     

Polyphenols decreased liver NADPH oxidase activity,increased muscle mitochondrial biogenesis and decreased gastrocnemius age-dependent autophagy in aged rats

Abstract

This study explored major systems of reactive oxygen species (ROS) production and their consequences on oxidative stress, mitochondriogenesis and muscle metabolism in aged rats, and evaluated the efficiency of 30-day oral supplementation with a moderate dose of a red grape polyphenol extract (RGPE) on these parameters. In the liver of aged rats, NADPH oxidase activity was increased and mitochondrial respiratory chain complex activities were altered, while xanthine oxidase activity remained unchanged. In muscles, only mitochondrial activity was modified with aging. The oral intake of RGPE decreased liver NADPH oxidase activity in the aged rats without affecting global oxidative stress, suggesting that NADPH oxidase was probably not the dominant detrimental source of production of O₂·^? in the liver. Interestingly, RGPE supplementation increased mitochondrial biogenesis and improved antioxidant status in the gastrocnemius of aged rats, while it had no significant effect in soleus. RGPE supplementation also decreased age-dependent autophagy in gastrocnemius of aged rats. These results extended existing findings on the beneficial effects of RGPE on mitochondriogenesis and muscle metabolism in aged rats.     

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT₁ receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT₁ receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT₁ receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar¹-Ile⁴-Ile⁸]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT₁ receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT₁ receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic β-cells: evidence for regulation by Rac1

Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated β-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic β-cells culminating in the onset of diabetes. Although the mitochondrial electron transport chain is felt to be the primary source of ROS, several lines of recent evidence suggest that phagocyte-like NADPH oxidase plays a central role in cytokine-mediated ROS generation and apoptosis of β-cells. However, the precise mechanisms underlying the regulation of NADPH oxidase remain unknown. To address this, insulin-secreting INS 832/13 cells were treated with cytomix (IL-1β, IFN-γ, and TNF-α; 10 ng/ml each) for different time intervals (0-24 h). A significant, time-dependent increase in NADPH oxidase activation/intracellular ROS production, p47(phox) subunit, but not p67(phox) subunit, expression of the phagocyte-like NADPH oxidase were demonstrable under these conditions. Furthermore, siRNA-p47(phox) transfection or exposure of INS 832/13 cells to apocynin, a selective inhibitor of NADPH oxidase, markedly attenuated cytomix-induced ROS generation in these cells. Cytomix-mediated mitochondrial dysfunction in INS 832/13 cells was evident by a significant loss of mitochondrial membrane potential (MMP) and upregulated caspase 3 activity. Cytomix treatment also caused a transient (within 15 min) activation of Rac1, a component of the NADPH oxidase holoenzyme. Furthermore, GGTI-2147 and NSC23766, known Rac1 inhibitors, not only attenuated the cytomix-induced Rac1 activation but also significantly prevented loss of MMP (NSC23766 > GGTI-2147). However, NSC23766 had no effect on cytomix-induced NO generation or caspase 3 activation, suggesting additional regulatory mechanisms might underlie these signaling steps. Together, these findings suggested that Rac1-mediated regulation of phagocyte-like NADPH oxidase contributes to cytokine-mediated mitochondrial dysfunction in the β-cell.     

Time-Dependent Stabilization of Hypoxia Inducible Factor-1α by Different Intracellular Sources of Reactive Oxygen Species

Intratumoral hypoxia is a major obstacle in the development of effective cancer chemotherapy, decreasing the efficacy of anti-neoplastic drugs in several solid tumours. The hypoxic environment, through its master regulator hypoxia inducible factor-1 (HIF-1), is able to maintain an anti-apoptotic potential through activation of critical genes associated with drug resistance. Besides affecting metabolism and motility of tumour cells, hypoxia also paradoxically increases production of reactive oxygen species (ROS), which contribute to stabilize HIF-1 through a redox-mediated inhibition of its proteolysis. Here we reported that 1% O₂ hypoxia increases the resistance of human metastatic melanoma cells to conventional chemotherapy with etoposide, and that the increase in chemoresistance strongly depends on ROS delivery due to hypoxia. We reported a biphasic redox-dependent role of HIF-1, involving mitochondrial complex III and NADPH oxidase as oxidants sources, synergising in enhancing survival to chemotherapy. The feed-forward loop engaged by hypoxia involves first an HIF-1-dependent vascular endothelial growth factor-A (VEGF-A) autocrine production and, in the later phase, activation of NADPH oxidase from VEGF/VEGFR2 interaction, finally leading to a further redox-dependent long lasting stabilization of HIF-1. We therefore identified a redox-dependent circuitry linking hypoxia-driven ROS to VEGF-A secretion and to enhanced melanoma cell survival to etoposide chemotherapy.