Mitochondria-derived Reactive Oxygen Species Production Research Articles

Trichostatin A Targets the Mitochondrial Respiratory Chain, Increasing Mitochondrial Reactive Oxygen Species Production to Trigger Apoptosis in Human Breast Cancer Cells

AimHistone deacetylase inhibitors (HDACIs)-based therapies have stimulated interest via their anti-tumor activities, including apoptosis induction, cell cycle arrest, cell differentiation, and autophagy. However, the mechanisms of HDACI-associated anti-tumor activity are not yet clearly defined. The aim of this study was to explore the key events of Trichostatin A (TSA), a classic HDACI agent, against breast cancer cells.MethodsThe MCF-7, MDA-MB-231 and MCF-10A cell lines were evaluated with colony-forming and cell viability assays. Apoptosis and cell cycle distribution were detected by flow cytometry. Mitochondrial function was measured with biochemical assays, flow cytometry and transmission electron microscopy.ResultsTSA inhibited breast cancer cell viability and proliferation, without affecting MCF-10A cell. TSA-induced breast cancer cell apoptosis was initiated by G2-M arrest and depended on mitochondrial reactive oxygen species (ROS) produced subsequent to reduced mitochondrial respiratory chain activity. The enhanced mitochondrial ROS production and apoptosis in cancer cells were markedly attenuated by antioxidants, such as N-acetyl cysteine (NAC), reduced glutathione (GSH) and Vitamin C.ConclusionThe present study demonstrated that TSA-induced cell death by arresting cell cycle in G2-M phase and was dependent on production of mitochondria-derived ROS, which was derived from impaired mitochondrial respiratory chain.

3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines

Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.

Cobalt Protoporphyrin Induces HO-1 Expression Mediated Partially by FOXO1 and Reduces Mitochondria-Derived Reactive Oxygen Species Production

BackgroundReactive oxygen species arise in the mitochondria as byproducts of respiration and oxidase activity and have important roles in many physiological and pathophysiological conditions. The level of reactive oxygen species is regulated by a number of enzymes and physiological antioxidants, including HO-1, Sod2, catalase and COX-2, etc. And HO-1 against oxidative stress requires an increase in stress-responsive genes, such as Sod2 and catalase. Especially for the activity of HO-1, cobalt protoporphyrin is known to be a potent and effective inducer in many tissues. The transcription factor, FOXO1 is resistant to oxidative stress through downregulating reactive oxygen species production. Previous study showed that FOXO1 induces HO-1 expression by binding to HO-1 promoter. The question whether cobalt protoporphyrin induces HO-1 expression mediated by FOXO1 and subsequently lessens reactive oxygen species production remains to be elucidated.ResultsCobalt protoporphyrin enhances the expression of FOXO1 and facilitates FOXO1 binding to HO-1 promoter and increasing its transcriptional activity without influencing the FOXO1 protein stability. CoPP induces HO-1 and other oxidative stress-responsive genes expression, such as catalase, cytochrome c, Sod2, and COX-2, and decreases mitochondria-derived reactive oxygen species production, which are mediated partially by FOXO1.ConclusionsCobalt protoporphyrin induces HO-1 and other oxidative stress-responsive genes expression mediated partially by FOXO1, and has an important role in reducing cellular reactive oxygen species level. Cobalt protoporphyrin may be a more promising therapeutic agent to upregulate some antioxidantive genes.

Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

Endothelial injury related to oxidative stress is a key event in cardiovascular diseases, such as hypertension and atherosclerosis. The activation of the redox-sensitive Kv1.5 potassium channel mediates mitochondrial reactive oxygen species (ROS)-induced apoptosis in vascular smooth muscle cells and some cancer cells. Kv1.5 channel is therefore taken as a new potential therapeutic target for pulmonary hypertension and cancers. Although Kv1.5 is abundantly expressed in vascular endothelium, there is little knowledge of its role in endothelial injury related to oxidative stress. We found that DPO-1, a specific inhibitor of Kv1.5, attenuated H2O2-evoked endothelial cell apoptosis in an in vivo rat carotid arterial model. In human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells (HPAECs), angiotensin II and oxLDL time- or concentration-dependently enhanced Kv1.5 protein expression in parallel with the production of intracellular ROS and endothelial cell injury. Moreover, siRNA-mediated knockdown of Kv1.5 attenuated, whereas adenovirus-mediated Kv1.5 cDNA overexpression enhanced oxLDL–induced cellular damage, NADPH oxidase and mitochondria-derived ROS production and restored the decrease in protein expression of mitochondria uncoupling protein 2 (UCP2). Collectively, these data suggest that Kv1.5 may play an important role in oxidative vascular endothelial injury.

Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Persistent Oxidative Stress Due to Absence of Uncoupling Protein 2 Associated with Impaired Pancreatic β-Cell Function

Uncoupling protein (UCP) 2 is a widely expressed mitochondrial protein whose precise function is still unclear but has been linked to mitochondria-derived reactive oxygen species production. Thus, the chronic absence of UCP2 has the potential to promote persistent reactive oxygen species accumulation and an oxidative stress response. Here, we show that Ucp2-/- mice on three highly congenic (N >10) strain backgrounds (C57BL/6J, A/J, 129/SvImJ), including two independently generated sources of Ucp2-null animals, all exhibit increased oxidative stress. Ucp2-null animals exhibit a decreased ratio of reduced glutathione to its oxidized form in blood and tissues that normally express UCP2, including pancreatic islets. Islets from Ucp2-/- mice exhibit elevated levels of numerous antioxidant enzymes, increased nitrotyrosine and F4/80 staining, but no change in insulin content. Contrary to results in Ucp2-/- mice of mixed 129/B6 strain background, glucose-stimulated insulin secretion in Ucp2-/- islets of each congenic strain was significantly decreased. These data show that the chronic absence of UCP2 causes oxidative stress, including in islets, and is accompanied by impaired glucose-stimulated insulin secretion.

Rac1‐dependent ROS production mediates TNF‐alpha‐induced apoptosis in intestinal epithelial cells

Reactive oxygen species (ROS) play a key role in cellular signaling pathways regulating cell proliferation, motility and apoptosis. We have shown that the small GTPase Rac1 regulates apoptosis via c-Jun N-terminal kinase (JNK) activation in intestinal epithelial cells (IEC-6). However, the mechanism by which Rac1 regulates JNK-mediated-apoptosis is unclear. The current studies test the hypothesis that Rac1-mediated ROS production is involved in TNF-α-induced apoptosis. First, mitochondrial ROS production increased during TNF-α-induced apoptosis, as judged by the ROS probe dihydrorhodamine123 (DHR123). The anti-oxidant, N-Acetyl-L-cysteine (NAC, 0.1mM) and mitochondrial electron transport chain complex I inhibitor rotenone (0.5μM) dramatically attenuated apoptosis as judged by DNA fragmentation ELISA assay. Inhibition of Rac1 by a Rac1 antagonist, NSC23766 (30μM), decreased TNF-α-induced mitochondrial permeability change and mitochondria-derived ROS production. In addition, an early burst of cytosolic ROS was inhibited by NSC23766, dominant negative Rac1, diphenyleneiodonium (NADPH oxidase inhibitor, DPI) and NAC. DPI inhibited TNF-α-induced apoptosis as judged by morphological changes, DNA fragmentation and JNK1/2 activation. Moreover, all ROS inhibitors inhibited caspase-3 activity. Thus, these results indicate that a Rac1-dependent-NADPH-oxidase-like system is involved in the initiation of TNF-α-induced apoptosis in intestinal epithelial cells. Supported by Thomas A. Gerwin Endowment.

Mitochondrial state 3 to 4 respiration transition during Fas-mediated apoptosis controls cellular redox balance and rate of cell death

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill defined. We show that ROS levels play a novel role in moderating the rate of cell death in Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or FCCP (mitochondrial uncoupler) and Fas activating antibody (CH11), facilitated rapid cell death. ATP levels, DEVDase activity and cytochrome c release were not account for the synergistic killing effect. However, a decrease in cellular ROS production was associated with CH11 treatment and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS levels. Thus, decreased ROS production is correlated with accelerated cell death. A transition from state 3 to state 4 mitochondrial respiration following apoptotic stimuli accounted for an attenuated membrane potential and as a results mitochondria-derived ROS production capacity diminished. Similar observations were demonstrated in isolated rat liver mitochondria. Transfection with mitochondrial targeted catalase inhibited mitochondrial ROS production and potentiated cell death. These data show that ROS production is important in receptor-mediated apoptosis and may play a pivotal role in cell survival.

Membrane depolarization of isolated rat liver mitochondria attenuates permeability transition pore opening and oxidant production.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.

Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O −⋅ 2) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O −⋅ 2 and H 2O 2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine- N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O −⋅ 2 production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H 2O 2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.