Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species

Regardless of rapid progression in the field of autophagy, it remains a challenging task to understand the cross talk with apoptosis. In this study, we overexpressed Ulk1 in HeLa cells and evaluated the apoptosis-inducing potential of the Ulk1 gene in the presence of cisplatin. The gain of function of Ulk1 gene showed a decline in cell viability and colony formation in HeLa cells. The Ulk1-overexpressing cells showed higher apoptotic attributes by an increase in the percentage of annexin V, escalated expression of Bax/Bcl2 ratio, and caspase-9, -3/7 activities. Further, reactive oxygen species (ROS) generation was found to be much higher in HeLa-Ulk1 than in the mock group. Scavenging the ROS by N-acetyl-L-cysteine increased cell viability and colony number as well as mitochondrial membrane potential (MMP). Our data showed that Ulk1 on entering into mitochondria inhibits the manganese dismutase activity and intensifies the mitochondrial superoxide level. The Ulk1-triggered autophagy (particularly mitophagy) resulted in a fall in ATP; thus the nonmitophagic mitochondria overwork the electron-transport cycle to replenish energy demand and are inadvertently involved in ROS overproduction that led to apoptosis. In this present investigation, our results decipher a previously unrecognized perspective of apoptosis induction by a key autophagy protein Ulk1 that may contribute to identification of its tumor-suppressor properties through dissecting the connection among cellular bioenergetics, ROS, and MMP.     

Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production

Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflammatory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflammatory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated peripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). However, the drug resistance of MTX in malignancies remains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, including cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC overexpression was shown to prevent TNFα-induced apoptosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug resistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation together with disrupting the mitochondrial membrane potential (ΔΨ_m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss of ΔΨ_m, through reducing intracellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintains ΔΨ_m, the cytochrome c release and activations of caspase 9 and 3 following MTX treatment. The results demonstrate that MTX-induced apoptosis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resistant to MTX-induced apoptosis by reducing intracellular ROS production.     

2-Hydroxycurcuminoid induces apoptosis of human tumor cells through the reactive oxygen species-mitochondria pathway

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI₅₀ value of 7 μM, while the parent compounds, HCA and curcumin, displayed GI₅₀ values of 12 and 30 μM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.     

Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis

Observations of apoptosis in virtual anaerobiosis have raised doubts on the significance of reactive oxygen species in the cascade of events of programmed cell death. This work presents evidence that cells and mitochondrial preparations produce similar levels of hydrogen peroxide under either aerobic or virtually anaerobic conditions. These levels are relevant to the increased production of radicals induced by a ceramide analog that promotes apoptosis. This ceramide acts at center o of mitochondrial complex III.     

Restraining reactive oxygen species in Listeria monocytogenes promotes the apoptosis of glial cells

Objectives: Listeria monocytogenes is a facultative anaerobic foodborne pathogen that can traverse the blood–brain barrier and cause brain infection. L. monocytogenes infection induces host cell apoptosis in several cell types. In this study, we investigated the apoptosis of human glioma cell line U251 invaded by L. monocytogenes and evaluated the function of bacterial reactive oxygen species (ROS) during infection.

Methods: Bacterial ROS level was reduced by carrying out treatment with N-acetyl cysteine (NAC) and diphenyleneiodonium chloride (DPI). After infection, the apoptosis of U251 cells was examined by flow cytometry assay and propidium iodide staining.

Results: DPI and NAC efficiently decreased ROS level in L. monocytogenes without affecting bacterial growth. Moreover, the apoptosis of glial cells was enhanced upon invasion of DPI- and NAC-pretreated L. monocytogenes.

Discussion: Results indicate that the apoptosis of glial cells can be induced by L. monocytogenes, and that the inhibition of bacterial ROS increases the apoptosis of host cells.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition

Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

Macrophage-induced preadipocyte survival depends on signaling through Akt, ERK1/2, and reactive oxygen species

Obesity is associated with adipose tissue remodeling, characterized by macrophage accumulation, adipocyte hypertrophy, and apoptosis. We previously reported that macrophage-conditioned medium (MacCM) protects preadipocytes from apoptosis, due to serum withdrawal, in a platelet-derived growth factor (PDGF)-dependent manner. We have now investigated the role of intracellular signaling pathways, activated in response to MacCM versus PDGF, in promoting preadipocyte survival. Exposure of 3T3-L1 preadipocytes to J774A.1-MacCM or PDGF strongly stimulated Akt and ERK1/2 phosphorylation from initially undetectable levels. Inhibition of the upstream regulators of Akt or ERK1/2, i.e. phosphoinositide 3-kinase (PI3K; using wortmannin or LY294002) or MEK1/2 (using UO126 or PD98509), abrogated the respective phosphorylation responses, and significantly impaired pro-survival activity. J774A.1-MacCM increased reactive oxygen species (ROS) levels by 3.4-fold, and diphenyleneiodonium (DPI) or N-acetyl cysteine (NAC) significantly inhibited pro-survival signaling and preadipocyte survival in response to J774A.1-MacCM. Serum withdrawal itself also increased ROS levels (2.1-fold), and the associated cell death was attenuated by DPI or NAC. In summary, J774A.1-MacCM-dependent 3T3-L1 preadipocyte survival requires the Akt and ERK1/2 signaling pathways. Furthermore, ROS generation by J774A.1-MacCM is required for Akt and ERK1/2 signaling to promote 3T3-L1 preadipocyte survival. These data suggest potential mechanisms by which macrophages may alter preadipocyte fate.     

Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

Synchronized generation of reactive oxygen species with the cell cycle

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amounts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G1 phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.