DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.     

Catechins induce oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H

2

O

2

)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using

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P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H

2

O

2

and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H

2

O

2

generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Mequindox induced cellular DNA damage via generation of reactive oxygen species

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS.     

Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.     

Heme induces neutrophil migration and reactive oxygen species generation through signaling pathways characteristic of chemotactic receptors

Hemolysis or extensive cell damage can lead to high concentrations of free heme, causing oxidative stress and inflammation. Considering that heme induces neutrophil chemotaxis, we hypothesize that heme activates a G protein-coupled receptor. Here we show that similar to heme, several heme analogs were able to induce neutrophil migration in vitro and in vivo. Mesoporphyrins, molecules lacking the vinyl groups in their rings, were not chemotactic for neutrophils and selectively inhibited heme-induced migration. Moreover, migration of neutrophils induced by heme was abolished by pretreatment with pertussis toxin, an inhibitor of Galpha inhibitory protein, and with inhibitors of phosphoinositide 3-kinase, phospholipase Cbeta, mitogen-activated protein kinases, or Rho kinase. The induction of reactive oxygen species by heme was dependent of Galpha inhibitory protein and phosphoinositide 3-kinase and partially dependent of phospholipase Cbeta, protein kinase C, mitogen-activated protein kinases, and Rho kinase. Together, our results indicate that heme activates neutrophils through signaling pathways that are characteristic of chemoattractant molecules and suggest that mesoporphyrins might prove valuable in the treatment of the inflammatory consequences of hemorrhagic and hemolytic disorders.     

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.     

Neuregulin induces sustained reactive oxygen species generation to mediate neuronal differentiation

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate NRG-induced differentiation through the MAPK signaling pathway. Here we demonstrate that NRG induces an increase in the intracellular concentration of reactive oxygen species (ROS). N-acetylcysteine, a ROS scavenger, inhibited NRG-induced activation of Ras and Erk and PC12-ErbB-4 cell differentiation. These results suggest that ROS production is involved in NRG-mediated neuronal differentiation and that ROS can regulate activation of Ras and Erk. Constitutively active Ras enhanced ROS production and dominant negative Ras inhibited NRG-induced ROS production, suggesting, a positive regulatory loop between Ras and ROS. The mitogen, EGF, induced short-term ROS production whereas NRG and NGF, which induce cell differentiation, induced prolonged ROS production. These results strongly suggest that the kinetics of ROS production may determine whether the cells will differentiate or proliferate.     

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.     

Arisostatins A induces apoptosis through the activation of caspase-3 and reactive oxygen species generation in AMC-HN-4 cells

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.     

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.     

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.     

Oxygen radicals and reactive oxygen species in reproduction

Free radicals and reactive oxygen species play a number of significant and diverse roles in reproductive biology. In common with other biological systems, mechanisms have evolved to minimize the damaging effects that these highly reactive molecules can have on reproductive integrity. Conversely, however, recent findings illustrate the constructive roles that oxygen radicals and reactive oxygen species play in a number of important junctures in the development of germ cells and the obligate endocrine support they receive for the successful propagation of the species. Specifically addressed in this review are some aspects of sperm development and action, the uterine environment, oocyte maturation and ovulation, and corpus luteum function and regression.     

High glucose induces vascular endothelial growth factor production in human synovial fibroblasts through reactive oxygen species generation

Background

Diabetes is an independent risk factor of osteoarthritis (OA). Angiogenesis is essential for the progression of OA. Here, we investigated the intracellular signaling pathways involved in high glucose (HG)-induced vascular endothelial growth factor (VEGF) expression in human synovial fibroblast cells.

Methods

HG-mediated VEGF expression was assessed with qPCR and ELISA. The mechanisms of action of HG in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the VEGF promoter.

Results

Stimulation of OA synovial fibroblasts (OASF) with HG induced concentration- and time-dependent increases in VEGF expression. Treatment of OASF with HG increased reactive oxygen species (ROS) generation. Pretreatment with NADPH oxidase inhibitor (APO or DPI), ROS scavenger (NAC), PI3K inhibitor (Ly294002 or wortmannin), Akt inhibitor, or AP-1 inhibitor (curcumin or tanshinone IIA) blocked the HG-induced VEGF production. HG also increased PI3K and Akt activation. Treatment of OASF with HG increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the VEGF promoter.

Conclusions

Our results suggest that the HG increases VEGF expression in human synovial fibroblasts via the ROS, PI3K, Akt, c-Jun and AP-1 signaling pathway.

General significance

We link high glucose on VEGF expression in osteoarthritis.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Ornithine decarboxylase prevents dibenzoylmethane-induced apoptosis through repressing reactive oxygen species generation

Dibenzoylmethane (DBM) belongs to the flavonoid family and is a minor constituent of the root extract of licorice and the β-diketone analogue of curcumin. It exhibits antimutagenic, anticancer, and chemopreventive effects. Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in growth, proliferation, and transformation. Our previous studies showed ODC overexpression prevented etoposide-, paclitaxel-, and cisplatin-induced apoptosis. Here, we investigated one mechanism of DBM-induced apoptosis and the antiapoptotic effects of ODC during DBM treatment. We found that DBM induced apoptosis, promoted reactive oxygen species (ROS) generation, and disrupted the mitochondrial membrane potential (Δψ(m). N-acetylcysteine, a ROS scavenger, reduced DBM-induced apoptosis, which led to the loss of Δψ(m) due to reduced ROS. Overexpression of ODC in parental cells had the same effects as the ROS scavenger. The results demonstrated that DBM-induced apoptosis was a ROS-dependent pathway and ODC overexpression blocked DBM-induced apoptosis by inhibiting intracellular ROS production.     

Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2

A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC₅₀ ranging from 1.8 to 6.1 μM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.     

Cyclo(phenylalanine‐proline) induces DNA damage in mammalian cells via reactive oxygen species

Cyclo(phenylalanine‐proline) is produced by various organisms such as animals, plants, bacteria and fungi. It has diverse biological functions including anti‐fungal activity, anti‐bacterial activity and molecular signalling. However, a few studies have demonstrated the effect of cyclo(phenylalanine‐proline) on the mammalian cellular processes, such as cell growth and apoptosis. In this study, we investigated whether cyclo(phenylalanine‐proline) affects cellular responses associated with DNA damage in mammalian cells. We found that treatment of 1 mM cyclo(phenylalanine‐proline) induces phosphorylation of H2AX (S139) through ATM‐CHK2 activation as well as DNA double strand breaks. Gene expression analysis revealed that a subset of genes related to regulation of reactive oxygen species (ROS) scavenging and production is suppressed by the cyclo(phenylalanine‐proline) treatment. We also found that cyclo(phenylalanine‐proline) treatment induces perturbation of the mitochondrial membrane, resulting in increased ROS, especially superoxide, production. Collectively, our study suggests that cyclo(phenylalanine‐proline) treatment induces DNA damage via elevation of ROS in mammalian cells. Our findings may help explain the mechanism underlying the bacterial infection‐induced activation of DNA damage response in host mammalian cells.     

Apoptosis induction of U937 human leukemia cells by diallyl trisulfide induces through generation of reactive oxygen species

Background

Diallyl trisulfide (DATS) is one of the major constituents in garlic oil and has demonstrated various pharmacological activities, including antimicrobial, antihyperlipidemic, antithrombotic, and anticancer effects. However, the mechanisms of antiproliferative activity in leukemia cells are not fully understood. In this study, the apoptotic effects of DATS were investigated in human leukemia cells.

Results

Results of this study indicated that treatment with DATS resulted in significantly inhibited leukemia cell growth in a concentration- and time-dependent manner by induction of apoptosis. In U937 cells, DATS-induced apoptosis was correlated with down-regulation of Bcl-2, XIAP, and cIAP-1 protein levels, cleavage of Bid proteins, activation of caspases, and collapse of mitochondrial membrane potential. The data further demonstrated that DATS increased intracellular reactive oxygen species (ROS) generation, which was attenuated by pretreatment with antioxidant N-acetyl-l-cysteine (NAC), a scavenger of ROS. In addition, administration of NAC resulted in significant inhibition of DATS-induced apoptosis by inhibiting activation of caspases.

Conclusions

The present study reveals that the cytotoxicity caused by DATS is mediated by generation of ROS and subsequent activation of the ROS-dependent caspase pathway in U937 leukemia cells.