Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 microM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 microM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H(2)O(2) treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 microM to 500 microM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.     

The influence of reactive oxygen species on cell cycle progression in mammalian cells

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.     

Regulation of reactive oxygen species in stem cells and cancer stem cells

Stem cells are defined by their ability to self-renew and their multi-potent differentiation capacity. As such, stem cells maintain tissue homeostasis throughout the life of a multicellular organism. Aerobic metabolism, while enabling efficient energy production, also generates reactive oxygen species (ROS), which damage cellular components. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer, and various anti-oxidative and anti-stress mechanisms of stem cells have been characterized. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, i.e., regulation of the cell cycle. An active area of current research is redox regulation in various cancer stem cells, the malignant counterparts of normal stem cells that are viewed as good targets of cancer therapy. In contrast to cancer cells, in which ROS levels are increased, some cancer stem cells maintain low ROS levels, exhibiting redox patterns that are similar to the corresponding normal stem cell. To fully elucidate the mechanisms involved in stem cell maintenance and to effectively target cancer stem cells, it is essential to understand ROS regulatory mechanisms in these different cell types. Here, the mechanisms of redox regulation in normal stem cells, cancer cells, and cancer stem cells are reviewed.     

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.     

The changes of reactive oxygen species and glutathione by MG132, a proteasome inhibitor affect As4.1 juxtaglomerular cell growth and death

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O₂^? and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (ΔΨ_m) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and death.     

Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species

We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2′,7′-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q₂) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q₁ as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decyl-ubiquinone. This behaviour relates the prooxidant CoQ₁ activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH-DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron-sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.     

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

A triazole-conjugated benzoxazone induces reactive oxygen species and promotes autophagic apoptosis in human lung cancer cells

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.     

Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.     

Effects of reactive oxygen species on prostacyclin production in perinatal rat lung cells

A differentiation-arrested primary cell culture model was used to examine the role of reactive oxygen species in the control of prostacyclin (PGI2) production in the perinatal rat lung. Coincubation of the lung cells with arachidonic acid (AA) and xanthine (X, 0.25 mM) plus xanthine oxidase (XO, 10 mU/ml) or with AA and glucose (25 mM) plus glucose oxidase (25 mU/ml) augmented the AA-induced PGI2 output. Superoxide dismutase (10 U/ml) did not alter the X + XO effect, whereas catalase (10 U/ml) eliminated both X + XO and glucose plus glucose oxidase effects. H2O2 (1-200 microM) showed a dose-related biphasic augmentation with peak stimulation at 20 microM. Catalase again blocked this effect, but dimethylthiourea, a hydroxyl radical scavenger, did not. A 20-min pretreatment of the cells with X + XO, glucose plus glucose oxidase, or H2O2, however, diminished the capacity of the cells to convert exogenous AA to PGI2. This pretreatment effect was also blocked by catalase. The responses were similar in lung cells obtained from day 20 rat fetuses (term = 22 days) and 1-day-old newborn rats. Lactate dehydrogenase release was not detected during treatment periods but increased significantly after exposure to reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vanadate-induced cell growth regulation and the role of reactive oxygen species

While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (*OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2*-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +*OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2*-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.     

Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells

Cross talk between unrelated cell surface receptors, such as G-protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK), is a crucial signaling mechanism to expand the cellular communication network. We investigated the ability of the GPCR formyl peptide receptor-like 1 (FPRL1) to transactivate the RTK epidermal growth factor receptor (EGFR) in CaLu-6 cells. We observed that stimulation with WKYMVm, an FPRL1 agonist isolated by screening synthetic peptide libraries, induces EGFR tyrosine phosphorylation, p47^phox phosphorylation, NADPH-oxidase-dependent superoxide generation, and c-Src kinase activity. As a result of EGFR transactivation, phosphotyrosine residues provide docking sites for recruitment and triggering of the STAT3 pathway. WKYMVm-induced EGFR transactivation is prevented by the FPRL1-selective antagonist WRWWWW, by pertussis toxin (PTX), and by the c-Src inhibitor PP2. The critical role of NADPH-oxidase-dependent superoxide generation in this cross-talk mechanism is corroborated by the finding that apocynin or a siRNA against p22^phox prevents EGFR transactivation and c-Src kinase activity. In addition, WKYMVm promotes CaLu-6 cell growth, which is prevented by PTX and by WRWWWW. These results highlight the role of FPRL1 as a potential target of new drugs and suggest that targeting both FPRL1 and EGFR may yield superior therapeutic effects compared with targeting either receptor separately.     

The role of reactive oxygen species in cell growth: lessons from root hairs

Reactive oxygen species (ROS) play a diversity of roles in plants. In recent years, a role for NADPH oxidase-derived ROS during cell growth and development has been discovered in a number of plant model systems. These studies indicate that ROS are required for cell expansion during the morphogenesis of organs such as roots and leaves. Furthermore, there is evidence that ROS are required for root hair growth where they control the activity of calcium channels required for polar growth. The role of ROS in the control of root hair growth is reviewed here and results are highlighted that may provide insight into the mechanism of plant cell growth in general.