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.     

IgE-induced mast cell survival requires the prolonged generation of reactive oxygen species

We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca(2+)) entry. However, whereas IgE+Ag more potently stimulates Ca(2+) entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca(2+) entry than IgE+Ag initially, they all trigger a more prolonged Ca(2+) influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca(2+) influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H(2)O(2) to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase

Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.     

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.     

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species

c-Jun NH(2)-terminal kinase (JNK) is activated by a number of cellular stimuli such as inflammatory cytokines and environmental stresses. Reactive oxygen species also cause activation of JNK; however, the signaling cascade that leads to JNK activation remains to be elucidated. Because recent reports showed that expression of Cas, a putative Src substrate, stimulates JNK activation, we hypothesized that the Src kinase family and Cas would be involved in JNK activation by reactive oxygen species. An essential role for both Src and Cas was demonstrated. First, the specific Src family tyrosine kinase inhibitor, PP2, inhibited JNK activation by H(2)O(2) in a concentration-dependent manner but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Second, JNK activation in response to H(2)O(2) was completely inhibited in cells derived from transgenic mice deficient in Src but not Fyn. Third, expression of a dominant negative mutant of Cas prevented H(2)O(2)-mediated JNK activation but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Finally, the importance of Src was further supported by the inhibition of both H(2)O(2)-mediated Cas tyrosine phosphorylation and Cas.Crk complex formation in Src-/- but not Fyn-/- cells. These results demonstrate an essential role for Src and Cas in H(2)O(2)-mediated activation of JNK and suggest a new redox-sensitive pathway for JNK activation mediated by Src.     

Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

TNFalpha and reactive oxygen species in necrotic cell death

Death receptors, including the TNF receptor-1 （TNF-RI）, have been shown to be able to initiate caspase-independent cell death. This form of ＂necrotic cell death＂ appears to be dependent on the generation of reactive oxygen species. Recent data have indicated that superoxide generation is dependent on the activation of NADPH oxidases, which form a complex with the adaptor molecules RIP1 and TRADD. The mechanism of superoxide generation further establishes RIP1 as the central molecule in ROS production and cell death initiated by TNFa and other death receptors. A role for the sustained JNK activation in necrotic cell death is also suggested. The sensitization of virus-infected cells to TNFα indicates that necrotic cell death may represent an alternative cell death pathway for clearance of infected cells.     

JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species

c-Jun N-terminal kinase (JNK), or stress-activated protein kinase, is an important member of the mitogen-activated protein kinase superfamily, the members of which are readily activated by many environmental stimuli. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important groups of free radicals that are capable of eliciting direct damaging effects or acting as critical intermediate signaling molecules, leading to oxidative and nitrosative stress and a series of biological consequences. Recently there has been an increasing amount of research interest focusing on the regulatory role of JNK activation in ROS-and RNS-induced cellular responses. In this review we will first summarize and discuss some recent findings regarding the signaling mechanisms of ROS-or RNS-mediated JNK activation. Second, we will talk about the role of JNK in ROS-or RNS-mediated cell death (both apoptosis and necrosis). Finally, we will analyze the emerging evidence for the involvement of ROS and RNS as mediators in tumor necrosis factor alpha-induced apoptosis. Taken together, the accumulating knowledge about the ROS/RNS-induced JNK signaling pathway has greatly advanced our understanding of the complex processes deciding the cellular responses to environmental stress.     

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.     

Regulation of reactive oxygen species generation in cell signaling

Reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H₂O₂) are thought to be byproducts of aerobic respiration with damaging effects on DNA, protein, and lipid. A growing body of evidence indicates, however, that ROS are involved in the maintenance of redox homeostasis and various cellular signaling pathways. ROS are generated from diverse sources including mitochondrial respiratory chain, enzymatic activation of cytochrome p450, and NADPH oxidases further suggesting involvement in a complex array of cellular processes. This review summarizes the production and function of ROS. In particular, how cytosolic and membrane proteins regulate ROS generation for intracellular redox signaling will be detailed.     

Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue

The importance of reactive oxygen species for herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid)-induced cell death in roots was investigated. This was in order to understand its mode of action in grass species grown in the dark. Under these dark conditions, quinclorac suppressed the shoot and root growth of maize (Zea mays L. cv. Honey Bantam) in a concentration-dependent manner (50muM), although the inhibition level was less than that observed under growth conditions in the light. Analysis of cell viability using Evans blue or fluorescein diacetate-propidium iodide (FDA-PI) staining showed that the maize root cells significantly lost their viability after 14h root treatment with 10muM quinclorac, but not 10muM 2,4-dichlorophenoxyacetic acid (2,4-D). Determination of reactive oxygen species (ROS) in maize roots using a superoxide anion (O(2)(-))-specific indicator, dihydroethidium (DHE), indicated that 50muM quinclorac induced a high level of O(2)(-) production in maize roots after 14h root treatment than that of either the control (non-treated) or with 50muM 2,4-D. Moreover, either cell death or ethane evolution, an indicator of lipid peroxide formation, in maize root segments was significantly enhanced by 50muM quinclorac, but not by 50muM 2,4-D. On the other hand, the 50muM 2,4-D treatment induced much higher ethylene and cyanide production in the root segments than with the 50muM quinclorac. These results suggest that quinclorac-induced cell death in maize roots may be caused by ROS and lipid peroxidation, but not by ethylene and its biosynthetic pathway-related substances including cyanide, which have been thought to be the causative factor of quinclorac-induced phytotoxicity in susceptible grass weeds such as Echinochloa, Digitaria, and Setaria.     

JNK activation limits dendritic cell maturation in response to reactive oxygen species by the induction of apoptosis

Dendritic cells (DC) sense infection in their local microenvironment and respond appropriately in order to induce T cell immunity. This response is mediated in part via the mitogen-activated protein kinase (MAPK) pathways. Hydrogen peroxide is present frequently in the inflammatory DC milieu and is known to activate MAPK. Therefore this study examines the role of hydrogen peroxide, both alone and in combination with lipopolysaccharide (LPS), in the regulation of activation of two key MAPK, p38 and JNK, regulation of phenotype, and regulation of apoptosis in human monocyte-derived DC. At low concentrations, hydrogen peroxide activates p38, but does not alter DC phenotype. At higher concentrations, hydrogen peroxide activates both p38 and JNK. Activation of JNK, which is associated with inhibition of tyrosine phosphatases in DC, is linked to the induction of DC apoptosis. An upstream JNK inhibitor (CEP11004) and a competitive JNK inhibitor (SP600125) both partially protected the DC from the proapoptotic effects of hydrogen peroxide. Unexpectedly, hydrogen peroxide and LPS synergize in inducing JNK activation and DC apoptosis. JNK-mediated apoptosis may limit damaging immune responses against neoepitopes generated by modification of self-antigens by reactive oxygen species present at sites of inflammation.     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

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.     

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.     

Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.