Khz (Fusion of Ganoderma lucidum and Polyporus umbellatus Mycelia) Induces Apoptosis by Increasing Intracellular Calcium Levels and Activating JNK and NADPH Oxidase-Dependent Generation of Reactive Oxygen Species

Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47^phox and p67^phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca²⁺]_i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47^phox and p67^phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca²⁺]_i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Cigarette smoke particle-phase extract induces HO-1 expression in human tracheal smooth muscle cells: role of the c-Src/NADPH oxidase/MAPK/Nrf2 signaling pathway

Heme oxygenase-1 (HO-1) is known as an oxidative stress protein that is up-regulated by various stimuli. HO-1 has been shown to protect cells against oxidative damage. Cigarette smoke is a potential inflammatory mediator that causes chronic obstructive pulmonary disease and asthma. In this study, we report that cigarette smoke particle-phase extract (CSPE) is an inducer of HO-1 expression mediated through various signaling pathways in human tracheal smooth muscle cells (HTSMCs). CSPE-induced HO-1 protein, mRNA expression, and promoter activity were attenuated by pretreatment with a ROS scavenger (N-acetyl-l-cysteine) and inhibitors of c-Src (PP1), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs for Src, p47^phox, NOX2, p42, p38, JNK2, or NF-E2-related factor 2 (Nrf2). CSPE-stimulated translocation of p47^phox and Nrf2, ROS production, and NADPH oxidase activity was attenuated by transfection with siRNAs for Src, p47^phox, and NOX2 or pretreatment with PP1, DPI, or APO. Furthermore, CSPE-induced NOX2, c-Src, and p47^phox complex formation was revealed by immunoprecipitation using an anti-NOX2, anti-p47^phox, or anti-c-Src Ab followed by Western blot against anti-NOX2, anti-p47^phox, or anti-c-Src Abs. These results demonstrate that CSPE-induced ROS generation is mediated through a c-Src/NADPH oxidase/MAPK pathway and in turn initiates the activation of Nrf2 and ultimately induces HO-1 expression in HTSMCs.     

Advanced Glycation End Products Induce Human Corneal Epithelial Cells Apoptosis through Generation of Reactive Oxygen Species and Activation of JNK and p38 MAPK Pathways

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.     

Induction of COX-2/PGE2/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation

Exposure to cigarette smoke extract (CSE) leads to airway and lung inflammation through an oxidant-antioxidant imbalance. Cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂) have been shown to play critical roles in respiratory inflammation. Here, we show that COX-2/PGE₂/IL-6 induction is dependent on Toll-like receptor 4 (TLR4)/NADPH oxidase signaling in human tracheal smooth muscle cells (HTSMCs). CSE induced COX-2 expression in vitro in HTSMCs and in vivo in the airways of mice. CSE also directly caused an increase in TLR4. Moreover, CSE-regulated COX-2, PGE₂, and IL-6 generation was inhibited by pretreatment with TLR4 Ab; inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride and apocynin), p38 MAPK (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (helenalin); a ROS scavenger (N-acetyl-l-cysteine); and transfection with siRNA of TLR4, MyD88, TRAF6, Src, p47^phox, p38, p42, JNK2, or p65. CSE-induced leukocyte numbers in BAL fluid were also reduced by pretreatment with these inhibitors. Furthermore, CSE induced p47^phox translocation and TLR4/MyD88/TRAF6 and c-Src/p47^phox complex formation. We found that PGE₂ enhanced IL-6 production in HTSMCs and leukocyte count in BAL fluid. In addition, treatment with nicotine could induce COX-2, PGE₂, and IL-6 generation in in vivo and in vitro studies. These results demonstrate that CSE-induced ROS generation was mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiated the activation of MAPKs and NF-κB, and ultimately induced COX-2/PGE₂/IL-6-dependent airway inflammation.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

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.     

Ca2+/Calmodulin-Dependent Protein Kinase II Contributes to Hypoxic Ischemic Cell Death in Neonatal Hippocampal Slice Cultures

We have recently shown that p38MAP kinase (p38MAPK) stimulates ROS generation via the activation of NADPH oxidase during neonatal hypoxia-ischemia (HI) brain injury. However, how p38MAPK is activated during HI remains unresolved and was the focus of this study. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) plays a key role in brain synapse development, neural transduction and synaptic plasticity. Here we show that CaMKII activity is stimulated in rat hippocampal slice culture exposed to oxygen glucose deprivation (OGD) to mimic the condition of HI. Further, the elevation of CaMKII activity, correlated with enhanced p38MAPK activity, increased superoxide generation from NADPH oxidase as well as necrotic and apoptotic cell death. All of these events were prevented when CaMKII activity was inhibited with KN93. In a neonatal rat model of HI, KN93 also reduced brain injury. Our results suggest that CaMKII activation contributes to the oxidative stress associated with neural cell death after HI.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

BAPTA-AM,an intracellular calcium chelator,inhibits RANKL-induced bone marrow macrophages differentiation through MEK/ERK,p38 MAPK and Akt,but not JNK pathways

To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca²⁺ concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca²⁺]_i was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1 h after RANKL-inducing, and the increased response of [Ca²⁺]_i was completely abolished and sustained to at least 8 h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca²⁺]_i which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Flos Puerariae Extract Prevents Myocardial Apoptosis via Attenuation Oxidative Stress in Streptozotocin-Induced Diabetic Mice

Background

Diabetic cardiomyopathy (DCM) suggests a direct cellular insult to myocardium. Apoptosis is considered as one of the hallmarks of DCM. Oxidative stress plays a key role in the pathogenesis of DCM. In this study, we explored the prevention of myocardial apoptosis by crude extract from Flos Puerariae (FPE) in experimental diabetic mice.

Methods

Experimental diabetic model was induced by intraperitoneally injection of streptozotocin (STZ, 50 mg/kg/day) for five consecutive days in C57BL/6J mice. FPE (100, 200 mg/kg) was orally administrated once a day for ten weeks. Cardiac structure changes, apoptosis, superoxide production, NADPH oxidase subunits expression (gp91^phox, p47^phox, and p67^phox), and related regulatory factors were assessed in the heart of mice.

Results

Diabetic mice were characterized by high blood glucose (≥11.1 mmol/L) and reduced body weight. In the end of the experiment, aberrant myofilament structure, as well as TUNEL positive cardiac cells coupled with increased Bax/Bcl-2 ratio and Caspase-3 expression was found in diabetic mice. Moreover, ROS formation, the ratio of NADP⁺/NADPH and NADPH oxidase subunits expression of gp91^phox and p47^phox, lipid peroxidation level was significantly increased, while antioxidant enzyme SOD and GSH-Px activity were reduced in the myocardial tissue of diabetic mice. In contrast, treatment with FPE resulted in a normalized glucose and weight profile. FPE administration also preserved myocardial structure and reduced apoptotic cardiac cell death in diabetic mice. The elevated markers of oxidative stress were significantly reversed by FPE supplementation. Further, FPE treatment markedly inhibited the increased Bax/Bcl-2 ratio and Caspase-3 expression, as well as suppressed JNK and P38 MAPK activation in the heart of diabetic mice.

Conclusions

Our data demonstrate for the first time that FPE may have therapeutic potential for STZ-induced diabetic cardiomyopathy through preventing myocardial apoptosis via attenuation oxidative stress. And this effect is probably mediated by JNK and P38 MAPK signaling pathway.     

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1. Consistent with this, Ask-1 overexpression considerably sensitizes keratinocytes to UVB-induced mitochondrial apoptosis. Although the JNK pathway is also stimulated after UVB, the killing effect of Ask-1 overexpression is reverted by p38 MAPK inhibition, suggesting that Ask-1 exerts its lethal effects mainly through the p38 MAPK pathway. Moreover, p38alpha(-/-) murine embryonic fibroblasts are protected from UVB-induced apoptosis even if JNK activation is fully preserved. These results argue for an important role of the UVB-generated ROS as mediators of the Ask-1-p38 MAPK pathway that, by culminating in apoptosis, restrains the propagation of potentially mutagenic keratinocytes.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.