The p38 pathway partially mediates caspase-3 activation induced by reactive oxygen species in Fanconi anemia C cells

Because Fanconi anemia (FA) cells display hypersensitivity to oxidative stress and reactive oxygen species (ROS) that act as second messengers in cellular signaling, we investigated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation in two FA-C lymphocyte cell lines (HSC536/N and PD149L) and one FA-A cell line (HSC99) exposed to interferon (IFN)-gamma or H2O2. IFN-gamma induced accumulation of ROS and activation of JNK and p38 in HSC536/N and PD149L but not in HSC99 cells. Higher concentrations of H2O2 were needed to induce moderate intracellular levels of ROS and phosphorylation of MAPKs in FA-A than in FA-C cells. Pre-incubation with dehydroascorbic acid resulted in reduced intracellular ROS levels and inhibition of MAPK activation induced by the above treatments. To define the functional role of JNK and p38 in IFN-gamma signaling, the effects of pharmacological inhibition of the MAPKs on induction of IFN-gamma and anti-Fas antibody responses were determined. Treatment of HSC536/N cells with p38-specific inhibitors partially inhibited caspase-3 activation while pre-incubation with specific inhibitors of JNK had no effect. Altogether, these results suggest that FA-C cells are hypersensitive to IFN-gamma and are more sensitive to oxidative stress than FA-A cells and that IFN-gamma and anti-Fas antibody mediate signals for apoptosis in FA-C cells via p38 but not JNK pathways.     

Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2

The generation of reactive oxygen species (ROS) has been implicated in the perturbation of endothelial function and cell death. However, the specific signaling pathways which mediate and modifying this response have not been fully elucidated. Therefore, in this study we tested the hypothesis that activation of JAK2 is involved in the aortic endothelial cell (EC) response to ROS. When ECs were exposed to HG (25 mM) for 6 h or ROS (i.e., H(2)O(2) (100 microM)) for 1 h and returned to normal medium we found a decrease in cell density and morphologic signs of apoptosis. Furthermore, incubation of ECs with HG and H(2)O(2) also resulted in the tyrosine phosphorylation of JAK2. In addition, pretreatment of ECs with AG-490, an inhibitor of JAK2, prevented nuclear fragmentation, whereas inhibitors of Jun kinase (SP 600125), MAP kinase (PD 98059), Src kinase (PP2) or PI-3 kinase (wortmannin) were without effect. Finally, immunoblot analysis of caspase-3 and PARP cleavage confirmed a role for activation of JAK2 in both HG- or ROS-induced apoptosis, based on inhibition by either AG-490 or adenoviral transfection with a dominant-negative JAK2 mutant. In conclusion the activation of JAK2 plays a pivotal role in oxidant stress-induced commitment of ECs to apoptosis, based on studies with HG and H(2)O(2).     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H2O2) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H2O2 exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

Abrogation of the cell death response to oxidative stress by the c-Abl tyrosine kinase inhibitor STI571

Normal aerobic metabolism is associated with the production of reactive oxygen species (ROS) and, consequently, the induction of apoptosis and necrosis. The cell death response to oxidative stress is thought to contribute to aging, neurological degeneration, and other disorders. ROS-induced apoptosis and necrosis involves activation of the cytoplasmic c-Abl tyrosine kinase and thereby signaling to mitochondria. Herein, we show that STI571, an inhibitor of Bcr-Abl in chronic myelogenous leukemia, blocks activation of c-Abl in the response of mouse embryo fibroblasts and human U-937 myeloid leukemia cells to hydrogen peroxide (H(2)O(2)). Immunofluorescence microscopy and subcellular fractionation studies demonstrate that STI571 decreases H(2)O(2)-induced targeting of c-Abl to mitochondria in the two cell types by 59 to 85%. The results also show that STI571 attenuates H(2)O(2)-induced loss of the mitochondrial transmembrane potential. In concert with these effects, STI571 inhibits the death response to H(2)O(2) exposure by 40 to 80% depending on the cell type. These findings indicate that inhibition of c-Abl signaling by STI571 attenuates mitochondrial dysfunction and cell death in the cellular response to oxidative stress.     

JWA as a novel molecule involved in oxidative stress-associated signal pathway in myelogenous leukemia cells

Previous data showed that JWA might be a novel environmental responsive gene regulated by environmental stressors such as heat shock and oxidative stress. However, the molecular mechanism underlying JWA gene function involved in oxidative stress is still unknown. In this study, the potential role of JWA was further investigated in hydrogen peroxide (H2O2) induced DNA damage and cell apoptosis in K562 cells. Series of the oxidative stress models were established to observe if JWA was involved in DNA damage or cell apoptosis induced by H2O2 exposure. These results indicated that the inhibitory effect on K562 cells' viability induced by H2O2 was concentration and time dependent. JWA was more sensitive to H2O2 (0.01 mmol/L) than the heat-shock proteins (hsp70 and hsp27), and its expression pattern was similar to that of hsp70. In addition, JWA, hsp70, hsp27, and p53 were overexpressed and the expression patterns of JWA, hsp70, and p53 were similar during cell apoptosis. H2O2 led to the cleavage and activation of procaspase-3. In conclusion, these results suggested that JWA might be an effective environmental responsive gene that functions as a parallel with hsp70 in oxidative stress-responsive pathways in K562 cells. Like hsp70, JWA might enhance intracellular defenses and function against H2O2-induced oxidative stress in leukemia cells. At the same time, JWA was involved in the p53-associated signal pathways of oxidative stress-induced apoptosis, which is also caspase-3 dependent.     

NOX4‐ and Nrf2‐mediated oxidative stress induced by silver nanoparticles in vascular endothelial cells

It has been widely reported that silver nanoparticles (AgNPs) induce oxidative stress in various cell lines. However, the mechanism for this effect and its consequences for cellular signaling are poorly understood. In this study, human umbilical vein endothelial cells (HUVECs) were used to assess the toxicity and investigate the associated molecular mechanisms caused by exposure to AgNPs. We demonstrated that AgNP exposure significantly and dose‐dependently decreased the cell viability, induced reactive oxygen species (ROS) generation and led to early apoptosis in HUVECs. Our findings showed that AgNPs induced excess ROS production that affected the signaling pathways by a mechanism that depended on activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity through upregulation of NADPH oxidase 4 (NOX4) protein expressions. Moreover, AgNPs could disrupt the inactivation of the nuclear factor erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant response, which is considered another important element for oxidative stress caused by AgNPs in HUVECs. The redox imbalance between NOX4 and Nrf2 was an important cause for the ROS overproduction that led to cell injury in HUVECs. The results provided insight into the mechanisms of oxidative stress induced by AgNPs in vascular endothelial cells.     

Big mitogen-activated protein kinase 1 protects cultured rat aortic smooth muscle cells from oxidative damage

Oxidative stress is considered a major mediator of arteriosclerosis. In vascular smooth muscle cells, oxidative stress-induced cell death (including apoptosis) is probably related to arterial calcification in arteriosclerosis. Big mitogen-activated protein kinase-1 / extracellular signal-regulated kinase 5 (BMK1/ERK5) is a newly identified member of the mitogen-activated protein kinases family. Like Src tyrosine kinase, BMK1/ERK5 is known to be sensitive to oxidative stress; however, its pathophysiological significance is poorly understood. In this study, we investigated the involvement of BMK1 and Src in H(2)O(2)-induced cell death using cultured rat aortic smooth muscle cells (RASMCs). Cell apoptosis was evaluated by using the TdT-mediated dUTP nick end labeling (TUNEL) method, and BMK1 and Src activities were determined by Western blotting. The main results are as follows: 1) BMK1 and Src were activated by H(2)O(2) in a time- and concentration-dependent manner in RASMCs; 2) BMK1 activation by H(2)O(2) was attenuated both in Src-knockdown RASMCs and in RASMCs pretreated with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src family kinases inhibitor; and 3) H(2)O(2)-induced cell death was increased in BMK1- and Src-knockdown RASMCs as well as in PP2-treated RASMCs. These findings suggested that Src and BMK1 may play defensive and resistive roles against oxidative stress-induced death in RASMCs.     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

Ampelopsin prevents apoptosis induced by H2O2 in MT-4 lymphocytes

Human immunodeficiency virus (HIV) infection can result in oxidative stress through production of reactive oxygen species (ROS). Simultaneously, oxidative stress is able to activate the replication of virus and lead to the apoptosis of T lymphocytes which is the defense of the immune function. Ampelopsin, belonging to the flavonoids, is a purified component from the root of a Chinese medicinal herb. Our previous studies revealed that ampelopsin could protect sensitive cells against HIV-1 infection and reduce HIV-1 antigen P24 expression. In this study, we determined whether ampelopsin, as an antioxidant, has protective effects on oxidant stress-induced apoptosis in MT-4 cells, a CD4 T lymphocyte cell line. The results indicate that ampelopsin scavenged hydroxyl radicals (.OH) and superoxide radicals (O(2).-) in a concentration-dependent manner. It significantly increased MT-4 cells viability after treatment with H(2)O(2) and inhibited H(2)O(2)-induced DNA laddering. The data from flow cytometry analysis showed that ampelopsin remarkably decreased the percentage of apoptotic cells induced by H(2)O(2). In addition, activation of caspase-3 was detected during the course of apoptosis induction. Western blot analysis showed that ampelopsin inhibited the cleavage of caspase-3 induced by H(2)O(2). All these findings might shed new light on the understanding of the anti-AIDS functions of ampelopsin by protecting T cells of persons infected with HIV.     

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease.     

Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H₂O₂)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H₂O₂ induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H₂O₂ induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases.     

小分子脾酪氨酸激酶抑制剂临床研究进展

脾酪氨酸激酶(Syk)是一种细胞质非受体蛋白酪氨酸激酶,主要在造血细胞中表达.Syk不仅在免疫受体信号中扮演至关重要的角色,同时还调节其他不同的生物功能,包括细胞的黏附、增殖、破骨细胞成熟、血小板活化等方面.近年来Syk在血液肿瘤、自身免疫、炎症疾病等方向已经成为极具吸引力的药物靶标.通过介绍Syk的结构与激活机制,及...     

Resolvin D1-mediated NOX2 inactivation rescues macrophages undertaking efferocytosis from oxidative stress-induced apoptosis

Effective clearance of apoptotic cells by macrophages, termed efferocytosis, is pre-requisite for successful resolution of inflammation, and drives macrophage emigration to the draining lymph node, thereby promoting restoration of tissue homeostasis. During efferocytosis, engulfment of apopototic cells induces generation of reactive oxygen species in abundance. Macrophage apoptosis is an important feature of chronic inflammatory diseases including atherosclerosis. In the present study, we found that resolvin D1 (RvD1), one of endogenous pro-resolving lipid mediators derived from docosahexaenoic acid, prevented apoptosis of murine macrophage-like RAW264.7 cells engulfing apoptotic T cells. The inhibitory effect of RvD1 on efferocytosis-induced oxidative burst appears to be mediated by the inactivation of NADPH oxidase (NOX), a key enzyme involved in intracellular ROS production. In RvD1-treated macrophages, efferocytosis-induced phosphorylation of p47^phox and association between p47^phox and gp91^phox were downregulated, resulting in abrogation of generation of superoxide anion and hydrogen peroxide. Furthermore, RvD1-mediated suppression of NOX activation was found to be dependent on cAMP-activated protein kinase (PKA) signaling. Besides inhibiting NOX activation, RvD1 rescued macrophages from oxidative stress-induced apoptosis by upregulating the expression of Bcl-xL and Bcl-2. However, knockdown of the RvD1 receptor, lipoxin A receptor/formyl-peptide receptor (ALX/FPR2), abolished the ability of RvD1 to activate cAMP-PKA signaling, to suppress NOX activation and to increase the expression of anti-apoptotic proteins, suggesting that ALX/FPR2 mediates the protective effect of RvD1 on effeocytosis-induced oxidative stress. Taken together, these findings indicate that RvD1 rescues macrophages from oxidative stress-induced apoptosis during efferocytosis through PKA-mediated repression of NOX activation and upregulation of anti-apoptotic protein expression.     

Scutellarin protects PC12 cells from oxidative stress-induced apoptosis

The present study investigated the effects of scutellarin on oxidative stress-induced cell apoptosis in PC12 cells. Exposure of cells to hydrogen peroxide (H₂O₂) triggered a typical apoptosis, as evidenced by DNA fragmentation, DNA loss and externalization of phosphatidylserine (PS). This treatment also caused significant elevation of oxidative stress characterized by intracellular accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), a product of lipid peroxidation. Preincubation of cells with scutellarin significantly inhibited the fragmentation and loss of DNA, the externalization of PS, and decreased the percentage of cell apoptosis. Also, intracellular accumulations of ROS and MDA resulting from H₂O₂ exposure were significantly reduced by scutellarin. These findings suggest that scutellarin exerts significant protection against oxidative stress-induced apoptosis, which might be beneficial for the prevention and treatment of oxidative stress-mediated disorders.     

Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 microM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 microM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages.