Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways

Arsenic (As), a well-known high toxic metal, is an important environmental and industrial contaminant, and it induces oxidative stress, which causes many adverse health effects and diseases in humans, particularly in inorganic As (iAs) more harmful than organic As. Recently, epidemiological studies have suggested a possible relationship between iAs exposure and neurodegenerative disease development. However, the toxicological effects and underlying mechanisms of iAs-induced neuronal cell injuries are mostly unknown. The present study demonstrated that iAs significantly decreased cell viability and induced apoptosis in Neuro-2a cells. iAs also increased oxidative stress damage (production of malondialdehyde (MDA) and ROS, and reduction of Nrf2 and thioredoxin protein expression) and induced several features of mitochondria-dependent apoptotic signals, including: mitochondrial dysfunction, the activations of PARP and caspase cascades, and the increase in caspase-3 activity. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these iAs-induced responses. iAs also increased the phosphorylation of JNK and ERK1/2, but did not that p38-MAPK, in treated Neuro-2a cells. NAC and the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) abrogated iAs-induced cell cytotoxicity, caspase-3/-7 activity, and JNK and ERK1/2 activation. Additionally, exposure of Neuro-2a cells to iAs triggered endoplasmic reticulum (ER) stress identified through several key molecules (GRP 78, CHOP, XBP-1, and caspase-12), which was prevented by NAC. Transfection with GRP 78- and CHOP-specific si-RNA dramatically suppressed GRP 78 and CHOP expression, respectively, and attenuated the activations of caspase-12, -7, and -3 in iAs-exposed cells. Therefore, these results indicate that iAs induces ROS causing neuronal cell death via both JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-triggered apoptosis pathways.     

Identification of molecular signatures predicting the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs)

There are multiple lines of evidence showing that environmental toxicants including pesticides may contribute to neuronal cell death. Fipronil (FPN) is a phenylpyrazole insecticide that acts on insect GABA receptors. Although the action of FPN is restricted to insect neuronal or muscular transmitter systems, a few studies have assessed the effects of this neurotoxicant on neuronal cell death distinct from an insect. To determine the mechanisms underlying FPN-induced neuronal cell death, we evaluated the ability of this chemical to induce oxidative stress and studied the involvement of mitogen activated protein kinases (MAPKs) in FPN-induced apoptosis stress in human neuroblastoma SH-SY5Y (SH-SY5Y) cells. Exposure of SH-SY5Y cells to FPN led to the production of reactive oxygen species (ROS) and apoptotic cell death via activation of caspase-9 and caspase-3. Interestingly, the antioxidant, N-acetyl-cysteine (NAC) attenuated apoptotic cell death and ROS production induced by FPN. These results indicated that oxidative stress plays a central role in FPN-induced cytotoxicity. Mitochondrial complex I activity was also inhibited by FPN treatment. These finding indicate that FPN triggers intrinsic apoptosis via the mitochondrial signaling pathway that is initiated by the generation of ROS. Furthermore, FPN treatment induced phosphorylation of MAPK members. Activation of these protein kinases by FPN was involved in the onset of apoptosis as inhibitors specific to these kinases protect against FPN-induced cell death as well as ROS generation. Our data indicate that FPN-induced apoptosis is mediated primarily by the generation of ROS and activation of MAPK members followed by activation of the intrinsic apoptotic pathway.     

A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.     

Oxidative stress-driven mechanisms of nordihydroguaiaretic acid-induced apoptosis in FL5.12 cells

Nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) enzyme inhibitor, induces apoptosis independently of its activity as a LOX inhibitor in murine pro-B lymphocytes (FL.12 cells) by a mechanism that is still not fully understood. Glutathione depletion, oxidative processes and mitochondrial depolarization appear to contribute to the apoptosis induced by NDGA. The current data demonstrate that NDGA (20 microM)-induced apoptosis in FL5.12 cells is partially protected by N-acetylcysteine (NAC) (10 mM) and dithiothreitol (DTT) (500 microM) pretreatment, confirming a role for oxidative processes. In addition, the treatment of FL5.12 cells with NDGA led to an increase in phosphorylation and activation of the MAP kinases ERK, JNK and p38. Although pretreatment with ERK inhibitors (PD98059 or U0126) abolished ERK phosphorylation in response to NDGA, neither inhibitor had any effect on NDGA-induced apoptosis. SP600125, a JNK inhibitor, did not have any effect on NDGA-induced phosphorylation of JNK nor apoptosis. Pretreatment with the p38 inhibitor SB202190 attenuated NDGA-induced apoptosis by 30% and also abolished p38 phosphorylation, compared to NDGA treatment alone. NAC, but not DTT, also decreased the phosphorylation of p38 and JNK supporting a role for oxidative processes in activating these kinases. Neither NAC nor DTT blocked the phosphorylation of ERK suggesting that this activation is not related to oxidative stress. The release of cytochrome c and activation of caspase-3 induced by NDGA were inhibited by NAC. SB202190 slightly attenuated caspase-3 activation and had no effect on the release of cytochrome c. These data suggest that several independent mechanisms, including oxidative reactions, activation of p38 kinase and cytochrome c release contribute to NDGA-induced apoptosis.     

Chlorpyrifos induces neuronal cell death via both oxidative stress and Akt activation downstream-regulated CHOP-triggered apoptotic pathways

Chlorpyrifos (CPF) is one of the most abundant and widely used organophosphate pesticides for agricultural, industrial, and household purposes in the world. Epidemiological studies have reported that CPF can induce neurotoxic impairments in mammalian, which is linked to an important risk factor for development of neurodegenerative diseases (NDs). However, limited information is available on CPF-induced neurotoxicity, with the underlying exact mechanism remains unclear. In this study, CPF exposure (10–400 μM) significantly reduced Neuro-2a cell viability and induced apoptotic events, including the increase in caspase-3 activity, apoptotic cell population, and cleavage of caspase-3/−7 and PARP. Exposure of Neuro-2a cells to CPF also triggered CHOP activation. Transfection with CHOP-specific siRNA markedly suppressed the expression of CHOP, and attenuated cytotoxicity and apoptotic events in CPF-exposed Neuro-2a cells. Furthermore, CPF exposure obviously evoked the phosphorylation of Akt as well as ROS generation in a time-dependent manner. Pretreatment with LY294002 (an Akt inhibitor) effectively attenuated the CPF-induced Akt phosphorylation, CHOP activation, and apoptotic events, but not that ROS production. Of note, buffering the ROS generation with antioxidant N-acetylcysteine effectively prevented the CPF-induced ROS generation, CHOP activation, and apoptotic events, but not that the Akt phosphorylation. Collectively, these findings indicate that CPF exposure exerts neuronal cytotoxicity via the independent pathways of ROS generation and Akt activation downstream-regulated CHOP-triggered apoptosis, ultimately leading to neuronal cell death.     

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl⁺ chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl⁺ cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl⁺ cells.     

Podophyllotoxin Induces ROS-Mediated Apoptosis and Cell Cycle Arrest in Human Colorectal Cancer Cells via p38 MAPK Signaling

Podophyllotoxin (PT), a lignan compound from the roots and rhizomes of Podophyllum peltatum, has diverse pharmacological activities including anticancer effect in several types of cancer. The molecular mechanism of the anticancer effects of PT on colorectal cancer cells has not been reported yet. In this study, we sought to evaluate the anticancer effect of PT on human colorectal cancer HCT116 cells and identify the detailed molecular mechanism. PT inhibited the growth of cells and colony formation in a concentration-dependent manner and induced apoptosis as determined by the annexin V/7-aminoactinomycin D double staining assay. PT-induced apoptosis was accompanied by cell cycle arrest in the G2/M phase and an increase in the generation of reactive oxygen species (ROS). The effects of PT on the induction of ROS and apoptosis were prevented by pretreatment with N-acetyl-L-cysteine (NAC), indicating that an increase in ROS generation mediates the apoptosis of HCT116 cells induced by PT. Furthermore, Western blot analysis showed that PT upregulated the level of phospho (p)-p38 mitogen-activated protein kinase (MAPK). The treatment of SB203580, a p38 inhibitor, strongly prevented the apoptosis induced by PT, suggesting that PT-induced apoptosis involved the p38 MAPK signaling pathway. In addition, PT induced the loss of mitochondrial membrane potential and multi-caspase activation. The results suggested that PT induced cell cycle arrest in the G2/M phase and apoptosis through the p38 MAPK signaling pathway by upregulating ROS in HCT116 cells.     

Aflatoxin G1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells

Our previous studies showed that Aflatoxin G₁ (AFG₁) could induce lung adenocarcinoma, and that the cancer cells originated from alveolar type II cells (AT-II cells). Recently, we found AFG₁ induced structural impairment in rat AT-II cells, which may account for an early event in lung tumorigenesis. However, the mechanism of AFG₁-induced AT-II cell damage remains unclear. DNA damage and apoptosis induced by oxidative stress are well accepted causes of cell damage. Thus, we explore whether AFG₁ activates the reactive oxygen species (ROS)/MAPK/apoptosis pathway to cause cell damage in human AT-II cells like the cell line (A549). We found AFG₁ induced oxidative stress by increasing ROS generation and caused DNA double-strand breaks (DSBs) by up-regulating γH2AX expression. AFG₁ also triggered apoptosis in A549 cells by regulating Fas/FasL, caspase-8, Bax, Bcl-2, and activating caspase-3. Pre-treatment with antioxidant n-acetyl-l-cysteine (NAC) reduced ROS generation and DNA DSBs, inhibited apoptosis, and increased cell viability in AFG₁-treated cells. Furthermore, we found AFG₁ activated ROS-mediated JNK and p38 pathways to induce cell apoptosis in A549 cells. In conclusion, our results indicate that AFG₁ induces oxidative DNA damage and triggers apoptosis through ROS-mediated JNK and p38 signaling pathways in A549 cells, which may contribute to AFG₁-induced AT-II cell damage.     

The role of mitochondria-mediated intrinsic death pathway in gingerdione derivative I6-induced neuronal apoptosis

Neuronal death induced by I6 displayed apoptotic characteristics but the precise mechanism has not been fully elucidated. In the present studies, I6 at 24 h after intraperitoneal administration significantly decreased the density of surviving neurons and increased caspase-3 activity in frontal cortex, suggesting that peripherally administered I6 may cross BBB to induce CNS toxicity. In rat embryonic primary cortical cells, I6-induced reduction of mitochondrial viability and neuronal apoptosis was inhibited by vitamin E. In addition, I6-induced reactive oxygen species (ROS) caused the disruption of mitochondria membrane potential (MMP), the release of cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), resulting in activation of mitochondrial-mediated intrinsic death pathway. Pre-treatment with antioxidant vitamin E or N-acetylcysteine (NAC) completely abolished the I6-induced generation of ROS, loss of MMP, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced I6-induced neuronal death as well as caspase-3 activation and PARP cleavage. These results suggest that I6 induces neuronal death by promoting intracellular ROS production to cause a loss of MMP that result in release of cytochrome c and activation of mitochondria-mediated intrinsic death pathway.     

N-乙酰半胱氨酸影响p38有丝分裂原活化蛋白激酶对顺铂诱导急性肾损伤的保护作用

目的研究N-乙酰半胱氨酸(N-acetylcysteine,NAC)对顺铂(cisplatin,CDDP)诱导大鼠急性肾损伤(acute kidney in-jury,AKI)后组织细胞凋亡的影响和与p38有丝分裂原活化蛋白激酶(p38 mitogen activated protein kinase,p38MAPK)的关系。方法静脉注射CDDP制备大鼠AKI模型。大鼠随机分为正常对照组、AKI模型对照组、NAC低剂量组(50 mg.kg-1)、NAC中剂量组(100 mg.kg-1)、NAC高剂量组(200 mg.kg-1)、特异性p38MAPK抑制剂SB203580组(10mg.kg-1)。大鼠预先连续给药3 d,给予CDDP,再继续给药5 d。TUNEL法进行细胞凋亡检查。试剂盒测定肾脏组织caspase-3。Western blot测定caspase-3、Bax、Bcl-2、磷酸化p38MAPK(phosphorylated p38MAPK,p-p38MAPK)表达。结果与正常对照组相比,CDDP诱导AKI模型组肾组织凋亡细胞增加,caspase-3、Bax、p-p38MAPK表达升高,Bcl-2表达降低(P<0.01)。与AKI模型组相比,NAC与SB203580减少凋亡细胞、降低肾脏组织caspase-3、Bax、p-p38MAPK表达和增加Bcl-2表达(P<0.01)。结论 NAC可有效防治CD-DP诱导大鼠AKI,并与p38MAPK相关。     

Mitochondria-targeted peptide antioxidants: Novel neuroprotective agents

Increasing evidence suggests that mitochondrial dysfunction and oxidative stress play a crucial role in the majority of neurodegenerative diseases. Mitochondria are a major source of intracellular reactive oxygen species (ROS) and are particularly vulnerable to oxidative stress. Oxidative damage to mitochondria has been shown to impair mitochondrial function and lead to cell death via apoptosis and necrosis. Because dysfunctional mitochondria will produce more ROS, a feed-forward loop is set up whereby ROS-mediated oxidative damage to mitochondria favors more ROS generation, resulting in a vicious cycle. It is now appreciated that reduction of mitochondrial oxidative stress may prevent or slow down the progression of these neurodegenerative disorders. However, if mitochondria are the major source of intracellular ROS and mitochondria are most vulnerable to oxidative damage, then it would be ideal to deliver the antioxidant therapy to mitochondria. This review will summarize the development of a novel class of mitochondria-targeted antioxidants that can protect mitochondria against oxidative stress and prevent neuronal cell death in animal models of stroke, Parkinson's disease, and amyotrophic lateral sclerosis.     

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.     

Cadmium induces apoptotic cell death in WI 38 cells via caspase-dependent Bid cleavage and calpain-mediated mitochondrial Bax cleavage by Bcl-2-independent pathway

Previous reports have demonstrated that cadmium (Cd) may induce cell death via apoptosis, but the mechanism responsible for cellular death is not clear. In this study, we investigated the signaling pathways implicated in Cd-induced apoptosis in lung epithelial fibroblast (WI 38) cells. Apoptotic features were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, propidium iodide staining and DNA laddering. A treatment of cadmium caused the caspase-8-dependent Bid cleavage, the release of cytochrome c (Cyt c), activation of caspase-9 and -3, and PARP cleavage. A caspase-8 specific inhibitor prevented the Bid cleavage, caspase-3 activation and cell death. Alternatively, we observed that full-length Bax was cleaved into 18-kDa fragment (p18/Bax); this was initiated after 12 h and by 36 h the full-length Bax protein was totally cleaved to the p18/Bax, which caused a drastic release of Cyt c from mitochondria. The p18/Bax was detected exclusively in the mitochondrial fraction, and it originated from mitochondrial full-length Bax, but not from the cytosol full-length Bax. Cd also induced the activation of the mitochondrial 30-kDa small subunit of calpain that was preceded by Bax cleavage. Cd induced the upregulation of Bcl-2 and the degradation of p53 protein. N-acetyl cysteine effectively inhibited the Cd-induced DeltaPsim reduction, indicating ROS acts upstream of mitochondrial membrane depolarization. Taken together, our results suggest that Cd-induced apoptosis was thought to be mediated at least two pathways; caspase-dependent Bid cleavage, and the other is calpain-mediated mitochondrial Bax cleavage. Moreover, we found that the function of Bid and Bax was not dependent of Bcl-2, and that ROS can also contribute in the Cd-induced cell death.     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells

Tetrandrine, which is isolated from Chinese herb Stephania tetrandrae, possesses anti-inflammatory, immunosuppressive, and cytoprotective properties. Though it was previously shown that tetrandrine causes a G1 blockade and apoptosis in various cell types, however, the mechanism by which tetrandrine initiates apoptosis remains poorly understood. In present study, we investigated the mechanisms of apoptosis induced by tetrandrine in U937 leukemia cells. Tetrandrine inhibited U937 cell growth by inducing apoptosis. After treatment of U937 cells with tetrandrine (10microM) for 24h, alteration of cell morphology, chromatin fragmentation, cytochrome c release, and caspase activation were observed. Tetrandrine also induced early oxidative stress, which resulted in activation of JNK, but not ERK and p38 MAPK. A broad-spectrum caspase inhibitor and antioxidants significantly blocked tetrandrine-induced caspase-3 activation. However, inhibition of the JNK activity with SP600125 did not block tetrandrine-induced apoptosis. Tetrandrine-induced apoptosis of U937 cells also required activity of PKC-delta, because pretreatment with a specific PKC-delta inhibitor greatly blocked tetrandrine-induced caspase-3 activation. In addition, the apoptotic response to tetrandrine was significantly attenuated in dominant-negative PKC-delta transfected MCF-7 cells, suggesting that PKC-delta plays an important role in tetrandrine-induced apoptosis and can induce caspase activation. These results suggest that tetrandrine induces oxidative stress, JNK activation, and caspase activation. However, JNK activation by ROS is not involved in the tetrandrine-induced apoptosis. In addition, tetrandrine induces caspase-dependent generation of a catalytically active fragment of PKC-delta, and this fragment also appears to play a role in the activation of caspases.     

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells

CMS-9, a phospholipase A₂ (PLA₂) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca²⁺ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca²⁺ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA₂ activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca²⁺/ROS-evoked p38 MAPK and JNK activation.     

Geraniin induces apoptosis of human breast cancer cells MCF-7 via ROS-mediated stimulation of p38 MAPK

Geraniin, a typical ellagitannin isolated from Phyllanthus urinaria Linn, has been found to possess a range of bioactive properties. In the present study, we found that Geraniin showed potent anti-proliferative effects on human breast cancer MCF-7 cells. The IC₅₀ values were 9.94, 17.98 and 42.32?µM after 72-, 48- and 24-h treatment, respectively. Meanwhile, Geraniin could remarkably disrupt mitochondrial membrane potential and arrest S phase cell cycle. Western-blot analysis showed that Geraniin induced phosphorylation of the anti-apoptotic Bcl-2, and the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in MCF-7 cells. Moreover, Geraniin treatment activated p38 mitogen-activated protein kinase (p38 MAPK) and the effect was blunted in MCF-7 cells with the treatment of a specific p38 inhibitor SB203580. Geraniin could generate intracellular reactive oxygen species (ROS), activate p38 MAPK then induce the apoptosis in MCF-7 cells, such phenomena was abrogated by pretreatment with N-acetyl-l-cysteine. In general, these results support the conclusion that Geraniin-induced apoptosis is mediated via ROS-mediated stimulation of p38 MAPK signaling.     

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As-induced cardiovascular burden.     

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.