Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.     

Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.     

Rescue of CH31 B cells from antigen receptor-induced apoptosis by inhibition of p38 MAPK

CH31 B lymphomas represent a model for antigen-induced deletional tolerance of immature B lymphocytes, because cross-linking the B cell antigen receptor (BCR) induces G(1) phase arrest and apoptosis. We have recently demonstrated that BCR cross-linking leads to a transient activation of p38 mitogen-activated protein kinase (MAPK) in CH31 B cells. In this paper, we functionally characterize the role of p38 MAPK in BCR-induced apoptosis as well as evaluate the regulation of additional MAPKs by the BCR. We demonstrate that JNK and ERK activities are not affected by BCR cross-linking, suggesting that these MAPKs are not directly involved in initiating the apoptotic cascade. By contrast, we show that pretreatment of CH31 B cells with the highly specific p38 MAPK inhibitor SB203580 ablated both BCR-induced p38 MAPK activity and apoptosis. Pretreatment of CH31 cells with an inactive SB203580 analog, SB202474, did not prevent apoptosis. These findings establish a key role for p38 MAPK in antigen receptor-mediated apoptosis of CH31 B cells.     

Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 microM) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38(MAPK)) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38(MAPK)-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H(2)O(2) accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38(MAPK) phosphorylation. In a similar manner, p38(MAPK) phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38(MAPK) activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.     

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils

Many neutrophil responses, including chemotaxis, exocytosis, respiratory burst activity and chemokine synthesis, are mediated by p38 MAPK. MAPK-activated protein kinase-2 (MK2) is activated by p38 MAPK in human neutrophils. The present study tested the hypothesis that MK2 mediates multiple p38 MAPK-dependent responses in human neutrophils by comparing the effect of the p38 MAPK inhibitor, SB203580, with an MK2 inhibitory peptide. Both SB203580 and MK2 inhibitory peptide attenuated respiratory burst activity, exocytosis, and chemotaxis. Lipopolysaccharide (LPS)-induced IL-8 production was inhibited by SB203580, but not by the MK2 inhibitory peptide. Inhibition of chemotaxis and respiratory burst activity by SB203580 was less than that of MK2 inhibitory peptide. Inhibition of extracellular signal-regulated kinase (ERK) activity by PD98059 attenuated superoxide release and chemotaxis, and simultaneous treatment with SB203580 and PD98059 demonstrated additive inhibition. ERK phosphorylated MK2 in vitro and activated MK2 in f-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These data suggest that MK2 mediates both ERK- and p38 MAPK-dependent neutrophil responses.     

Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines

Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) are major signaling molecules activated in human neutrophils stimulated by cytokines. Both molecules were cleaved at the N-terminal portion in neutrophils undergoing apoptosis induced by in vitro culture alone or treatment with TNF and/or cycloheximide. The cleavage of both molecules was inhibited by G-CSF and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a caspase inhibitor, both of which can inhibit neutrophil apoptosis. In a cell-free system, ERK and p38 MAPK were not cleaved by recombinant caspase-3 or caspase-8 while gelsolin was cleaved by caspase-3 under the same condition. The cleavage of both molecules appears to be specific to mature neutrophils, since it was not detected in immature cells (HL-60 and Jurkat) undergoing apoptosis, indicating that proteases responsible for the cleavage of both molecules may develop during differentiation into mature neutrophils. Concomitant with the cleavage of ERK and p38 MAPK, GM-CSF- and TNF-induced superoxide release, adherence, and phosphorylation of ERK and p38 MAPK were decreased in neutrophils undergoing apoptosis. In addition, GM-CSF- and TNF-induced superoxide release and adherence were inhibited by PD98059 MAPK/ERK kinase inhibitor) as well as SB203580 (p38 MAPK inhibitor), suggesting possible involvement of ERK and p38 MAPK in superoxide release and adherence induced by these cytokines. These findings indicate that ERK and p38 MAPK are cleaved and degraded in neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines.     

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.     

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Differential requirement of members of the MAPK family for CCL2 expression by hepatic stellate cells

Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38(MAPK), and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38(MAPK)), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.     

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Role of p38 MAPK in CYP2E1-dependent arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-kappaB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-kappaB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-kappaB activation.