JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.     

A peptide inhibitor of c-Jun NH2-terminal kinase reduces myocardial ischemia-reperfusion injury and infarct size in vivo

The c-Jun NH(2)-terminal kinase (JNK) pathway of the mitogen-activated protein kinase (MAPK) signaling cascade regulates cell function and survival after stress stimulation. Equally robust studies reported dichotomous results suggesting both protective and detrimental effects of JNK during myocardial ischemia-reperfusion (I/R). The lack of a highly specific JNK inhibitor contributed to this controversy. We recently developed a cell-penetrating, protease-resistant peptide inhibitor of JNK, d-JNKI-1. Here we report on the effects of d-JNKI-1 in myocardial I/R. d-JNKI-1 was tested in isolated-perfused adult rat hearts. Increased activation of JNK, p38-MAPK, and extracellular signal-regulated kinase-1/2 (ERK1/2), as assessed by kinase assays and Western blotting, occurred during I/R. d-JNKI-1 delivered before onset of ischemia prevented the increase in JNK activity while not affecting ERK1/2 and p38-MAPK activation. JNK inhibition reduced ischemic injury, as manifested by increased time to contracture (P < 0.05) and decreased left ventricular end-diastolic pressure during ischemia (P < 0.01), and enhanced posthypoxic recovery of systolic and diastolic function (P < 0.01). d-JNKI-1 reduced mitochondrial cytochrome-c release, caspase-3 activation, and the number of apoptotic cells determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (P < 0.05), indicating suppression of the mitochondrial machinery of apoptosis. d-JNKI-1 delivered at the time of reperfusion did not improve functional recovery but still prevented apoptosis. In vivo, d-JNKI-1 reduced infarct size after coronary artery occlusion and reperfusion by approximately 50% (P < 0.01). In conclusion, d-JNKI-1 is an important compound that can be used in preclinical models to investigate the role of JNK signaling in vivo. Inhibition of JNK during I/R is cardioprotective in anesthetized rats in vivo.     

Microtubule inhibitors elicit differential effects on MAP kinase (JNK, ERK, and p38) signaling pathways in human KB-3 carcinoma cells

Microtubule inhibitors are widely used in cancer chemotherapy, but the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. Because members of the mitogen-activated protein kinase (MAPK) family have been implicated in regulation of cell survival and cell death, we examined the extent and kinetics of activation of JNK, ERK, and p38 MAPKs in response to treatment of KB-3 carcinoma cells with several microtubule inhibitors. All four agents tested (vinblastine, vincristine, Taxol, and colchicine) caused significant (6- to 13-fold) activation of JNK, concomitant inactivation of ERK, and a reduction in basal p38 MAPK activity. JNK activation and ERK inactivation occurred prior to caspase 3 activation. The microtubule inhibitors also induced phosphorylation of Raf-1 kinase. SEK-1, upstream of JNK, was also activated and phosphorylated in response to the microtubule inhibitors, and sustained phosphorylation of three endogenous JNK substrates (c-Jun, ATF-2, and JunD) was observed. By comparison, the antitumor agent doxorubicin induced activation of JNK and p38 but had no effect on ERK activity or Raf-1. These data demonstrate that microtubule inhibitors elicit distinct and specific effects on MAPK-mediated signaling pathways and suggest in particular that coordinate and reciprocal alterations in JNK and ERK activities are important facets of the cellular response to microtubule disruption.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

Attenuation of ischemia/reperfusion induced MAP kinases by N-acetyl cysteine,sodium nitroprusside and phosphoramidon

Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P³⁸ MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.     

Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis

Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus–host interaction.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart. Cross-talk between cardiac hypertrophic signaling pathways

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiomyocytes. Both necessary and sufficient roles have been described for the mitogen activated protein kinase(1) (MAPK) signaling pathway, specific protein kinase C (PKC) isoforms, and calcineurin. Here we investigate the interdependence between calcineurin, MAPK, and PKC isoforms in regulating cardiomyocyte hypertrophy using three separate approaches. Hearts from hypertrophic calcineurin transgenic mice were characterized for PKC and MAPK activation. Transgenic hearts demonstrated activation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK factors. Calcineurin transgenic hearts demonstrated increased activation of PKCalpha, beta(1), and theta, but not of epsilon, beta(2), or lambda. In a second approach, cultured cardiomyocytes were infected with a calcineurin adenovirus to induce hypertrophy and the effects of pharmacologic inhibitors or co-infection with a dominant negative adenovirus were examined. Calcineurin-mediated hypertrophy was prevented with PKC inhibitors, Ca(2+) chelation, and attenuated with a dominant negative SEK-1 (MKK4) adenovirus, but inhibitors of ERK or p38 activation had no effect. In a third approach, we examined the activation of MAPK factors and PKC isoforms during the progression of load-induced hypertrophy in aortic banded rats with or without cyclosporine. We determined that inhibition of calcineurin activity with cyclosporine prevented PKCalpha, theta, and JNK activation, but did not affect PKCepsilon, beta, lambda, ERK1/2, or p38 activation. Collectively, these data indicate that calcineurin hypertrophic signaling is interconnected with PKCalpha, theta, and JNK in the heart, while PKCepsilon, beta, lambda, p38, and ERK1/2 are not involved in calcineurin-mediated hypertrophy.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Effect of nitric oxide donor and gamma irradiation on MAPK signaling in murine peritoneal macrophages

Irradiation (IR) of cells is known to activate enzymes of mitogen activated protein kinase (MAPK) family. These are known to be involved in cellular response to stress and are determinants of cell death or survival. When radiotherapy is delivered to malignant cells, macrophages, being radioresistant, survive, get activated, and produce large amounts of nitric oxide. As a result of activation they recognize and phagocytose tumor and normal cell apoptotic bodies leading to tumor regression. In this study, the MAPK signaling in peritoneal macrophages was investigated which plays an important role in its various functions, in an environment which is predominantly nitric oxide, as is after IR. The behavior of macrophages in such an environment was also looked at. The three MAPK (ERK1/2, p38, and JNK) respond differently to Sodium nitroprusside (SNP) alone or IR alone. All the three were activated following IR but only JNK was activated following SNP treatment. Surprisingly, when both the stresses were given simultaneously or one after the other, this differential response was lost and there was a complete inhibition of phosphorylation of all the three MAPKs, irrespective of the order of the two insults (IR and SNP). The noteworthy observation was that despite the complete inhibition of MAPK signaling there was no effect on either the viability or the phagocytic efficiency of peritoneal macrophages.     

c-Jun N-terminal kinases (JNK) antagonize cardiac growth through cross-talk with calcineurin-NFAT signaling

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.     

Olaquindox-induced apoptosis is suppressed through p38 MAPK and ROS-mediated JNK pathways in HepG2 cells

We investigated mitogen-activated protein kinase (MAPK) pathways as well as reactive oxygen species (ROS) in olaquindox-induced apoptosis. Exposure of HepG2 cells to olaquindox resulted in the phosphorylation of p38 MAPK and c-Jun N-terminal kinases (JNK). To confirm the role of p38 MAPK and JNK, HepG2 cells were pretreated with MAPKs-specific inhibitors prior to olaquindox treatment. Olaquindox-induced apoptosis was significantly potentiated by the JNK inhibitor (SP600125) or the p38 MAPK inhibitor (SB203580). Furthermore, we observed that olaquindox treatment led to ROS generation and that olaquindox-induced apoptosis and ROS generation were both significantly reduced by the antioxidants, superoxide dismutase and catalase. In addition, the levels of phosphorylation of JNK, but not p38 MAPK, were significantly suppressed after pretreatment of the antioxidants, while inhibition of the activations of JNK or p38 MAPK had no effect on ROS generation. This result suggested that ROS may be the upstream mediator for the activation of JNK. Conclusively, our results suggested that apoptosis in response to olaquindox treatment in HepG2 cells might be suppressed through p38 MAPK and ROS–JNK pathways.