Tangeretin, a citrus flavonoid, inhibits PGDF-BB-induced proliferation and migration of aortic smooth muscle cells by blocking AKT activation

Tangeretin, a natural polymethoxylated flavone concentrated in the peel of citrus fruits, is known to have antiproliferative, antiinvasive, antimetastatic and antioxidant activities. However, the effect of tangeretin on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of tangeretin on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Tangeretin significantly inhibited proliferation, DNA synthesis and migration of PDGF-BB-stimulated RASMCs without inducing cell death. Treatment with tangeretin-induced cell-cycle arrest in the G?/G? phase was associated with down-regulation of cyclin D1 and cyclin E in addition to up-regulation of p27(kip1). We also showed that tangeretin inhibited PDGF-BB-induced phosphorylation of AKT, while it had no effect on the phosphorylation of phospholipase Cγ (PLCγ), PDGF receptor β-chain (PDGF-Rβ) and extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs). An in vitro kinase assay revealed that tangeretin inhibited AKT activity in a dose-dependent manner. Moreover, treatment of LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, had similar effects than that of tangeretin on the expression of p27(kip1) and cyclin D1, as well as cell migration in PDFG-BB-stimulated RASMCs. Taken together, these findings suggest that tangeretin could suppress PDGF-BB-induced proliferation and migration of RASMCs through the suppression of PI3K/AKT signaling pathway, and may be a potential candidate for preventing or treating vascular diseases, such as atherosclerosis and restenosis.     

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Interleukin-1beta (IL-1beta) has been implicated in the pathogenesis of inflammatory diseases of the airway. In this study, we investigated the regulation of MUC2 and MUC5AC expression and of their regulatory mechanisms through cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Cells activated by IL-1beta showed increased COX-2, MUC2, and MUC5AC expressions at both the mRNA and protein levels. Mucin production was blocked by the selective COX-2 inhibitor NS398, and PGE(2) directly induced MUC2 and MUC5AC expression at both the mRNA and protein levels in a dose-dependent manner. These results suggest a role for PGE(2) in IL-1beta-induced mucin synthesis in NCI-H292 cells. To investigate the roles of molecules upstream of COX-2 in mucin regulation, we examined the role of mitogen-activated protein kinases (MAPKs). Cells activated by IL-1beta showed increased extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, and IL-1beta-induced MUC2 and MUC5AC production was blocked by the ERK pathway inhibitor PD98059 or the p38 inhibitor SB203580. The inhibition of both MAPKs reduced IL-1beta-induced COX-2 expression and PGE(2) synthesis. Furthermore, the addition of PGE(2) to cells overcame the inhibitory effects of both MAPK inhibitors in IL-1beta-induced mucin production. These results indicate that in human pulmonary epithelial cells, IL-1beta activates ERK or p38 to induce COX-2 production, which in turn induces MUC2 and MUC5AC production.     

Byakangelicol,isolated from Angelica dahurica,inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Mitogen-activated protein kinase signalling pathways in IL-1 beta-dependent rat airway smooth muscle proliferation

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen-activated protein kinase (MAPK) pathway in IL-1beta induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen-activated and extracellular regulated kinase kinase, MEK-1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c-jun N-terminal kinase, JNK, inhibitor) on IL-1beta-induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL-1beta in a dose-dependent manner. p38, JNK and ERK MAPKs were activated by IL-1beta in a time-dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 microM) had no effect on IL-1beta-induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose-dependently inhibited IL-1beta-dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL-1beta-dependent proliferation of rat ASM.     

Protein kinase calpha but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1beta: involvement of sequential activation of tyrosine kinase, nuclear factor-kappaB-inducing kinase, and IkappaB kinase 2

IL-1beta induced an increase in ICAM-1 expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C inhibitor (D609) attenuated IL-1beta-induced ICAM-1 expression. IL-1beta produced an increase in PKC activity and this effect was abolished by D609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited IL-1beta-induced response. TPA, a PKC activator, stimulated ICAM-1 expression as well, this effect being inhibited by tyrosine kinase inhibitors. Treatment of cells with IL-1beta resulted in stimulation of p44/42 MAPK, p38, and JNK. However, neither the mitogen activated protein kinase kinase inhibitor PD 98059 nor the p38 inhibitor SB 203580 affected IL-1beta-induced ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by IL-1beta and these effects were inhibited by tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity as well, these effects being inhibited by tyrosine kinase inhibitors. Dominant-negative PKCalpha, NIK, or IKK2, but not IKK1 mutant, inhibited IL-1beta- or TPA-induced ICAM-1 promoter activity. IKK activity was stimulated by either IL-1beta or TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. Taken together, IL-1beta activates phosphatidylcholine-specific phospholipase C and induces activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of NIK, IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression. However, activation of p44/42 MAPK, p38, and JNK is not involved.     

Regulation of cyclooxygenase-2 expression by phospholipase D in human amnion-derived WISH cells

Prostaglandins (PGs) are known to play a key role in the initiation of labor, but the mechanisms regulating their synthesis in amnion are largely unknown. In this study, the regulatory mechanisms for PGE(2) production during phospholipase D (PLD) and p38-dependent activation of WISH cells were investigated. We found that the stimulation of WISH cells with interleukin (IL)-1 beta elicited dose-dependent synthesis of cyclooxygenase-2 (COX-2) mRNA, protein, and their products, PGE(2). Moreover, the treatment of [(3)H]myristate-labeled cells in the presence of 1-butanol caused the dose-dependent formation of [(3)H]phosphatidylbutanol (PBt), a product specific to PLD activity. Pretreating the cells with 1-butanol and Ro 31-8220 inhibited the IL-1 beta-induced COX-2 expression, but 3-butanol did not affect this response. In addition, evidence that PLD was involved in the stimulation of COX-2 expression was provided by the observations that COX-2 expression was stimulated by the dioctanoyl phosphatidic acid (PA) and that the prevention of PA dephosphorylation by 1-propranolol potentiated COX-2 expression by IL-1 beta. Moreover, IL-1 beta stimulation of the cells caused the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and IL-1 beta-induced COX-2 expression was inhibited by the pretreatment of WISH cells with a p38 inhibitor, in contrast ERK upstream inhibitor had no effect. Furthermore, Ro 31-8220 inhibited IL-1 beta-induced p38 phosphorylation but not ERK phosphorylation. The results of this study indicate that in human amnion cells, IL-1 beta might activate PLD through an upstream protein kinase C to elicit p38 and finally induce COX-2 expression.     

Ent-pimara-8(14), 15-dien-19-oic acid isolated from the roots of Aralia cordata inhibits induction of inflammatory mediators by blocking NF-kappaB activation and mitogen-activated protein kinase pathways

Macrophages play central roles in the innate immune system. The roots of Aralia cordata are widely used in Oriental medicine as a remedy for arthritis. During our program to screen medicinal plants for potential anti-inflammatory compounds, ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) was isolated from the roots of A. cordata. We examined the effect of PA on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. PA was found to significantly inhibit the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and interleukin-6 (IL-6), as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6. Furthermore, we examined whether mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are involved in LPS-induced RAW 264.7 cells. We found that a p38 inhibitor (SB203580) and an ERK 1/2 inhibitor (PD98059) significantly affected LPS-induced IL-6 production. In contrast, a JNK 1/2 inhibitor (SP600125) and PI3K inhibitor (wortmannin or LY294002) did not block the induction of IL-6 production by LPS. The LPS-induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) was inhibited by PA, but not the phosphorylation of JNK 1/2 and AKT (Ser473). Moreover, PA suppressed I kappaB alpha degradation, NF-kappaB activation and luciferase activity. These results suggest that PA isolated from A. cordata has a potential regulatory effect on inflammatory iNOS, COX-2 and IL-6 expression through blockade of the phosphorylation of MAPKs following I kappaB alpha degradation and NF-kappaB activation.     

Role of c-jun N-terminal kinase in the induced release of GM-CSF,RANTES and IL-8 from human airway smooth muscle cells

1. Human airway smooth muscle cells (HASMC) contribute to airway inflammation in asthma by virtue of their capacity to produce several inflammatory mediators including IL-8, GM-CSF and RANTES. The intracellular signal pathway underlying the production of these cytokines in HASMC is not entirely elucidated. 2. We examined the role of the mitogen-activated protein kinase (MAPK) c-jun N-terminal kinase (JNK) in TNFalpha- and IL-1beta-induced GM-CSF, RANTES and IL-8 production in HASMC by using a novel specific inhibitor for JNK (SP600125). 3. Confluent HASMC were treated with TNFalpha or IL-1beta (10 ng ml(-1)) for 24 h in the presence or absence of SP600125 (1-100 micro M). JNK activity was determined by a kinase assay. Phosphorylation of JNK, p38 MAPK and ERK was examined by Western blotting. Culture supernatants were assayed for GM-CSF, RANTES and IL-8 content by ELISA. 4. Maximum TNFalpha- or IL-1beta-induced phosphorylation of JNK in HASMC occurred after 15 min and returned to baseline levels after 4 h. SP600125 inhibited TNFalpha- and IL-1beta-induced JNK activity in HASMC as shown by the reduced phosphorylation of its substrate c-jun. Furthermore, GM-CSF, RANTES and to a lesser extent IL-8 release from HASMC treated with TNFalpha and IL-1beta was inhibited dosedependently by SP600125. 5. JNK activation is involved in TNFalpha- and IL-1beta-induced GM-CSF, RANTES and IL-8 production from HASMC. JNK may therefore represent a critical pathway for cytokine production in HASMC.     

Docosahexaenoic acid enhances cyclooxygenase-2 induction by facilitating p44/42, but not p38, mitogen-activated protein kinase activation in rat vascular smooth muscle cells

The effect of docosahexaenoic acid (DHA) on cyclooxygenase expression induced by interleukin (IL)-1beta and phorbol 12-myristate 13-acetate (PMA) in rat vascular smooth muscle cells (VSMCs) was investigated in order to clarify the cellular mechanism of cardiovascular protective effects. DHA and eicosapentaenoic acid slightly enhanced IL-1beta-induced cyclooxygenase (COX)-2, but not COX-1, expression, whereas arachidonic acid had no effect. DHA also slightly enhanced PMA-induced COX-2 expression. DHA stimulated both rapid and prolonged activation of p44/42, but not p38, mitogen-activated protein kinase (MAPK) induced by IL-1beta and PMA. These results suggest that DHA enhances the COX-2 expression by selectively facilitating p44/42 MAPK activation in VSMCs.     

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8?h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E₂, a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.     

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8?h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E(2), a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.     

Role of mitogen-activated protein kinase family in serum-induced leukaemia inhibitory factor and interleukin-6 secretion by bone marrow stromal cells

1: In the haematopoietic microenvironment, bone marrow stromal cells play an important role in regulating haematopoiesis by expressing various cytokines, including leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6). However, the intracellular signal that regulates cytokine secretion in bone marrow stromal cells has not been determined. The aim of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) family in serum-induced secretion of LIF and IL-6 by bone marrow stromal cells. 2: Transformed human bone marrow stromal cells (HS-5) were stimulated with foetal calf serum (FCS) to produce LIF and IL-6. FCS also induced activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). 3: Both PD98059 (MAPK/ERK kinase inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated FCS-induced LIF protein production and gene expression. SB203580 decreased IL-6 production and gene expression, but PD98059 had no effect on IL-6 production and gene expression. 4: Expression of a dominant-negative mutant form of JNK1 that blocked FCS-induced JNK activity had no effect on protein production and gene expression of these cytokines. 5: These findings demonstrate that both ERK and p38 MAPK are involved in FCS-induced LIF secretion, whereas only p38 MAPK is important for IL-6 secretion, and that FCS-induced activation of JNK has no effect on the production of LIF and IL-6. We conclude that, in spite of their similar biological effects, they are differentially regulated at the level of MAPK activity in bone marrow stromal cells.     

COX-2 is associated with cadmium-induced ICAM-1 expression in cerebrovascular endothelial cells

In order to get insight into the mechanism of cadmium (Cd)-induced brain injury, we investigated the effects of Cd on the induction of COX-2 and ICAM-1 in bEnd.3 mouse brain endothelial cells (EC). Cd stimulated PGE(2) release in a time and dose dependent manner, which was accompanied by increase of COX-2 expression. The thiol-reducing antioxidant N-acetylcyteine attenuated Cd-induced PGE(2) production and COX-2 expression. Cd increased phosphorylation of p38 MAPK, but not of JNK and ERK1/2. A blockade of p38 MAPK pathway abrogated Cd-induced COX-2 expression and PGE(2) production. Cd-induced ICAM-1 expression and leukocyte-EC adhesion were diminished by non-steroidal anti-inflammatory drugs such as indomethacin and NS-398, which was reversed by addition of PGE(2). Together, these data suggest that Cd induces COX-2 expression through the activation of p38 MAPK, an oxidative stress-sensitive cellular signaling molecule, and induction of COX-2 is associated with ICAM-1 expression in brain endothelial cells following Cd exposure.     

Fluoride-induced IL-8 release in human epithelial lung cells: relationship to EGF-receptor-, SRC- and MAP-kinase activation

Exposure of human epithelial lung cells to fluorides is known to induce a marked increase in the release of interleukin (IL)-8, a chemokine involved in neutrophil recruitment. In the present study, the involvement of mitogen-activating protein kinases (MAPKs), the role of upstream activation of Src family kinases (SFKs), epidermal growth factor receptor (EGFR) activation and the interrelationships between these pathways in fluoride-induced IL-8 were examined in a human epithelial lung cell line (A549). Sodium fluoride strongly activated MAPK, in particular JNK1/2 and p38. The ERK1/2-inhibitor PD98059, the p38-inhibitor SB202190 and the JNK1/2-inhibitor SP600125 partially inhibited the fluoride-induced IL-8 response. Combinations of these inhibitors reduced the responses nearly to basal levels. Treatment with siRNA against JNK2 also reduced the IL-8 response to fluoride. Furthermore, fluoride activated SFKs, which was abolished by the SFK-inhibitor PP2. PP2 substantially inhibited the increased levels of IL-8, and partially reduced the fluoride-induced activation of ERK1/2, p38 and JNK1/2. Fluoride exposure also led to a phosphorylation of the EGFR, that was partially inhibited by PP2. AG1478, an EGFR-inhibitor, partially reduced the fluoride-induced IL-8 response and the phosphorylation of JNK1/2 and ERK1/2, but less the phosphorylation of p38. The effects of AG1478 were less than that of PP2. In conclusion, our findings suggest that the fluoride-induced IL-8 release involves the combined activation of ERK1/2, JNK1/2 and p38, and that the phosphorylation of these kinases, and in particular JNK1/2 and ERK1/2, partly, is mediated via a SFK-dependent EGFR-linked pathway. SFK-dependent, but EGFR-independent mechanisms seem important, and especially for phosphorylation of p38.     

Indomethacin induces apoptosis in 786-O renal cell carcinoma cells by activating mitogen-activated protein kinases and AKT

Studies on chemoprevention of cancer are generating increasing interest. The anti-neoplastic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) involves cyclooxygenase (COX)-dependent and COX-independent mechanisms. Evidence suggests that mitogen-activated protein kinases (MAPKs) may mediate apoptotic signaling induced by anti-neoplastic agents. While many reports have revealed the existence of MAPK activation in apoptosis induced by various stimuli, the signaling transduction pathways used by NSAIDs to trigger apoptosis in human renal cell carcinoma (RCC) remain largely unknown. Treatment of RCC 786-O cells with indomethacin resulted in growth regression and apoptosis. Caspase-dependent apoptosis was evidenced by the detection of enzymatic activities of caspase-3, caspase-6, and caspase-9 and suppression of toxicity using a caspase inhibitor. Indomethacin treatment was associated with increased expression of glucose-regulated protein 78 (GRP78) and C/EBP homologus protein (CHOP) and activation of ATF-6, characteristics of endoplasmic reticulum stress. In addition, the concomitant induction of peroxisome proliferator-activated receptor (PPAR), especially PPAR-beta, was apparent in treated cells. Western blotting revealed the activation of extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) with indomethacin treatment. Selective inhibitors of ERK, p38 MAPK, and JNK suppressed the induction of GRP78, CHOP, and PPAR-beta, attenuated indomethacin-induced cytotoxicity and reduced increased caspase activity. LY294002, a phosphoinositide-3 kinase (PI3K)/AKT inhibitor, and Trolox, an antioxidant, suppressed indomethacin-induced cytotoxicity and caspase activation. Furthermore, Trolox attenuated indomethacin-induced increased phosphorylation in ERK, p38 MAPK, JNK, and AKT. In conclusion, our findings establish a mechanistic link between the oxidative stress, PI3K/AKT pathway, MAPK pathway and indomethacin-induced cellular alterations and apoptosis in 786-O cells.