P38 Kinase Pathways Research Articles

Salinomycin and Other Polyether Ionophores Are a New Class of Antiscarring Agent

Although scarring is a component of wound healing, excessive scar formation is a debilitating condition that results in pain, loss of tissue function, and even death. Many tissues, including the lungs, heart, skin, and eyes, can develop excessive scar tissue as a result of tissue injury, chronic inflammation, or autoimmune disease. Unfortunately, there are few, if any, effective treatments to prevent excess scarring, and new treatment strategies are needed. Using HEK293FT cells stably transfected with a TGFβ-dependent luciferase reporter, we performed a small molecule screen to identify novel compounds with antiscarring activity. We discovered that the polyether ionophore salinomycin potently inhibited the formation of scar-forming myofibroblasts. Salinomycin (250 nm) blocked TGFβ-dependent expression of the cardinal myofibroblast products α smooth muscle actin, calponin, and collagen in primary human fibroblasts without causing cell death. Salinomycin blocked phosphorylation and activation of TAK1 and p38, two proteins fundamentally involved in signaling myofibroblast and scar formation. Expression of constitutively active mitogen activated kinase kinase 6, which activates p38 MAPK, attenuated the ability of salinomycin to block myofibroblast formation, demonstrating that salinomycin targets the p38 kinase pathway to disrupt TGFβ signaling. These data identify salinomycin and other polyether ionophores as novel potential antiscarring therapeutics.

S100B is required for high glucose-induced pro-fibrotic gene expression and hypertrophy in mesangial cells.

The advanced glycation end‑product (AGE)‑receptor for AGE (RAGE) axis induces transforming growth factor‑β (TGF‑β) expression, cell hypertrophy and increases extracellular matrices that are indicated in the pathogenesis of diabetic nephropathy (DN). RAGE binds to numerous ligands besides AGE, including S100B. In the present study, the roles of S100B in high glucose‑induced p21WAF1, extracellular matrices, TGF‑βl and cell hypertrophy in mouse mesangial (MES13) cells were investigated. High glucose (30 mM) time‑dependently (24‑72 h) induced S100B expression. High glucose and exogenous S100B (1 µM) time‑dependently increased p21WAF1 gene transcription and protein expression, increased type IV collagen and fibronectin protein expression, and TGF‑β gene transcription and bioactivity. Exogenous S100B also time‑dependently activated the extracellular regulated kinases (ERK1/2), p38 kinase and c‑Jun N‑terminal kinase (JNK) signaling pathways. Exogenous S100B‑induced TGF‑β gene transcription and bioactivity were attenuated by SB203580 (p38 kinase inhibitor) and PD98059 (ERK1/2 inhibitor). Finally, the knockdown of S100B by small interfering RNA (siRNA) attenuated high glucose‑induced TGF‑β gene transcription and bioactivity, type IV collagen and fibronectin protein expression and p21WAF1 protein expression. Thus, S100B induced TGF‑β via the ERK1/2 and p38 kinase pathways in mesangial cells. Additionally, high glucose‑induced pro‑fibrotic genes (TGF‑β, type IV collagen and fibronectin) and cell hypertrophy‑related p21WAF1 are dependent on S100B.

Abstract 315: The Role of Mixed Lineage Kinase 3 in Inflammatory Cell-Fibroblast Communication

Mixed lineage kinase 3 (MLK3) is a ubiquitously expressed pro-inflammatory, pro-apoptotic mitogen activated protein kinase kinase kinase (MAP3K). MLK3 is a key regulator of the p38 and c-jun terminal kinase (JNK) pathways and has been studied in cancer and neurodegenerative disease. Although the p38 and JNK pathways have been studied in the cardiac function and disease, little is known regarding the role of MLK3 in the heart. Studies in our laboratory have indicated that MLK3 RNA is highly expressed in macrophages, cardiomyocytes and cardiac fibroblasts, suggesting an important role in cardiac function. Recently published work has indicated that MLK3 plays an important role in inflammatory cell motility, leading us to hypothesize that MLK3 is involved in inflammatory cell-fibroblast communication during cardiac disease. Knockout (KO) or inhibition of MLK3 using the novel small molecule inhibitor URMC-099 does not significantly affect heart rate, mean arterial pressure, systolic pressure, minimum or maximum dp/dt compared to wild type (WT) controls as measured by invasive hemodynamics at 3 months of age. Echocardiographic analysis indicates that MLK3 KO or inhibition does not affect cardiac architecture or cardiac function, indicated by fractional shortening or ejection fraction. However, upon transaortic constriction (TAC), MLK3 KO and URMC-099 treatment results in decreases in Mac-3 positive staining at 3 and 7 days post-TAC as well as decreases in CD-45 staining 7 days post-TAC compared to WT TAC operated, vehicle treated controls, suggesting that MLK3 KO and drug treatment may attenuate the early inflammatory response after TAC. Studies examining the relationship between the MLK3 mediated inflammatory response and subsequent fibrosis and cardiac dysfunction post-TAC are currently ongoing.

Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways.

Resveratrol has been shown to possess anticancer, anti-aging, and anti-inflammatory properties. Matrix metalloproteinases (MMPs) appear to be responsible for much of the extracellular matrix (ECM) degradation observed in the progression of cancer, aging and inflammation. We found that resveratrol significantly inhibited MMP-2 and MMP-9, and induced the expression of type II collagen and sex-determining region Y-box (SOX)-9 and the production of sulfated proteoglycans in HTB94 chondrosarcoma cells. Moreover, inhibition of MMPs with an MMP inhibitor further enhanced the effects of resveratrol. Phosphorylation of p38 was increased and phosphorylation of c-Jun N-terminal kinase (JNK) was inhibited by resveratrol. Treatment with SB203580, a p38 kinase inhibitor, enhanced the suppression of MMP-2 and MMP-9 by resveratrol and inhibited resveratrol-induced stimulation of type II collagen and SOX-9 expression and production of sulfated proteoglycans. Treatment with SP600125, a JNK inhibitor, attenuated the effects of resveratrol on MMP-2 and MMP-9, and accelerated resveratrol-induced effects on type II collagen, SOX-9 and sulfated proteoglycan production. Our results suggest that resveratrol inhibits MMP-induced differentiation via the p38 kinase and JNK pathways in HTB94 chondrosarcoma cells.

Molecular mechanisms underlying synergistic adhesion of sickle red blood cells by hypoxia and low nitric oxide bioavailability

AbstractThe molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD.

Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway

PurposeThe present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells.MethodsThe apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods.ResultsData showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose) polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase.ConclusionMidazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways.

TNF-α Modulation of Intestinal Epithelial Tight Junction Barrier Is Regulated by ERK1/2 Activation of Elk-1

Tumor necrosis factor (TNF-α) is a proinflammatory cytokine that plays a critical role in the pathogenesis of inflammatory bowel disease. TNF-α causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of MAP kinase pathways (ERK1/2 and p38 kinase) and the molecular processes involved. An in vitro intestinal epithelial model system consisting of Caco-2 monolayers and an in vivo mouse model system were used to delineate the cellular and molecular mechanisms involved in TNF-α effects on tight junction barrier. The TNF-α-induced increase in Caco-2 tight junction permeability was mediated by activation of the ERK1/2 signaling pathway, but not the p38 kinase pathway. Activation of the ERK1/2 pathway led to phosphorylation and activation of the ETS domain-containing transcription factor Elk-1. The activated Elk-1 translocated to the nucleus, where it bound to its binding motif on the myosin light chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. In addition, in vivo intestinal perfusion studies also indicated that the TNF-α-induced increase in mouse intestinal permeability requires ERK1/2-dependent activation of Elk-1. These studies provide novel insight into the cellular and molecular processes that regulate the TNF-α-induced increase in intestinal epithelial tight junction permeability.

FXR-induced secretion of FGF15/19 inhibits CYP27 expression in cholangiocytes through p38 kinase pathway

Cholangiocytes, bile duct lining cells, actively adjust the amount of cholesterol and bile acids in bile through expression of enzymes and channels involved in transportation and metabolism of the cholesterol and bile acids. Herein, we report molecular mechanisms regulating bile acid biosynthesis in cholangiocytes. Among the cytochrome p450 (Cyp) enzymes involved in bile acid biosynthesis, sterol 27-hydroxylase (Cyp27) that is the rate-limiting enzyme for the acidic pathway of bile acid biosynthesis expressed in cholangiocytes. Expression of other Cyp enzymes for the basic bile acid biosynthesis was hardly detected. The Cyp27 expression was negatively regulated by a hydrophobic bile acid through farnesoid X receptor (FXR), a nuclear receptor activated by bile acid ligands. Activated FXR exerted the negative effects by inducing an expression of fibroblast growth factor 15/19 (FGF15/19). Similar to its repressive function against cholesterol 7α-hydroxylase (Cyp7a1) expression in hepatocytes, secreted FGF15/19 triggered Cyp27 repression in cholangiocytes through interaction with its cognate receptor fibroblast growth factor receptor 4 (FGFR4). The involvements of FXR and FGFR4 for the bile acid-induced Cyp27 repression were confirmed in vivo using knockout mouse models. Different from the signaling in hepatocytes, wherein the FGF15/19-induced repression signaling is mediated by c-Jun N-terminal kinase (JNK), FGF15/19-induced Cyp27 repression in cholangiocytes was mediated by p38 kinase. Thus, the results collectively suggest that cholangiocytes may be able to actively regulate bile acid biosynthesis in cholangiocytes and even hepatocyte by secreting FGF15/19. We suggest the presence of cholangiocyte-mediated intrahepatic feedback loop in addition to the enterohepatic feedback loop against bile acid biosynthesis in the liver.

Mitogen‐activated protein kinase p38 and retinoblastoma protein signalling is required for DNA damage‐mediated formation of senescence‐associated heterochromatic foci in tumour cells

DNA-damaging agents are able to induce irreversible cell growth arrest and senescence in some types of tumour cells, thus contributing to the static feature of cancer. However, senescent tumour cells may re-enter the cell cycle, leading to tumour relapse. Understanding the mechanisms that control the viability of senescent cells may be critical for tumour suppression. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which contribute to the stability of the senescent state. However, it is unclear whether SAHF formation is universal in tumour cells. We report that the DNA-damaging agents doxorubicin and 7-ethyl-10-hydroxycamptothecin were able to induce the formation of SAHF in some tumour cell types, and this induction was accompanied by activation of the retinoblastoma protein pathway. By contrast, tumour cells in which the retinoblastoma protein pathway could not be activated by doxorubicin or 7-ethyl-10-hydroxycamptothecin failed to form SAHF. In parallel, tumour cells with deficient retinoblastoma protein were also unable to form SAHF. In addition, we show that the mitogen-activated protein kinase p38 pathway was involved in tumour cell SAHF formation in response to doxorubicin and 7-ethyl-10-hydroxycamptothecin. Furthermore, HMG box transcription factor 1 (HBP1), a downstream target of the mitogen-activated protein kinase p38-mediated senescence pathway, was required for SAHF formation. Taken together, the results of the present study highlight the roles of the mitogen-activated protein kinase p38/retinoblastoma protein pathway in tumour cell SAHF formation in response to DNA-damaging agents, and provide new insights into the mechanisms of DNA damage-mediated tumour suppression.

Thymoquinone-induced reactive oxygen species causes apoptosis of chondrocytes via PI3K/Akt and p38kinase pathway

Thymoquinone (TQ), a bioactive ingredient of the volatile oil of black seed (Nigella sativa), has been shown to possess anti-neoplastic and anti-inflammatory effects on a variety of tumours. However, the precise mechanism of action is not clear in normal cells such as primary chondrocytes. So, we have investigated the effects of TQ on the apoptosis of chondrocytes with a focus on reactive oxygen species (ROS) production. In in vitro experiments, chondrocytes were cultured with increasing concentrations of TQ for 24 h or with 20 µmol/L TQ for the indicated time periods, and various experiments were performed to detect the apoptotic effects caused by TQ. The results showed that TQ significantly increases apoptosis. Apoptosis was dose- and time-dependently expressed, and the generation of ROS also dramatically increased in a dose-dependent manner. Pretreatment of N-acetyl-L-cysteine (NAC), an inhibitor of ROS, inhibited both TQ-induced apoptosis and ROS generation. Also, TQ up-regulated phosphorylation of phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinases ([MAPKs] p38kinase, ERK-1/-2, and JNKinase), and these effects were prevented by pretreatment of NAC. However, pretreatment with inhibitors of PI3K/Akt and MAPKs did not inhibit TQ-caused ROS generation. Among the inhibitors of PI3K/Akt, p38kinase, ERK-1/-2, and JNKinase, pretreatment with LY294002 and SB203580 abolished TQ-induced apoptosis, but PD98059 and SP600125 did not have any effect on TQ-caused apoptosis. These findings suggest that TQ-induced ROS generation regulates apoptosis by modulating PI3K/Akt and p38kinase pathways.

Mechanism of IL-1β Modulation of Intestinal Epithelial Barrier Involves p38 Kinase and Activating Transcription Factor-2 Activation

The defective intestinal epithelial tight junction (TJ) barrier has been postulated to be an important pathogenic factor contributing to intestinal inflammation. It has been shown that the proinflammatory cytokine IL-1β causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of the p38 kinase pathway and the molecular processes involved. In these studies, the in vitro intestinal epithelial model system (Caco-2 monolayers) was used to delineate the cellular and molecular mechanisms, and a complementary in vivo mouse model system (intestinal perfusion) was used to assess the in vivo relevance of the in vitro findings. Our data indicated that the IL-1β increase in Caco-2 TJ permeability correlated with an activation of p38 kinase. The activation of p38 kinase caused phosphorylation and activation of p38 kinase substrate, activating transcription factor (ATF)-2. The activated ATF-2 translocated to the nucleus where it attached to its binding motif on the myosin L chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. Small interfering RNA induced silencing of ATF-2, or mutation of the ATF-2 binding motif prevented the activation of MLCK promoter and MLCK mRNA transcription. Additionally, in vivo intestinal perfusion studies also indicated that the IL-1β increase in mouse intestinal permeability required p38 kinase-dependent activation of ATF-2. In conclusion, these studies show that the IL-1β-induced increase in intestinal TJ permeability in vitro and in vivo was regulated by p38 kinase activation of ATF-2 and by ATF-2 regulation of MLCK gene activity.

Protective effect of esculentoside A on lipopolysaccharide-induced acute lung injury in mice

BackgroundEsculentoside A (EsA) is a saponin isolated from the Chinese herb Phytolacca esculenta. In our study, we sought to investigate the protective effects of EsA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Materials and methodsTo determine the effects of EsA on the reduction of histopathologic changes in mice with ALI, inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung wet-to-dry weight ratio were measured in LPS-challenged mice, and lung histopathologic changes observed via paraffin section were assessed. Next, cytokine production induced by LPS in BALF was measured by enzyme-linked immunosorbent assay. To further study the mechanism of EsA protective effects on ALI, IκBa, p38, and extracellular signal receptor-activated kinase pathways were investigated in lung tissue of mice with ALI. ResultsIn the present investigation, EsA showed marked effects by reducing inflammatory infiltration, thickening of the alveolar wall, and pulmonary congestion. Levels of tumor necrosis factor α and interleukin 6 elevated by LPS were significantly decreased in BALF in EsA-pretreated ALI model. Furthermore, EsA significantly suppressed phosphorylation of IκBa, p38, and extracellular signal receptor-activated kinase. ConclusionsTaken together, our results suggest that EsA suppressed inflammatory responses in LPS-induced ALI through inhibition of the nuclear factor kappa B and mitogen activated protein kinase signaling pathways. EsA may be a promising potential preventive agent for ALI treatment.

Antagonizing Effects and Mechanisms of Afzelin against UVB-Induced Cell Damage

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes, resulting in skin inflammation, photoaging, and photocarcinogenesis. Adequate protection of skin against the harmful effects of UV irradiation is essential. Therefore, in this study, we investigated the protective effects of afzelin, one of the flavonoids, against UV irradiation in human keratinocytes and epidermal equivalent models. Spectrophotometric measurements revealed that the afzelin extinction maxima were in the UVB and UVA range, and UV transmission below 376 nm was <10%, indicating UV-absorbing activity of afzelin. In the phototoxicity assay using the 3T3 NRU phototoxicity test (3T3-NRU-PT), afzelin presented a tendency to no phototoxic potential. In addition, in order to investigate cellular functions of afzelin itself, cells were treated with afzelin after UVB irradiation. In human keratinocyte, afzelin effectively inhibited the UVB-mediated increase in lipid peroxidation and the formation of cyclobutane pyrimidine dimers. Afzelin also inhibited UVB-induced cell death in human keratinocytes by inhibiting intrinsic apoptotic signaling. Furthermore, afzelin showed inhibitory effects on UVB-induced release of pro-inflammatory mediators such as interleukin-6, tumor necrosis factor-α, and prostaglandin-E2 in human keratinocytes by interfering with the p38 kinase pathway. Using an epidermal equivalent model exposed to UVB radiation, anti-apoptotic activity of afzelin was also confirmed together with a photoprotective effect at the morphological level. Taken together, our results suggest that afzelin has several cellular activities such as DNA-protective, antioxidant, and anti-inflammatory as well as UV-absorbing activity and may protect human skin from UVB-induced damage by a combination of UV-absorbing and cellular activities.

Dimerumic acid attenuates receptor for advanced glycation endproducts signal to inhibit inflammation and diabetes mediated by Nrf2 activation and promotes methylglyoxal metabolism into d-lactic acid

This study was designed to evaluate the effects of dimerumic acid (DMA) on receptor for advanced glycation endproducts (RAGE) signal activation and THP-1 monocyte inflammation treated with S100b, a specific ligand of RAGE. We found that DMA inhibited inflammatory cytokine production via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and alleviated oxidative stress through attenuation of p47phox translocation to the membrane of S100b-treated THP-1 monocytes. We found that DMA activated Nrf2 mediated by the p38 kinase pathway in THP-1 monocytes. However, anti-inflammatory activity of DMA was attenuated by Nrf2 siRNA treatment. In an animal model, methylglyoxal (MG; 200mg/kg bw) was chosen to induce diabetes in Balb/C mice (6 weeks) in this work. The in vivo verification of anti-inflammation in peripheral blood mononuclear cells by DMA treatment was confirmed by tumor necrosis factor-α and interleukin-1β measurements. Oral glucose tolerance test, insulin tolerance test, hyperinsulinemia, and hyperglycemia were improved in MG-treated mice by DMA treatment and these effects were greater than those of silymarin and N-acetylcysteine. Furthermore, DMA increased hepatic glyoxalase mRNA and glutathione mediated by Nrf2 activation to metabolize MG into d-lactic acid, thereby reducing serum and hepatic AGE levels and suppressing inflammatory factor generation in MG-treated mice. However, DMA did not exert the antiglycation activity in MG–bovine serum albumin incubation. Taken together, the results indicate that DMA is a novel antioxidant and Nrf2 activator that lowers AGE levels and may prove to be an effective treatment for diabetes.

Resveratrol induces MMP-9 and cell migration via the p38 kinase and PI-3K pathways in HT1080 human fibrosarcoma cells

Trans-3,4',5-trihydroxystilbene (resveratrol) is a grape polyphenol present in various plants, food products, red wine and grapes. Resveratrol has anti-inflammatory, anticarcinogenic, anti-oxidant and anti-aging properties. Matrix metalloproteinases (MMPs) are key enzymes involved in the degradation of the extracellular matrix, and their expression may be regulated in cancer metastasis. In the present study, we aimed to evaluate the effect of resveratrol on MMPs and cell migration, and to understand the mechanism of action in HT1080 human fibrosarcoma cells. We found that resveratrol inhibited HT1080 cell viability at various concentrations as detected by the MTT assay and FACS analysis. However, resveratrol dramatically increased the activation and expression of MMP-9 in a dose-andtime-dependent manner, as determined by gelatin zymography assay and western blot analysis. We also discovered that resveratrol enhanced the migratory ability of HT1080 cells, as determined by the wound healing assay, and decreased the phosphorylation of p38 kinase. Moreover, the Akt kinase was inhibited by resveratrol in the HT1080 cells. The inhibition of p38 and Akt kinases with SB203580 and LY294002 further increased resveratrol-induced MMP-9 as well as cell migration in the HT1080 cells. Our results suggest that resveratrol regulates MMP-9 and migratory abilities through the p38 kinase and PI-3K pathways in HT1080 human fibrosarcoma cells.

Gallotannin causes differentiation and inflammation via ERK-1/-2 and p38 kinase pathways in rabbit articular chondrocytes

Gallotannin (GT) is a type of tannic acid, derived from plant polyphenols, that is an agonist of plant defense mechanisms. Tannins have two types of structure; condensed tannins are a polymer of flavonoid units, while hydrolysable tannins are carbohydrates. GT is used in medical agents for its anti‑viral, anti‑bacterial and anti‑parasitic effects. The present study investigated the effects of GT on differentiation and inflammation in rabbit articular chondrocytes. GT caused differentiation and inflammatory responses in the rabbit articular chondrocytes. GT treatment induced the expression of type Ⅱ collagen and sulfated proteoglycan, as determined by western blot analysis and alcian blue staining, respectively, in a dose‑ and time‑dependent manner. Additionally, treatment with GT increased the expression of cyclooxygenase‑2 (COX‑2) and the production of prostaglandin E2 (PGE2), as determined by western blot analysis and PGE2 assay. GT was confirmed to cause phosphorylation of ERK‑1/‑2 and p38 kinase. Inhibition of pERK with PD98059 promoted GT‑induced type Ⅱ collagen expression. However, the inhibition of p38 with SB203580 suppressed GT‑induced COX‑2 expression and PGE2 production. In summary, the results demonstrated that GT‑induced ERK‑1/‑2 and p38 kinase have opposite effects on differentiation and inflammation in rabbit articular chondrocytes.

Gallotannin regulates apoptosis and COX-2 expression via Akt and p38kinase pathway in human lung cancer cell line, A549

Gallotannin (GT) is derived from plant poly phenol and is associated with biological actions in a wide range of cells. In this study, we evaluated the effect of GT on apoptosis and cyclooxygenase-2 (COX-2) expression and attempted to shed light on the mechanism of action in A549 human lung carcinoma cells. We found that GT dramatically induced apoptosis as demonstrated by expression of p53 and active caspase-3 via western blot analysis and fragmented DNA as detected by DNA fragmentation and DAPI staining. We also observed that GT significantly causes COX-2 expression in a dose-dependent manner determined by western blot analysis. Phosphorylation of Akt and p38 was considerably increased by GT in A549 human lung carcinoma cells. Inhibition of Akt and p38kinase with LY294002 or SB203580 suppressed GT-induced apoptosis and COX-2 expression. Furthermore, we have shown that prevention of COX-2 with NS398 or indomethacin does not any effects on apoptosis induced by GT. Taken together, our present results suggest that GT regulates apoptosis and COX-2 expression through Akt and p38kinase pathway in A549, human lung carcinoma cells.

2′-Nitroflavone induces apoptosis and modulates mitogen-activated protein kinase pathways in human leukaemia cells

The cytotoxic activity of 2'-nitroflavone was evaluated in different haematological cancer cell lines and its mechanism of action was further studied in HL-60 cells. 2'-Nitroflavone arrested the cell cycle at the G(2)/M phase and induced an apoptotic response characterized by an increase in the sub-G1 fraction of cells, a typical DNA ladder fragmentation, chromatin condensation and the detection of cells stained with Annexin V. Apoptosis was dependent on the activation of at least caspase-8, caspase-9 and caspase-3. The involvement of the death receptor pathway was indicated by the upregulation of both the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5). We also showed that 2'-nitroflavone increased the expression levels of Bax and induced the release of cytochrome C to cytosol, suggesting the participation of the mitochondria-dependent pathway. When mitogen-activated protein kinases pathways were studied, it was found that p38 and c-Jun NH(2)-terminal kinase (JNK) pathways were activated by 2'-nitroflavone in HL-60 cells, whereas the phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 decreased significantly. In addition, whereas both pharmacological inhibition of JNK and downregulation of JNK expression by RNA interference reduced the nitroflavone growth-inhibitory activity and the apoptotic effect, contrasting results were obtained when the ERK1/2 pathway was inhibited, and no effect was observed in the presence of a specific inhibitor of p38 mitogen-activated protein kinase. These findings show for the first time the antitumour action of 2'-nitroflavone in haematological cancer cell lines and suggest that both JNK and ERK1/2 cascades are involved in the apoptotic response induced by 2'-nitroflavone in HL-60 cells.

Resveratrol Upregulates Nrf2 Expression To Attenuate Methylglyoxal-Induced Insulin Resistance in Hep G2 Cells

Oxidative stress can result in insulin resistance, a primary cause of type-2 diabetes. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite generated during glucose metabolism, has also been confirmed to cause pancreatic injury and induce inflammation, thereby resulting in insulin resistance. Recently, resveratrol has been reported to exert antioxidant properties, protecting cells from the generation of reactive oxygen species (ROS). The aim of this study was to evaluate resveratrol activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced insulin resistance in Hep G2 cells. Therefore, the molecular signaling events affecting resveratrol-mediated heme oxygenase-1 (HO-1) and glyoxalase expression levels were further investigated in this study. Our findings indicated that resveratrol activated the extracellular signal-regulated kinase (ERK) pathway but not the p38 or c-Jun N-terminal kinase (JNK) pathways, subsequently leading to Nrf2 nuclear translocation and elevation of HO-1 and glyoxalase expression levels. Moreover, resveratrol significantly elevated glucose uptake and protected against MG-induced insulin resistance in Hep G2 cells. In contrast, depletion of Nrf2 by small interfering RNA (si-RNA) resulted in the abrogation of HO-1 and glyoxalase expression in the MG-treated resveratrol group in Hep G2 cells. Administration of an appropriate chemopreventive agent, such as resveratrol, may be an alternative strategy for protecting against MG-induced diabetes.

Thymoquinone (TQ) regulates cyclooxygenase-2 expression and prostaglandin E2 production through PI3kinase (PI3K)/p38 kinase pathway in human breast cancer cell line, MDA-MB-231

Thymoquinone (TQ), a drug extracted from the black seeds of Nigella sativa, has been shown to exhibit anti-inflammatory, anti-oxidant, and anti-neoplastic effects in numerous cancer cells. The effects of TQ on cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in MDA-MB-231, however, remain poorly understood. Western blot analysis and immunofluorescence staining were performed to study the expression levels of inflammation regulatory proteins in MDA-MB-231. PGE2 assay was conducted to explore the TQ-induced production of PGE2. In this study, we investigated the effects of TQ on COX-2 expression and PGE2 production in MDA-MB-231. TQ significantly induced COX-2 expression and increased PGE2 production in a dose-dependent manner, as determined by a Western blot analysis and PGE2 assay. Furthermore, the activation of Akt and p38 kinase, respectively, was up-regulated in TQ treated cells. Inhibition of p38 kinase with SB203580 and PI3kinase (PI3K) with LY294002 abolished TQ-caused COX-2 expression and decreased PGE2 production. These results collectively demonstrate that TQ effectively modulates COX-2 expression and PGE2 production via PI3K and p38 kinase pathways in the human breast cancer cell line MDA-MB-231.