Selective MEK Inhibitor Research Articles

Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.

743 Rdea119, a Potent and Highly Selective MEK Inhibitor Ameliorates Murine Colitis

743 Rdea119, a Potent and Highly Selective MEK Inhibitor Ameliorates Murine Colitis

Akt1 inhibition by RNA interference sensitizes human non‐small cell lung cancer cells to cisplatin

Akt/protein kinase B signaling is very important for cancer cell survival and growth when cells are exposed to various apoptotic stimuli. Akt is constitutively activated in NSCLC cells and is a potential target for enhancing the cytotoxicity of chemotherapeutic agents in treatment of NSCLC. In our study, we investigated whether down-regulating Akt1 using RNAi techniques can enhance sensitivity to cisplatin in NSCLC cells. An siRNA targeting Akt1 significantly decreased the protein level of Akt1 and the activity of ERK. Treatment of these cells with 20 muM cisplatin increased apoptotic cell death approximately 2.6-fold compared to cells transfected with a scrambled siRNA. While Akt activity was slightly reduced, ERK activity was greatly increased in cells treated with cisplatin alone. Pretreatment of these cells with the selective MEK inhibitor U0126 effectively reduced the level of cisplatin-induced apoptosis. These results imply that cisplatin-induced MEK/ERK activation appears to mediate apoptotic cell death, but that constitutively activated Akt1 and/or ERK pathway may mediate resistance to cisplatin in NSCLC cells. Taken together, our data demonstrate that down-regulation of Akt1 using RNAi enhances the chemosensitivity of NSCLC cells to cisplatin.

Comparative Gene Profiling of Acute Myeloid Leukemia (AML) and Malignant Melanoma (MEL) Cell Lines Exposed to the MEK Inhibitor PD0325901 Reveals Common Effectors of the MEK/ERK Kinase Module.

The MEK/ERK kinase module is frequently deregulated in human malignancies, including AML and MEL, and represents a promising therapeutic target. We have recently demonstrated that PD0325901, a selective MEK inhibitor, potently inhibits the growth of both AML and MEL cell lines (IC50 5–20 nM), through a combination of inhibition of cell cycle progression and induction of apoptosis. In order to identify functionally relevant targets of the activity of the MEK/ERK kinase module, we compared the gene expression profiles of the PD0325901-sensitive cell lines OCI-AML3 (AML, harboring a NPM gene mutation) and M14 (MEL, harboring a BRAF mutation), after exposure to vehicle control or 10 nM PD0325901 for 6 and 24h, using the Affymetrix U133A 2.0 and U133 Plus 2.0 gene chips, respectively. All analyses were conducted using the dChip software. In both models, the unsupervised approach showed a tight clustering that was both treatment- and time-dependent; unsupervised and supervised analyses highlighted that the greatest PD0325901-induced variations in gene expression were observed at 24h and 6h in the OCI-AML3 and M14 models, respectively. Comparison of the results obtained in the OCI-AML3 and M14 cell lines showed that 5 genes were consistently modulated after 6h, including CDC25A, cyclin G2, and DUSP6. Cyclin G2 and DUSP6 were also modulated after 24h, together with cyclin D1, TIMP3, and SEMA6A. On the contrary, MAFB, selected in both models, displayed a discordant behavior, being down-regulated upon treatment in OCI-AML3 and up-regulated in M14: this is consistent with the role of MAFB as an important regulator of hematopoiesis, while it does not appear to play a role in melanogenesis. Given the ability of PD0325901 to cause apoptosis, partly through induction of mitochondrial dysfunction in both AML and MEL models, we next investigated whether MEK inhibition specifically modulated the expression of genes related to mitochondrial homeostasis: also in this specific context, the effects were more rapidly induced in M14 than in OCI-AML3, with the greatest changes observed within 6h in the M14 cell line. Two genes were down-regulated after 24h in both models: PPIF, supposedly involved in apoptosis induction, and GRPEL1, on which little is currently known. In addition to mitochondrion-specific changes in gene expression, expression of the mitochondrial protein CPT1A significantly decreased (2-fold), as assessed by WB, after 6 h of exposure of OCI-AML3 cells to 10 nM PD0325901, under which conditions ß-oxidation was also suppressed by the drug. Overall, these results indicate that MEK inhibition by PD0325901 results in the modulation of the expression of a small number of genes that are common to ontogenetically distant tumor models, thereby representing attractive candidates for a shared, functionally relevant, MAPK activation signature; moreover, MEK inhibition results in profound intracellular metabolic changes that might importantly contribute to the anti-neoplastic effect of PD0325901 in AML and, possibly, in other tumor models. MM and SC equally contributed to the work presented.

Central role of the MEK/ERK MAP kinase pathway in a mouse model of rheumatoid arthritis: Potential proinflammatory mechanisms

To evaluate the role of the MEK/ERK MAP kinase pathway in murine collagen-induced arthritis (CIA) using the selective MEK inhibitor PD184352. We examined the effects of the inhibitor in cytokine-stimulated synovial fibroblasts and in cytokine-induced arthritis in rabbits to investigate its antiinflammatory mechanisms. Murine CIA was used to assess the effects of the selective MEK inhibitor on paw edema, clinical scores, weight loss, histopathologic features, and joint levels of p-ERK. Western blotting and immunohistochemistry techniques were used to assess p-ERK in human and rabbit synovial fibroblasts and synovial tissue from rheumatoid arthritis (RA) patients. Interleukin-1alpha (IL-1alpha)-stimulated stromelysin production in rabbit synovial fibroblasts was assessed by enzyme-linked immunosorbent assay. A rabbit IL-1alpha-induced arthritis model was used to assess the effects of the inhibitor on IL-1alpha-induced MEK activity, stromelysin production, and cartilage degradation. In the CIA model, PD184352 inhibited paw edema and clinical arthritis scores in a dose-dependent manner. Disease-induced weight loss and histopathologic changes were also significantly improved by treatment. Inhibition of disease-induced p-ERK levels in the joints was seen with the inhibitor. Levels of p-ERK in the synovium were higher in RA patients than in normal individuals. PD184352 reduced IL-1alpha-induced p-ERK levels in human RA synovial fibroblasts. The production of p-ERK and stromelysin was also inhibited in IL-1alpha-stimulated rabbit synovial fibroblasts. We observed IL-1alpha-induced p-ERK in the synovial lining, subsynovial vasculature, and articular chondrocytes. IL-1alpha-induced stromelysin production and proteoglycan loss from the articular cartilage were reduced by PD184352. These data demonstrate the inhibition of murine CIA by PD184352, support the hypothesis that antiinflammatory activity contributes to the mechanism of action of the inhibitor, and suggest that a selective inhibitor may effectively treat RA and other inflammatory disorders.

Potentiation of the antitumor effects of both selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors in human hepatic cancer cells by inhibition of the MEK/ERK pathway

The molecular mechanisms behind the anti-neoplastic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are not completely understood and cannot be explained by the inhibition of the cyclooxygenase (COX) enzymes COX-1 and COX-2 alone. We previously reported that both the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor CAY10404 exhibit anti-tumor effects in human hepatoma cells. NSAID inhibitors have many COX-independent actions and, among others, the mitogen-activated protein kinase (MAPK) pathways are targets for NSAIDs. Here, we examined the role of MEK/ERK1/2 signaling in the anti-neoplastic effects of both selective COX-1 and COX-2 inhibitors in two human hepatoma cell lines. Treatment of hepatoma cells with the selective COX-1 inhibitor SC-560, as well as with the selective COX-2 inhibitor CAY10404, was associated with activation of ERK1/2 in a time- and dose-dependent manner. Treatment with COX-1 and COX-2 inhibitors in the presence of the selective MEK1/2 inhibitor U0126 effectively suppressed ERK1/2 activation and combinations of either SC-560 or CAY10404 with U0126 resulted in synergistic effects on cell growth inhibition and induction of apoptosis. In HuH-6 hepatoma cells the combination-induced apoptosis was associated with caspase-9 and -3 activation, PARP cleavage, release of cytochrome c from the mitochondria into the cytosol and down-regulation of survivin and β-catenin levels. In conclusion, our study showed that growth inhibitory concentrations of selective COX-1 and COX-2 inhibitors increased ERK1/2 phosphorylation in hepatoma cells, and that inhibition of the MEK/ERK signaling pathway potentiates the antitumor activity of both types of inhibitors. Therefore, our results provide preclinical support for a combined chemotherapeutic approach with selective NSAIDs and MEK inhibitors for the treatment of hepatocellular carcinoma.

Geniposide, a novel agonist for GLP-1 receptor, prevents PC12 cells from oxidative damage via MAP kinase pathway

Alzheimer's disease (AD) is the most common form of dementia. Glucagon-like peptide-1 (GLP-1) gives a new genre in therapeutic targets for intervention in AD with its neurotrophic and neuroprotective functions. In previous work, we identified that geniposide is a novel agonist for GLP-1 receptor, which shows neurotrophic characteristics to induce the neuronal differentiation of PC12 cells. The aim of this study is to determine whether geniposide prevents neurons from oxidative damage, and to explore its signaling pathways. The results demonstrated that geniposide increased the expression of anti-apoptotic proteins, including Bcl-2 and heme oxygenase-1 (HO-1), to antagonize the oxidative damage in PC12 cells induced by hydrogen peroxide. LY294002 (a PI3K inhibitor) inhibited the effect of geniposide increasing of Bcl-2 level by activation of MAPK, MEK and c-Raf phosphorylation in hydrogen peroxide treated PC12 cells. U0126 (a selective inhibitor of MEK) also attenuated the enhancement of geniposide on Bcl-2 level by inhibiting the phosphorylation of p90RSK in the hydrogen peroxide treated PC12 cells. All these data demonstrate that geniposide, an agonist for GLP-1 receptor, regulates expression of anti-oxidative proteins including HO-1 and Bcl-2 by activating the transcriptor of p90RSK via MAPK signaling pathway in PC12 cells.

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.

Common Leukemic Signaling Pathways Identified by Comparative Analysis of GM-CSF and FLT3 Activated Receptor Mutations.

As a strategy to identify key targets in myeloid leukemia we examined directly the overlap in signaling and downstream events induced by several activated receptor mutants associated with AML. We compared the events induced by a leukemic mutant (V449E) of the GM-CSF receptor with events associated with the activating FLT3 internal tandem duplication mutant (FLT3-ITD) and the D835Y kinase domain mutant (FLT3-TKD). Receptor mutants were introduced by retroviral transduction into the FDB-1 bi-potential myeloid cell line model (McCormack MP et al., Blood. 95:120–127, 2000). We used biochemical analysis and pathway inhibitors to demonstrate that the GMR V449E mutant selectively activates the JAK2-STAT5 and p44/42 MAPK pathways which are central to the ability of this mutant to confer continued growth and arrested differentiation in the absence of growth factor. Like the GMR V449E mutant, the FLT3-ITD and -TKD mutants arrest granulocyte-macrophage differentiation and support continued growth in the absence of added growth factor. For both of these mutants we also observed constitutive JAK2 and STAT5 phosphorylation and sensitivity to a selective JAK2 inhibitor (JAK2 Inhibitor II, Merck) suggesting a potential key role for JAK2 in signalling from FLT3 activating mutants. Both FLT3 mutants activated the p44/42 MAPK pathway, resulted in Akt phosphorylation and were sensitive to PI3-kinase and MEK inhibitors. As activation of the p44/42 MAPK pathway is common to all mutants, and has recently been associated with suppressed differentiation in AML (Radomska HS et al, J Exp Med. 203:371–381, 2006), we used the selective MEK inhibitor, U0126 (Merck), to investigate the contribution of this pathway to maintenance of the differentiation block. Treatment of factor-independent FDB-1 cells expressing the GMR V449E mutant, or either FLT3 mutant, with U0126 for 48 hours induced morphological differentiation suggesting that the sustained activation of p44/42 MAPK contributes to maintaining the arrest in myeloid differentiation. To explore further the overlapping nature of signalling by the GMRV449E and FLT3-ITD activated mutants we utilised the Wilcoxan rank sum test to perform gene-set enrichment analysis. Analysis of 119 FLT3-ITD associated genes (126 probes) (Mizuki M et al, Blood 101: 3164–3173, 2003) revealed that this gene-set shows evidence of differential expression in response to GMR V449E signaling (p=2.8x10−7). In particular, we identify the tumor suppressor gene, Gadd45α, as a common gene, down-regulated in response to signalling via the FLT3-ITD, FLT3-TKD and GMR V449E activated receptors. Analysis of AML gene expression data (Valk PJ et al, New Engl. J. Med : 350:1617–1628, 2004) indicates that activation of Gadd45α maybe suppressed by multiple pathways in AML blasts.

MEK1 Blockade Sensitizes Multiple Myeloma Tumor Cells to Arsenic Trioxide (ATO)-Induced Apoptosis: A Functional and Molecular Study.

Preliminary data from clinical studies indicate that ATO has clinical activity as a single agent in human multiple myeloma (MM), and combination therapies are being investigated. We recently reported that PD184352 (PD) ( Pfizer), a highly selective inhibitor of MEK1, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines and in Acute Myelogenous Leukemia via multiple intrinsic apoptotic pathways activation. The aim of this study was to investigate whether the combined treatment with PD and ATO has cytotoxic effects on MM cells. We first analyzed the pharmacologic interactions between PD (2 μM) and ATO (1–2 μM) using a fixed-ratio experimental design on 7 human myeloma cell lines (HMCL) with varying p53 status (RPMI 8226, U266, XG-1, XG-6, JJN3, HS-SULTAN, NCI-H929) and found that the combined treatment resulted in the synergistic (Combination Index <1.0) induction of apoptosis in NCI-H929, XG-1, XG-6, RPMI 8226, SULTAN and JJN3 HMCL. Conversely, the combination of PD plus ATO had a slight antagonistic effect on apoptosis induction in U266 HMCL (Combination Index >1). Moreover, PD plus ATO-induced cytotoxicity on HMCL was maintained also in presence of IL-6 (20 ng/ml) or in a co-culture system with bone marrow stromal cells. Similarly to HMCL we found that the treatment with PD significantly enhanced the apoptosis of fresh purified MM cells induced by ATO (P< .01) in 8 out of 11 patients of MM analyzed. Conversely, PD treatment partially attenuated (n=2) or did not affect (n=1) the ATO cytotoxicity in normal bone marrow B cells. To investigate the molecular mechanisms by which PD plus ATO induced MM cell apoptosis first we compared the effect on caspase activation in myeloma cells expressing wild type (wt) p53 or mutated p53. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in PD plus ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase-9 and -3 in dual treated cells expressing wt p53. In addition, PD plus ATO induced a p53-dependent up regulation of Puma, Bax and Bak in HMCL with wt p53, and p53-specific siRNA significantly (P< .01) reduced the induction of apoptosis in dual treated cells. The caspase-8-mediated proteolytic activation of BID, a key protein involved in the cross-talk between the intrinsic and extrinsic apoptotic pathways, closely correlated with the caspase-9 activation and loss of mitochondrial membrane potential observed in dual treated HMCL with mutated p53. Finally, in the responsive HMCL both with wt or mutated p53, the combined treatment increased the level of the pro-apoptotic Bim (PD-mediated) and decreased its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated) causing an imbalance between these proteins that positively influenced the pro-apoptotic efficacy of the combination. In conclusion, our data indicate that the disruption of MEK pathway potentiates the apoptotic effect of ATO in MM cells through the activation of both extrinsic and intrinsic (caspase-8-BID-mediated) pathways in HMCL with mutated p53 or through the primarily activation of intrinsic pathway in HMCL with wt p53, with the positive contribution of Bim pathway. These findings suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of MM.

Neurotrophic property of geniposide for inducing the neuronal differentiation of PC12 cells

The emerging data show that the insulinotrophic hormone glucagon-like peptide-1(GLP-1) and its agonist extendin-4 have neurotrophic function to inducing neuronal differentiation of PC12 cells and prevent neurons damage challenged by oxidative stress. Here, with the model of high throughput screen for GLP-1 receptor agonists, we screen and identify that geniposide is a novel agonist for GLP-1 receptor. Furthermore, geniposide induces the neuronal differentiation of PC12 cells with resulting neurites outgrowth; we also observe an increase in expression of growth-associated protein-43. U0126, a selective MEK inhibitor, prevents neurites out growth and phosphorylation of mitogen-activated kinase proteins in PC12 cells induced by geniposide. All these results show that activation of GLP-1 receptor by geniposide to induce the neuronal differentiation of PC12 cells involves in MAPK signaling cascade.

Role of phosphorylation of ERK in induction and maintenance of LTP of the C‐fiber evoked field potentials in spinal dorsal horn

Previous works have shown that activation of extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway is essential for long-term potentiation (LTP) in hippocampus. In the present study, the role of the ERK/CREB pathway in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathologic pain, was investigated in adult rats. Western blotting analysis showed that the protein level of phosphorylated ERK (p-ERK) in ipsilateral spinal dorsal horn was transiently increased after LTP induction, starting at 15 min and returning to control at 60 min after tetanic stimulation and that the protein level of p-CREB increased at 30 min, persisting for at least 3 hr after LTP induction. Double immunofluorescence staining showed that p-ERK and p-CREB were only located in neurons but not in glial cells in the spinal dorsal horn after LTP induction. More importantly, we found that spinal application of PD 98059 (100 microM), a selective MEK inhibitor, at 30 min before tetanic stimulation blocked LTP induction and prevented the increase in p-ERK and p-CREB in spinal dorsal horn. When applied 15 min after LTP induction, PD98059 reversed established LTP. The drug, however, did not affect the spinal LTP, when applied at 30 min after LTP. Our results suggested that activation of ERK/CREB pathway in spinal dorsal neurons is necessary for induction and maintenance of long-term potentiation of the C-fiber evoked field potentials.

Long-Term Potentiation Is Mediated by Multiple Kinase Cascades Involving CaMKII or Either PKA or p42/44 MAPK in the Adult Rat Dentate Gyrus In Vitro

The induction of NMDA-receptor-dependent long-term potentiation (LTP) in adult CA1 is contingent on activation of Ca/calmodulin-dependent protein kinase II (CaMKII). However, little is known about kinase mediation of LTP in the dentate gyrus. In the present study, the involvement of the kinases CaMKII, PKA, and MAPK in the induction of LTP was studied in the dentate gyrus of adult rats. Individual application of selective inhibitors of CaMKII, MEK, or PKA did not inhibit induction of LTP. In contrast, coapplication of a CaMKII inhibitor with either a PKA or MEK inhibitor resulted in a strong block of LTP. Induction of LTP was blocked by the coapplication of the inhibitors CaMKII and PKA or MEK, both when they were applied 1 h before the induction stimulus and also when they were applied after the induction stimulus. Thus LTP is mediated by either of two parallel cascades, one involving CaMKII and the other PKA or MAPK. Moreover, these cascades are active for a certain period after the induction stimulus.

Green tea polyphenol stimulates cancer preventive effects of celecoxib in human lung cancer cells by upregulation of GADD153 gene

To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (-)-epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase-2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage-inducible 153 (GADD153) gene, in human lung cancer cell line PC-9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC-9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21(WAF1) and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC-9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K-1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC-9 cells in combination with N-(4-hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC-9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG.

Inhibition of angiotensin II‐ and endothelin‐1‐stimulated proliferation by selective MEK inhibitor in cultured rabbit gingival fibroblasts†

We investigated the implication of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the proliferation stimulated by angiotensin II (Ang II) and endothelin-1 (ET-1) in cultured rabbit gingival fibroblasts (CRGF). Ang II stimulated activation of ERK1/2 and the activation was inhibited by CV-11974, an AT1 antagonist, and saralasin, an AT1/AT2 antagonist, but not by PD123,319, an AT2 antagonist in the CRGF. Ang II-stimulated proliferation was inhibited by PD98059 or U0126, selective MEK inhibitors. Furthermore, ET-1 stimulated proliferation via G-protein-coupled ETA receptors, which were identified by Western blot analysis of membrane protein from the CRGF. ET-1 also stimulated activation of ERK1/2 and the activation was inhibited by BQ-123, an ETA inhibitor, and TAK044, an ETA/ETB inhibitor, but not by BQ-788, an ETB inhibitor. ET-1-stimulated proliferation was inhibited by PD98059 or U0126. These findings suggest that ERK1/2 play a role in the signaling process leading to proliferation stimulated by Ang II and ET-1 via G-protein-coupled receptors, AT1 and ETA in CRGF.

TNF-α is a mediator of the anti-inflammatory response in a human neonatal model of the non-septic shock syndrome

The anti-inflammatory/immunoparalytic phase of the systemic inflammatory response syndrome (SIRS) following major insult (surgery, thermal/traumatic injury) is of major clinical importance in the neonate, during which the risk of infection is particularly great. Here, the mechanisms by which TNF-alpha production is suppressed in response to infection are largely unknown. We questioned whether TNF-alpha itself could be a critical mediator of this suppression. Monocytes, isolated from cord blood (n=3), were treated with LPS (100 ng/ml), TNF-alpha (10 ng/ml, +/- anti-TNF-alpha antibody) for 18 and 36 h. Cells were then restimulated with LPS (Gram -ve) or Pam-3-Cys (Gram +ve) for 24 h. This was also done in the presence of selective inhibitors of MAP kinases p38, MEK and JNK. TNF-alpha, IL-6, IL-10 and IL-8 were quantified by ELISA CD86 and HLA-DR expression were determined flow cytometrically. Cells stimulated with LPS for 24 h produced TNF-alpha (282 pg/ml), IL-10 (1,236 pg/ml), IL-6 (2,694 pg/ml) and IL-8 (2,144 pg/ml). In cells pre-exposed to TNF-alpha for 36 h, there was a significant suppression in TNF-alpha and IL-6 levels (9 and 221 pg/ml, respectively) (P<0.05) with minimal impact on IL-10 (1,206 pg/ml) and IL-8 levels (1,886 pg/ml). A similar effect was seen with Pam-3-Cys with a tenfold decrease in levels of TNF-alpha and IL-6 (86-->8.5 pg/ml and 458-->46 pg/ml, respectively) with no effect on IL-10 and IL-8 levels. Anti-TNF-alpha antibody negated this effect. Inhibition of p38 kinase reversed the TNF-alpha effect. Inhibition of the JNK and MEK kinases had no effect. A reduction in the expression of CD86 and HLA-DR was observed. This ex-vivo model of non-septic SIRS demonstrates that TNF-alpha, released during a major insult, can suppress subsequent monocyte responses to bacterial agents through p38 MAP kinase, making it a potential therapeutic target.

Inducible phosphorylation of cortactin is not necessary for cortactin-mediated actin polymerisation

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. It was originally identified as a substrate for the protein kinase Src; however, the role of tyrosine phosphorylation in the translocation of cortactin to the cell periphery and in the subsequent actin polymerisation is still unclear. Recently, two serine/threonine kinases, Pak1 and Erk, have been implicated in the regulation of cortactin. Therefore, we systematically investigated whether phosphorylation on either tyrosine or serine/threonine residues is necessary for cortactin function. In COS7 cells over-expressing Vav2 or treated with EGF, we could not detect tyrosine phosphorylation, although cortactin was translocated to cell periphery and induced membrane ruffle formation. In addition, the selective MEK inhibitor, PD98059, did not influence in vivo the ability of cortactin to bind to and induce membrane ruffles upon Vav2 over-expression or short-term EGF treatment. Finally, using a constitutively active Pak1 mutant, Pak1 T423E, we showed that Pak1 is not capable of phosphorylating cortactin either in vitro or in COS7 cells. These results suggest that cortactin-mediated actin polymerisation at cell periphery requires only Rac activation but neither tyrosine nor serine/threonine phosphorylation.

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes

1 Bisindolylmaleimide inhibitors of protein kinase C (PKC), such as GF109203X and Ro31-8220, have been used to investigate the roles of PKC isoforms in many cellular processes in cardiac myocytes, but these agents may also inhibit p90RSK activity. 2 In in vitro kinase assays utilising 50 microM [ATP], GF109203X and Ro31-8220 inhibited p90RSK isoforms (IC50 values for inhibition of RSK1, RSK2 and RSK3, respectively, were 610, 310 and 120 nM for GF109203X, and 200, 36 and 5 nM for Ro31-8220) as well as classical and novel PKC isoforms (IC50 values for inhibition of PKCalpha and PKCepsilon, respectively, were 8 and 12 nM for GF109203X, and 4 and 8 nM for Ro31-8220). 3 At physiological [ATP] (5 mM), both GF109203X and Ro31-8220 exhibited reduced potency as inhibitors of RSK2, PKCalpha and PKCepsilon (IC50 values of 7400, 310 and 170 nM, respectively, for GF109203X, and 930, 150 and 140 nM, respectively, for Ro31-8220), with the latter agent retaining its relatively greater potency. 4 To determine the effects of GF109203X and Ro31-8220 on p90RSK activity in cultured adult rat ventricular myocytes (ARVM), phosphorylation of the eukaryotic elongation factor 2 kinase (eEF2K) at Ser366, a known p90RSK target, was used as the index of such activity. Adenoviral expression of a constitutively active form of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) was used to induce PKC-independent p90RSK activation and downstream phosphorylation of eEF2K. 5 eEF2K phosphorylation was abolished by U0126 (1 microM), a selective inhibitor of MEK1, and was significantly reduced by GF109203X at > or =3 microM and by Ro31-8220 at > or =1 microM. At 1 microM, both agents inhibited PMA-induced PKC activity in ARVM. 6 These data show that GF109203X and Ro31-8220 inhibit various isoforms of PKC and p90RSK in vitro and in intact ARVM, with the former agent exhibiting relatively greater selectivity for PKC.

Inhibition effects of par-4 antisense oligodeoxynucleotide on apoptosis of PC12 cell induced by glutamate is mediated by ERK1/2

To investigate the inhibition effects of par-4 antisense oligodeoxynucleotide on apoptosis of PC12 cell induced by glutamate and its signal transduction mechanism. (1) Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (Par-4-AS-ODN) was transfected into PC12 cells before they were treated with glutamate. Mismatch oligodeoxynucleotide (MS-ODN) were also transfected into cells as controls. (2) Morphological observation and the detection of anti-apoptosis effects of par-4-AS-ODN on PC12 cells were done with the Laser Scanning confocal Microscope by double staining the cells with acridine orange/ethidium bromide (AO/EB), addition to with flow cytometry. (3) Western blot was used to detect the protein levels of par-4 and phosphorylated ERK(1/2) at threonine-202 and Tyrosine-204. (1) Par-4-AS-ODN significantly suppressed up-regulation of the par-4 protein levels induced by glutamate in PC12 cells. (2) Par-4-AS-ODN could resist the decrease of phosphorylated ERK(1/2) (Thr202/Tyr204) induced by glutamate in PC12 cells. (3) Par-4 AS-ODN could inhibit apoptosis of PC12 cells induced by glutamate. But its inhibition effect could be eliminated by PD98059, a selective MEK(1) inhibitor which could inhibit phosphorylation of ERK(1/2). Par-4 AS-ODN may inhibit apoptosis of PC12 cells induced by glutamate, and its inhibition effects may be medicated by the activation of ERK(1/2).