Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.
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Cisplatin treatment activates multiple signal transduction pathways, which can lead to several cellular responses including cell cycle arrest, DNA repair, survival, or apoptosis. We investigated the response of the mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun-N-terminal kinase 1 (JNK1), and p38, to cisplatin treatment in the ovarian carcinoma cell line SK-OV-3. Cisplatin caused a late and prolonged induction in a dose-dependent manner of both ERK1/2 and JNK1 activity. ERK1/2 and JNK1 activities continued to increase in magnitude up to 24 h following initiation of cisplatin treatment. In contrast, cisplatin treatment had no effect on p38 activity. Transplatin failed to induce either ERK1/2 or JNK1 at 24 h, which suggests that the activation of these kinases was dependent on cisplatin-specific DNA damage. Treatment with cycloheximide resulted in inhibition of cisplatin-induced ERK1/2 activation, demonstrating that ERK1/2 activity induced by cisplatin was dependent on de novo protein synthesis. Furthermore, inhibition of cisplatin-induced ERK1/2 activity by PD 98059 caused enhanced cisplatin cytotoxicity. Similar enhanced cytotoxic effects of cisplatin were also observed following treatment with PD 98059 in the ovarian carcinoma cell line UCI 101. These observations indicate that ERK1/2 activation induced by cisplatin partially protects cells from cisplatin cytotoxicity. Continued investigation into the mechanism by which the ERK pathway and other signal transduction pathways modulate the response to cisplatin may be helpful in the development of new strategies for improving the therapeutic use of platinum drugs. (+info)
Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils.
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Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA. (+info)
Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids.
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The cholesterol-lowering effects of tangerine peel extract and a mixture of two citrus flavonoids were tested. Male rats were fed a 1 g/100 g high-cholesterol diet for 42 d with supplements of either tangerine-peel extract or a mixture of naringin and hesperidin (0.5 g/100 g) to study the effects of plasma and hepatic lipids, hepatic enzyme activities, and the excretion of fecal neutral sterols. Both the tangerine-peel extract and mixture of two flavonoids significantly lowered the levels (mean +/- SE) of plasma (2.44 +/- 0. 59 and 2.42 +/- 0.31 mmol/L, vs. 3.80 +/- 0.28 mmol/L, P < 0.05), hepatic cholesterol (0.143 +/- 0.017 and 0.131 +/- 0.010 mmol/g vs. 0.181 +/- 0.003 mmol/g, P < 0.05), and hepatic triglycerides (0.069 +/- 0.007 and 0.075 +/- 0.006 mmol/g vs. 0.095 +/- 0.002 mmol/g, P < 0.05) compared to those of the control. The 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (1565.0 +/- 106. 0 pmol. min-1. mg protein-1 and 1783.0 +/- 282 pmol. min-1. mg protein-1 vs. 2487.0 +/- 210.0 pmol. min-1. mg protein-1, P < 0.05) and acyl CoA: cholesterol O-acyltransferase (ACAT) activities (548.0 +/- 65.0 and 615.0 +/- 80.0 pmol. min-1. mg protein-1 vs. 806.0 +/- 105.0 pmol. min-1. mg protein-1, P < 0.05) were significantly lower in the experimental groups than in the control. These supplements also substantially reduced the excretion of fecal neutral sterols compared to the control (211.1 +/- 26.7 and 208.2 +/- 31.6 mg/d vs. 521.9 +/- 53.9 mg/d). The inhibition of HMG-CoA reductase and ACAT activities resulting from the supplementation of either tangerine-peel extract or a combination of its bioflavonoids could account for the decrease in fecal neutral sterol that appears to compensate for the decreased cholesterol biosynthesis in the liver. (+info)
Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.
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The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation. (+info)
Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways.
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An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded. (+info)
Dietary flavonols quercetin and kaempferol are ligands of the aryl hydrocarbon receptor that affect CYP1A1 transcription differentially.
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Transcriptional activation of the human CYP1A1 gene (coding for cytochrome P450 1A1) is mediated by the aryl hydrocarbon receptor (AhR). In the present study we have examined the effect of the common dietary polyphenolic compounds quercetin and kaempferol on the transcription of CYP1A1 and the function of the AhR in MCF-7 human breast cancer cells. Quercetin caused a time- and concentration-dependent increase in the amount of CYP1A1 mRNA and CYP1A1 enzyme activity in MCF-7 cells. The increase in CYP1A1 mRNA caused by quercetin was prevented by the transcription inhibitor actinomycin D. Quercetin also caused an increase in the transcription of a chloramphenicol reporter vector containing the CYP1A1 promoter. Quercetin failed to induce CYP1A1 enzyme activity in AhR-deficient MCF-7 cells. Gel retardation studies demonstrated that quercetin activated the ability of the AhR to bind to an oligonucleotide containing the xenobiotic-responsive element (XRE) of the CYP1A1 promoter. These results indicate that quercetin's effect is mediated by the AhR. Kaempferol did not affect CYP1A1 expression by itself but it inhibited the transcription of CYP1A1 induced by the prototypical AhR ligand 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), as measured by a decrease in TCDD-induced CYP1A1 promoter-driven reporter vector activity, and CYP1A1 mRNA in cells. Kaempferol also abolished TCDD-induced XRE binding in a gel-shift assay. Both compounds were able to compete with TCDD for binding to a cytosolic extract of MCF-7 cells. Known ligands of the AhR are, for the most part, man-made compounds such as halogenated and polycyclic aromatic hydrocarbons. These results demonstrate that the dietary flavonols quercetin and kaempferol are natural, dietary ligands of the AhR that exert different effects on CYP1A1 transcription. (+info)
The extracellular-signal-regulated protein kinases (Erks) are required for UV-induced AP-1 activation in JB6 cells.
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Mitogen activated protein (MAP) kinase belongs to a large family of serine/threonine protein kinases, including extracellular-signal-regulated protein kinases (Erks), P38 kinase and c-Jun N-terminal kinases (JNKs). Although previous work has shown that both Erks and JNKs are activated in cells in response to ultraviolet (UV) irradiation, most studies have focused only on the role of JNKs in UV-induced AP-1 activation. Hence, the role of Erks in UV-induced AP-1 activity is not well defined. We here have investigated this issue by using MAP kinase kinase (MEK1) inhibitor PD098059 and a dominant negative Erk2, as well as wild-type Erk2, in a JB6 cell model. PD098059 inhibited UVB- or UVC-induced AP-1 activity and phosphorylation of MEK1 and Erks, but not JNKs, in JB6 Cl 41 cells. Overexpression of wild-type Erk2 in Cl 30.7b cells that contain small amounts of Erks caused a 46.6- or 138.1-fold increase of AP-1 activity by UVB and UVC, respectively; introduction of a dominant negative Erk2 into Cl 41 cells significantly blocked the UV-induced Erks activation as well as the AP-1 activation. In contrast, overexpression of wild-type Erk2 in Cl 30.7b cells and dominant negative Erk2 in Cl 41 cells did not show a marked influence on the phosphorylation of JNKs. These results demonstrate that activation of Erks, in addition to the previously reported JNKs, is required for UV-induced AP-1 activation. (+info)
Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation.
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We have examined fibroblast growth factor (FGF) receptor-1 mediated signal transduction in differentiation of endothelial cells (EC). The activated FGFR-1 couples to Ras through two adaptor proteins, FRS2 and Shc. In FGF-2 treated proliferating EC, FRS2 as well as Shc are tyrosine phosphorylated and interact with Grb2. In contrast, in FGF-2 treated differentiating cells, Shc, but not FRS2, is engaged in Grb2-interactions. Sustained MAP kinase activity has previously been implicated in differentiation. In FGF stimulated proliferating and differentiating endothelial cells, the MAP kinase Erk2 is activated in a sustained manner. Inhibition of MEK and MAP kinase activity by PD98059 treatment of cells, still allows EC tube formation. The FGFR-1 mediates activation of protein kinase C (PKC) through direct binding and activation of phospholipase C-gamma (PLC-gamma), and has also been shown to activate the cytoplasmic tyrosine kinase Src. Treatment of the cells with the PKC inhibitor bisindolylmaleimide does not prevent tube formation. In contrast, Src kinase activity is a prerequisite for EC differentiation, since treatment of the cells with PP1, a Src family specific inhibitor, abrogates tube formation. In differentiating EC, FGF-2 induces complex formation between Src and focal adhesion kinase (FAK). These data indicate that the Ras pathway is initiated via Shc or FRS2, dependent on the cellular program. Blocking the function of Src family kinases, attenuates differentiation. (+info)