c-Src Tyrosine Kinase Inhibitors Research Articles

Comprehensive in silico discovery of c-Src tyrosine kinase inhibitors in cancer treatment: A unified approach combining pharmacophore modeling, 3D QSAR, DFT, and molecular dynamics simulation

ObjectiveTo investigate c-Src, a non-receptor tyrosine kinase dysregulated in various cancer types including colon, breast, and pancreatic cancers, as a potential drug target for cancer therapy. MethodsLigand-based pharmacophore modeling and 3D-QSAR analysis on a dataset of 34c-Src tyrosine kinase inhibitors were employed. The established pharmacophore model (DDRRR_1) features two hydrogen bond donor (D) and three aromatic ring (R) features, exhibiting favorable parameters (R2 = 0.926; Q2 = 0.895; F value = 47.9). Hypothesis validation, enrichment analysis, and contour plot analysis were conducted, followed by virtual screening of a PubChem database using the optimized pharmacophore model and filtering based on the Lipinski rule of five. ResultsThe most promising inhibitors underwent multistep molecular docking, density Functional Theory (DFT) analysis, ADMET assessments, molecular dynamics simulation, and PCA. CID_70144047 emerged as the most promising hit with all the above favorable properties. ConclusionThe study provides a comprehensive approach for identifying novel c-Src tyrosine kinase inhibitors, highlighting CID_70144047 as a promising leads with potential therapeutic applications in cancer treatment.

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]5, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR]x (x = 6-9) and hybrid cyclic-linear peptides, [R6K]W6 and [R5K]W7, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]9 was found to be the most potent tyrosine kinase inhibitor. [WR]9 showed higher inhibitory activity (IC50 = 0.21 μM) than [WR]5, [WR]6, [WR]7, and [WR]8 with IC50 values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-33P]ATP. Consistent with the result above, [WR]9 inhibited other protein kinases such as Abl kinase activity with an IC50 value of 0.35 μM, showing 2.2-fold higher inhibition than [WR]5 (IC50 = 0.79 μM). [WR]9 also inhibited PKCa kinase activity with an IC50 value of 2.86 μM, approximately threefold higher inhibition than [WR]5 (IC50 = 8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]9 exhibited IC50 values of <0.25 μM against Akt1, Alk, and Btk. These data suggest that [WR]9 is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R6K]W6 and [R5K]W7 against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR](5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]5 or [WR]9 affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]9.

Use of QSAR Global Models and Molecular Docking for Developing New Inhibitors of c-src Tyrosine Kinase.

A prototype of a family of at least nine members, cellular Src tyrosine kinase is a therapeutically interesting target because its inhibition might be of interest not only in a number of malignancies, but also in a diverse array of conditions, from neurodegenerative pathologies to certain viral infections. Computational methods in drug discovery are considerably cheaper than conventional methods and offer opportunities of screening very large numbers of compounds in conditions that would be simply impossible within the wet lab experimental settings. We explored the use of global quantitative structure-activity relationship (QSAR) models and molecular ligand docking in the discovery of new c-src tyrosine kinase inhibitors. Using a dataset of 1038 compounds from ChEMBL database, we developed over 350 QSAR classification models. A total of 49 models with reasonably good performance were selected and the models were assembled by stacking with a simple majority vote and used for the virtual screening of over 100,000 compounds. A total of 744 compounds were predicted by at least 50% of the QSAR models as active, 147 compounds were within the applicability domain and predicted by at least 75% of the models to be active. The latter 147 compounds were submitted to molecular ligand docking using AutoDock Vina and LeDock, and 89 were predicted to be active based on the energy of binding.

2D QSAR studies of the inhibitory activity of a series of substituted purine derivatives against c-Src tyrosine kinase

A series of 34 substituted purine analogues derivatives were subjected to quantitative structure-activity relationship analyses as inhibitors of c-Src tyrosine kinase. Partial least squares regression was applied to derive QSAR models, which were further validated for statistical significance by internal and external validation. The best QSAR model developed had a good predictive correlation coefficient (r2) of 0.8319, a significant cross-validated correlation coefficient (q2) of 0.7550, and an r2 for the external test set (pred_r2) of 0.7983. It was developed from the PLS method with descriptors including the SsCH3E-index, H-Donor Count, T_2_Cl_3, and negative correlation with SsOHcount. The current study provides better insight into the future design of more potent c-Src tyrosine kinase inhibitors prior to synthesis.

Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells.

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca2+ concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [3H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

Conformation-independent QSAR on c-Src tyrosine kinase inhibitors

The main idea of this work was to find predictive quantitative structure–activity relationships (QSAR) for a wide set of c-Src tyrosine kinase inhibitors, by means of resorting to a conformation-independent representation of the chemical structure. In this way, our attempt was to avoid the availability of X-ray crystallographic structural information of the target. Therefore, in a set composed of 80 pyrrolo-pyrimidine derivatives, 1179 theoretical descriptors were simultaneously analyzed through linear regression models obtained with the replacement method variable subset selection technique. Alternatively, the flexible (activity dependent) descriptor approach was also applied in this study. The models were validated and tested through the use of an external test set of compounds, the leave-group-out cross validation method, Y-randomization and applicability domain analysis. Our results were compared with previously published ones based on docking analysis and 3D-QSAR. The obtained conformation-independent approach was in good agreement with experimental observations.

A 2D image-based method for modeling some c-Src tyrosine kinase inhibitors

The detailed application of multivariate image analysis method for evaluation of quantitative structure–activity relationship (MIA–QSAR) of some c-Src tyrosine kinase inhibitors is demonstrated here. Partial least squares (PLS) and genetic algorithm principal components general regression neural network (GA–PC–GRNN) methods were applied. The performance of PLS and GA–PC–GRNN were investigated by several validation methods. The resulted PLS model had a high statistical quality (R 2 = 0.951 and R CV 2 = 0.949) for predicting the activity of the compounds. For GA–PC–GRNN model, these values were R 2 = 0.850 and R CV 2 = 0.692. Applicability domain of developed models was considered using leverage and William plots were used to visualize it. PLS method was proved to be more predictive and accurate.

Cysteine 27 Variant of the δ-Opioid Receptor Affects Amyloid Precursor Protein Processing through Altered Endocytic Trafficking

Agonist-induced activation of the δ-opioid receptor (δOR) was recently shown to augment β- and γ-secretase activities, which increased the production of β-amyloid peptide (Aβ), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the δOR variant with a phenylalanine at position 27 (δOR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (δOR-Cys27) variant. For this study, we expressed these variants in human SH-SY5Y and HEK293 cells expressing exogenous or endogenous amyloid precursor protein (APP) and assessed the effects on APP processing. Expression of δOR-Cys27, but not δOR-Phe27, resulted in a robust accumulation of the APP C83 C-terminal fragment and the APP intracellular domain, while the total soluble APP and, particularly, the β-amyloid 40 levels were decreased. These changes upon δOR-Cys27 expression coincided with decreased localization of APP C-terminal fragments in late endosomes and lysosomes. Importantly, a long-term treatment with a subset of δOR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the δOR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the δOR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

Docking, 3D-QSAR studies and in silico ADME prediction on c-Src tyrosine kinase inhibitors

Docking simulations and three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis were performed on a wide set of c-Src inhibitors. The study was conducted using a structure-based alignment and by applying the GRID/GOLPE approach. The present 3D-QSAR investigation proved to be of good statistical value, displaying r 2, q 2 and cross-validation SDEP values of 0.94, 0.84 and 0.42, respectively. Moreover, such a model also proved to be capable of predicting the activities of an external test set of compounds. The availability of the 3D structure of the target made possible the interpretation of steric and electrostatic maps within the binding site environment and provided useful insight into the structural requirements for inhibitory activity against c-Src. Two regions whose occupation by hydrophobic portions of ligands would favourably affect the activity were clearly identified. Moreover, hydrogen bond interactions involving residues Met343, Asp406 and Ser347 emerged as playing a key role in determining the affinity of the active inhibitors toward c-Src. Furthermore, the inhibitors bearing a basic nitrogen provided enhanced potency through protonation and salt bridge formation with Asp350. A preliminary pharmacokinetic profile of the molecules under analysis was also drawn on the basis of Volsurf predictions.

Naturally Occurring Homoisoflavonoids as Potent Protein Tyrosine Kinases Inhibitors

Protein tyrosine kinases (PTK) have been recognized as promising therapeutic targets for a variety of human diseases. The development of new and more effective PTK inhibitors represents an attractive therapeutic strategy for cancer chemoprevention [1]. Here we report the identification of some potent c-Src tyrosine kinase inhibitors by an automated high-throughput screening (HTS) strategy based on the orthogonal-compound-mixing ELISA method [2]. Hematoxylin (1), the main component of the plant Heamatoxylon campechianum (Leguminosae) [3], stood out as one of the most remarkable c-Src inhibitors of a 32,200 compounds library. Further investigations revealed that hematoxylin is an ATP competitive broad-spectrum PTK inhibitor in vitro, with IC50 values ranging from nanomolar to micromolar level. In addition, hematoxylin is also noted to inhibit the phosphorylation of PTK and arrest the downstream signaling pathways in cellular level. Moreover, other ten new homoisoflavonoids, together with sixteen known homoisoflavonoids and chalcones, were isolated from the stems of H. campechianum upon bioassay-guided fractionation for the purpose of seeking bioactive hematoxylin analogues. The structure-activity relationship of these compounds was further performed by assessing the PTK inhibitory potency with concurrent molecular docking simulation. Hematoxylin and its natural analogues were substantially validated to function as a new class of PTK inhibitors.

Differential D 1 and D 5 Receptor Regulation and Degradation of the Angiotensin Type 1 Receptor

Renal sodium transport is increased by the angiotensin type 1 receptor (AT(1)R), which is counterregulated by dopamine via unknown mechanisms involving either the dopamine type 1 (D(1)R) or dopamine type 5 receptor (D(5)R) that belong to the D(1)-like receptor family of dopamine receptors. We hypothesize that the D(1)R and D(5)R differentially regulate AT(1)R protein expression and signaling, which may have important implications in the pathogenesis of essential hypertension. D(1)R and D(5)R share the same agonists and antagonists; therefore, the selective effects of either D(1)R or D(5)R stimulation on AT(1)R expression in human renal proximal tubule cells were determined using antisense oligonucleotides selective to either D(1)R or D(5)R. We also determined the role of receptor tyrosine kinase and the proteosome on the D(1)R/D(5)R-mediated effects on AT(1)R expression and internalization. In renal proximal tubule cells, D(5)R (not D(1)R) decreased AT(1)R expression (half-life: 0.47+/-0.18 hours) and AT(1)R-mediated extracellular signal-regulated kinase 1/2 phosphorylation (232+/-18.9 U with angiotensin II [10(-7) mol/L] versus 81+/-8.9 U with angiotensin II [10(-7) mol/L] and fenoldopam [D(1)R/D(5)R agonist; 10(-6) mol/L; P<0.05; n=6). The fenoldopam-induced decrease in AT(1)R expression was reversed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3,4-d) pyrimidine (c-Src tyrosine-kinase inhibitor) and clasto-lactacystin beta-lactone (proteasome inhibitor), demonstrating that the fenoldopam-mediated decrease in total cell AT(1)R expression is a result of a c-Src- and proteasome-dependent process. D(5)R stimulation decreases AT(1)R expression and is c-Src and proteasome dependent. The discovery of differential regulation by D(1)R and D(5)R opens new avenues for the development of agonists selective to either receptor subtype as targeted antihypertensive agents that can decrease AT(1)R-mediated antinatriuresis.

Angiotensin II Upregulates Acyl-CoA:Cholesterol Acyltransferase-1 via the Angiotensin II Type 1 Receptor in Human Monocyte-Macrophages

Angiotensin II (Ang II) is known to accelerate the progression of macrophage-driven atherosclerotic lesions. Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets, and promotes foam cell formation in atherosclerotic lesions. The present study explored the effect of Ang II on ACAT1 expression as a molecular mechanism of foam cell formation in primary cultured human monocyte-macrophages. Ang II significantly increased ACAT1 protein expression in a time- or concentration-dependent manner. Application of an Ang II type 1 (AT(1)) receptor agonist (L162313), but not an Ang II type 2 (AT(2)) receptor agonist (CGP42112A), mimicked the effects of Ang II treatment in inducing ACAT1 protein expression. ACAT activity and ACAT1 mRNA levels were also significantly increased by Ang II. Two-fold increases in ACAT1 protein expression and ACAT activity with Ang II treatment were completely inhibited by AT(1) receptor antagonists (candesartan, [Sar(1),Ile(8)]-Ang II), but not by an AT(2) receptor antagonist (PD123319). Treatment with a G-protein inactivator (GDP-beta-S), a c-Src tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), or a mitogen activated protein kinase (MAPK) kinase inhibitor (PD98059) significantly reduced Ang II-induced ACAT1 protein expression. Macrophage foam cell formation assessed using acetylated low-density lipoprotein (LDL)-induced CE accumulation was significantly enhanced by Ang II, which was completely inhibited by treatment with candesartan. These results suggested that Ang II enhances foam cell formation by upregulating ACAT1 expression predominantly through the actions of AT(1) receptor via the G protein/c-Src/PKC/MAPK pathway in human monocyte-macrophages.

Human Urotensin-II Potentiates the Mitogenic Effect of Mildly Oxidized Low-Density Lipoprotein on Vascular Smooth Muscle Cells: Comparison with Other Vasoactive Agents and Hydrogen Peroxide

Human urotensin-II (U-II) is the most potent vasoactive peptide identified to date, and may be involved in hypertension and atherosclerosis. We investigated the effects of the interactions between U-II or other vasoactive agents and mildly oxidized low-density lipoprotein (mox-LDL) or hydrogen peroxide (H2O2) on the induction of vascular smooth muscle cell (VSMC) proliferation. Growth-arrested rabbit VSMCs were incubated with vasoactive agents (U-II, endothelin-1, angiotensin-II, serotonin, or thromboxane-A2) in the presence or absence of mox-LDL or H2O2. [3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. On interaction with mox-LDL or H2O2, U-II induced the greatest increase in [3H]thymidine incorporation among these vasoactive agents. A low concentration of U-II (10 nmol/l) enhanced the potential mitogenic effect of low concentrations of mox-LDL (120 to 337%) and H2O2 (177 to 226%). U-II at 50 nmol/l showed the maximal mitogenic effect (161%), which was abolished by G protein inactivator (GDP-beta-S), c-Src tyrosine kinase inhibitor (radicicol), protein kinase C (PKC) inhibitor (Ro31-8220), extracellular signal-regulated kinase (ERK) kinase inhibitor (PD98059), or Rho kinase inhibitor (Y27632). Mox-LDL at 5 microg/ml showed the maximal mitogenic effect (211%), which was inhibited by free radical scavenger (catalase), intracellular and extracellular antioxidants (N-acetylcysteine and probucol), nicotinamide adenine dinucleotide phosphate oxidase inhibitor (diphenylene iodonium), or c-Jun N-terminal kinase (JNK) inhibitor (SP600125). These results suggested that U-II acts in synergy with mox-LDL in inducing VSMC DNA synthesis at the highest rate among these vasoactive agents. Activation of the G protein/c-Src/PKC/ERK and Rho kinase pathways by U-II together with the redox-sensitive JNK pathway by mox-LDL may explain the synergistic interaction between these agents.

Synthesis and anti-tyrosine kinase activity of 3-(substituted-benzylidene)-1, 3-dihydro-indolin derivatives: investigation of their role against p60 c-Src receptor tyrosine kinase with the application of receptor docking studies

A series of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thione derivatives were synthesized as modified congeners of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one series. All the synthesized compounds were examined for their in vitro anti-tyrosine kinase activity against p60 c-Src. The activity results revealed that compounds ( Z)-3-(4′-Dimethylamino-benzylidene)-1, 3-dihydro-indolin-2-thione (12) ( E)-3-(2′, 6′-Dichloro-benzylidene)-1, 3-dihydro-indolin-2-thione (13) and ( E)-3-(3′-Hydroxy-4′-methoxy-benzylidene)-1, 3-dihydro-indolin-2-thione ( 19) exhibited anti-tyrosine kinase activity with IC 50 value of 21.91, 21.20 and 30.92 μM, respectively. These results are comparable to PP1 [1- tert-Butyl-3-p-tolyl-1H-pyrazolo[3, 4-d]pyrimidine-4-yl-amine] (IC 50 = 0.17 μM), which is reported as a potent and selective p60 c-Src tyrosine kinase inhibitor. Some thio congeners are found to be more potent than oxo derivatives; however, no significant correlation was observed between the activity profiles of these two series. Docking program was used to investigate the docking mode of each compound at the active site. Among all of the compounds, only ( Z)-3-(2′-Chloro-benzylidene)-1, 3-dihydro-indolin-2-one (8) and ( E)-3-(3′-Nitro-benzylidene)-1, 3-dihydro-indolin-2-thione (16) were docked at the active site where the PP1 was embedded.

Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line.

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [(14)C]mannitol and [(14)C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells.

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.

Synergistic effect of urotensin II with mildly oxidized LDL on DNA synthesis in vascular smooth muscle cells.

The urotensin II (UII) found in coronary atheroma is the most potent vasoconstrictor known to date. Mildly oxidized LDL (moxLDL) contributes to atherogenesis and plaque formation. We assessed the effect of UII and its interaction with moxLDL and the oxidative components of moxLDL on vascular smooth muscle cell (VSMC) proliferation. Methods and Results-Growth-arrested VSMCs were incubated in serum-free medium with different concentrations of LDL, moxLDL, oxLDL, hydrogen peroxide, lysophosphatidylcholine, or 4-hydroxy-2-nonenal, with or without UII. [(3)H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. UII stimulated [(3)H]thymidine incorporation in a dose-dependent manner, with a maximal effect at a concentration of 50 nmol/L (161%). Low concentrations of UII potentiated the mitogenic effect of LDL (108% to 242%), oxLDL (129% to 302%), moxLDL (120% to 337%), hydrogen peroxide (177% to 226%), lysophosphatidylcholine (115% to 332%), and 4-hydroxy-2-nonenal (142% to 299%). The synergistic interaction between UII and moxLDL was partially inhibited by anti-Gq/11alpha antibody, the epidermal growth factor receptor tyrosine kinase inhibitor erbstatin A (10 micromol/L), and the intracellular free radical scavenger N-acetylcysteine (400 micromol/L) and was completely inhibited by the c-Src tyrosine kinase inhibitor radicicol (10 micromol/L), the protein kinase C (PKC) inhibitor Ro31-8220 (0.1 micromol/L), and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 micromol/L). Our results suggest that UII acts synergistically with moxLDL in inducing VSMC proliferation via the c-Src/PKC/MAPK pathway, which may explain the relatively rapid progression of atherosclerosis in patients with hypertension and hypercholesterolemia.

Biochemical and cellular effects of c-Src kinase-selective pyrido[2,3- d]pyrimidine tyrosine kinase inhibitors

Increased expression or activity of c-Src tyrosine kinase has been associated with the transformed phenotype in tumor cells and with progression of neoplastic disease. A number of pyrido[2,3- d]pyrimidines have been characterized biochemically and in cells as part of an assessment of their potential as anti-tumor agents. The compounds were ATP-competitive inhibitors of c-Src kinase with ic 50 values < 10 nM and from 6 to >100-fold selectivity for c-Src tyrosine kinase relative to basic fibroblast growth factor receptor (bFGFr) tyrosine kinase, platelet-derived growth factor receptor (PDGFr) tyrosine kinase, and epidermal growth factor receptor (EGFr) tyrosine kinase. The compounds yielded ic 50 values < 5 nM against Lck. Human colon tumor cell growth in culture was inhibited, as was colony formation in soft agar at concentrations < 1 μM. Phosphorylation of the c-Src cellular substrates paxillin, p130 cas, and Stat3 was also inhibited at concentrations < 1 μM. Autophosphorylation of EGFr tyrosine kinase or PDGFr tyrosine kinase was not inhibited by c-Src inhibitors, thus showing the selective nature of the compounds in cells. In a mitogenesis assay measuring thymidine incorporation stimulated by specific mitogens, the c-Src tyrosine kinase inhibitors reduced incorporated thymidine in a manner consistent with previously reported roles of c-Src in mitogenic signaling. Progression through the cell cycle was inhibited at G 2/M in human colon tumor cells treated with two of the c-Src-selective compounds, which is also consistent with earlier reports describing a requirement for active c-Src tyrosine kinase for G 2 to M phase progression. The compounds described here are selective inhibitors of c-Src tyrosine kinase and have antiproliferative effects in tumor cells consistent with inhibition of c-Src.