Src Tyrosine Kinase Inhibitor Research Articles

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane-dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel-filtered platelets, intact or loaded with calcium-sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine-positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine-negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha-granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine-positive platelets formation several-fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate-limiting, for subpopulation segregation. Inhibition of mitogen-activated protein kinase kinase (U0126) and glycoprotein IIb-IIIa (Monafram(®)) was without effect, whereas inhibitors of phosphatidylinositol 3-kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.

A QSAR Study on a Series of Indolin-2-Ones Acting as Non-Receptor Src Tyrosine Kinase Inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on a novel series of substituted 3-[3-(aminopropyl)-4,5,6,7-tetrahydro-1H-indol-2-ylmethylene]-1,3-dihydro-indole-2-ones as potent inhibitors of the non-receptor Src tyrosine kinase. The activity is found to be significantly correlated with calculated hydrophobicity and molar refractivity of the molecule. The correlation obtained suggests that the activity of the compounds is controlled by the hydrophobic and steric properties of the molecules. The internal and external validation of the correlation is judged by calculating r2cv (= 0.66) for the training set and r2pred (= 0.52) for test set. Using the correlation, some new compounds have been predicted possessing higher potencies than the compounds in the existing series. Keywords: Quantitative structure-activity relationship, Src tyrosine kinase inhibitors, Tyrosine kinase inhibitors, QSAR, hydrophobicity, tyrosine kinase, transmembrane receptor-linked kinase, RTKs, tetrahydroindole, aminopropyl, calculated molar refractivity (CMR), ClogP, R1-substituents, Protease Inhibitors, Integrase Inhibitors

Effect of Ketoconazole on the Pharmacokinetics of Oral Bosutinib in Healthy Subjects

Bosutinib (SKI-606), a dual inhibitor of Src and Abl tyrosine kinases, is being developed for the treatment of chronic myelogenous leukemia. The effect of coadministration of ketoconazole on the pharmacokinetic (PK) profile of bosutinib was evaluated in an open-label, randomized, 2-period, crossover study. Healthy subjects (fasting) received a single dose of oral bosutinib 100 mg alone and with multiple once-daily doses of oral ketoconazole 400 mg. PK sampling occurred through 96 hours. The least square geometric mean treatment ratios (90% confidence interval [CI]) of C(max(bosutinib+ketoconazole))/C(max(bosutinib alone)), AUC(T(bosutinib+ketoconazole))/AUC(T(bosutinib alone)), and AUC((bosutinib+ketoconazole))/AUC((bosutinib alone)) were assessed. Compared with bosutinib administered alone, coadministration with ketoconazole increased bosutinib C(max) 5.2-fold, AUC(T) 7.6-fold, and AUC 8.6-fold. Ketoconazole coadministration decreased the mean apparent clearance of bosutinib approximately 9-fold and increased the mean (SD) terminal half-life from 46.2 (16.4) hours to 69.0 (29.1) hours. The incidence of adverse events (AEs) was comparable between the 2 treatments. The most common AEs were headache, nausea, and increased blood creatinine. No safety-related discontinuations or serious AEs occurred. These PK results indicate that bosutinib is susceptible to interaction with potent CYP3A4 inhibitors.

Abstract A91: A phase I dose-escalation study of bosutinib plus capecitabine in selected advanced solid tumors.

Abstract Background: Bosutinib is an orally active, dual Src and Abl tyrosine kinase inhibitor that has demonstrated efficacy and acceptable safety in patients (pts) with metastatic breast cancer and chronic myelogenous leukemia. Capecitabine is a standard of care for metastatic breast cancer. Preclinical evidence suggests that the combination of a Src inhibitor and capecitabine may have improved activity. Methods: This phase 1, open-label study determined the maximum tolerated dose (MTD) of bosutinib plus capecitabine in pts with advanced colon, breast, pancreatic, cholangiocarcinoma, and glioblastoma tumors, and evaluated the safety and efficacy of this combination. Part 1 of this study incorporated an “up-and-down” dose escalation scheme to determine the MTD. The initial dose cohorts were 1) bosutinib 200 mg/day + capecitabine 750 mg/m2 BID on Days 1 to 14 of a 21-day cycle and 2) bosutinib 300 mg/day + capecitabine 625 mg/m2 BID on Days 1 to 14. Additional cohorts could be enrolled at higher or lower dose levels based on the number of dose-limiting toxicities (DLTs) during the first 21 days in the current cohort of 2 pts, the cumulative toxicity rate at that dose combination, and the current dose of bosutinib and capecitabine. The design allowed up to 9 dose combinations, of which 8 were evaluated. Results: A total of 32 pts were enrolled: median age was 62 years (range, 42–82 years) and 44% were male. Three pts experienced DLTs: 1 pt receiving bosutinib 400 mg/day + capecitabine 750 mg/m2 BID (grade 3 neurological pain), 1 pt receiving bosutinib 400 mg/day + 1,000 mg/m2 BID (grade 3 increased alanine aminotransferase [ALT] and rash), and 1 pt receiving bosutinib 200 mg/day + capecitabine 1,000 mg/m2 BID (grade 4 fatigue). Based on the observed toxicity, the MTD was determined to be bosutinib 300 mg/day + capecitabine 1,000 mg/m2 BID (n = 9 treated at the MTD). The most common treatment-related adverse events across all dose cohorts were diarrhea (66%), nausea (41%), palmar-plantar erythrodysesthesia syndrome (PPE; 31%), fatigue (28%), vomiting (25%), and decreased appetite (16%). Although diarrhea was common, 91% of events were grade 1/2, all pts had their diarrhea resolve (median cumulative duration of 10 days), and no pt discontinued treatment due to diarrhea. The most common grade 3/4 treatment-related events were increased ALT, increased aspartate aminotransferase (AST), and PPE (9% each). All grade 3/4 ALT and AST elevations resolved, with no pt discontinuing treatment due to ALT or AST elevation. Six (19%) deaths occurred during the study, including 5 deaths within 28 days of the last study dose (all due to cancer-related causes and/or disease progression); no drug-related deaths were reported. Best overall response included 2 (6%) pts with partial responses and 14 (44%) pts with stable disease, including 4 (13%) pts with stable disease for >24 weeks. Conclusions: The combination of bosutinib and capecitabine was well tolerated with a safety profile characterized by low-grade gastrointestinal events, similar to that previously observed for bosutinib monotherapy. Bosutinib plus capecitabine also demonstrated promising preliminary efficacy. The “up-and-down” dose-escalation design allowed estimation of a MTD contour, with more pts evaluated at near-MTD doses compared with standard 3+3 designs. A phase 2 evaluation in breast cancer pts was planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A91.

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant (32)P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.

Acidic Fibroblast Growth Factor (FGF) Potentiates Glial-mediated Neurotoxicity by Activating FGFR2 IIIb Protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.

Contractile Forces Contribute to Increased Glycosylphosphatidylinositol-anchored Receptor CD24-facilitated Cancer Cell Invasion

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.

Emerging treatment options for patients with castration‐resistant prostate cancer

Most prostate cancer-related deaths occur in patients with castration-resistant prostate cancer (CRPC). Recent preclinical and clinical studies have identified intracellular signaling pathways and changes in the tumor and bone microenvironment as potential key drivers of CRPC. This increased understanding of mechanisms associated with CRPC has driven the development of numerous new agents, many of which are poised to alter the current CRPC treatment landscape. A review of literature was conducted to identify ongoing and planned phase III studies of novel agents to treat CRPC. Multiple studies were identified, including novel androgen biosynthesis inhibitors (abiraterone, TAK-700), androgen-receptor inhibitors (MDV3100), angiogenesis inhibitors (aflibercept, tasquinimod), endothelin antagonists (zibotentan, atrasentan), a Src tyrosine kinase inhibitor (dasatinib), a novel radiotherapy (radium-223), and new immunotherapies (ipilimumab and ProstVac). In addition, both sipuleucel-T (an immunotherapy) and cabazitaxel (third-generation taxane) and the RANK-L inhibitor, denosumab, have recently been approved by the US Food and Drug Administration. Various combinations of these agents could theoretically be used to treat future patients with CRPC by targeting multiple signaling pathways as well as aspects of the tumor and bone microenvironments. Additional research will be needed to understand how to best use these agents and individualize care to optimize CRPC patient outcomes.

Na+/K+‐ATPase inhibition upregulates NMDA‐evoked currents in rat hippocampal CA1 pyramidal neurons

Na(+)/K(+)-ATPase and N-methyl-D-aspartate (NMDA) receptor in hippocampus play very important roles in the regulation of learning and memory. Here, we showed that dihydroouabain (DHO, 10(-5)-10(-3) M), a Na(+)/K(+)-ATPase inhibitor, significantly potentiated NMDA current in rat hippocampal CA1 pyramidal neurons, which was blocked by PP2 (the selective Src tyrosine kinase inhibitor) and PD-98059 [the selective inhibitor of the mitogen-activated protein kinases (MAPK) cascade]. These findings reported here uncover that Src mediates the cross-talk between Na(+)/K(+)-ATPase and NMDA receptor to transduce the signals from Na(+)/K(+)-ATPase to the MAPK cascade and provide new insights into therapeutic target for deeper understanding of the nature of cognitive disorder.

Effects of Src on proliferation and invasion of lung cancer cells

背景与目的Src蛋白在肿瘤的发生、发展和转移中具有重要作用。本研究旨在探讨Src蛋白在肺癌细胞中的表达活化情况及其对肺癌细胞增殖浸润的影响。方法采用Western blot和免疫沉淀法检测Src蛋白在肺癌细胞株中的表达和磷酸化情况；MT法检测抑制Src酪氨酸激酶活化对肺癌细胞体外增殖的影响；Boyden chamber法检测抑制Src酪氨酸激酶活化对肺癌细胞体外侵袭浸润的影响。结果本实验选用的肺癌细胞都存在pp60src的表达，非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞株Src蛋白的自主磷酸化水平（SrcpY418）明显增高，而人小细胞肺癌细胞株SBC5中几乎检测不到SrcpY418。抑制Src酪氨酸激酶活化对肺癌细胞体外增殖呈现出不同的反应，亚微摩尔Src蛋白酪氨酸激酶抑制剂对PC-9和A549细胞体外增殖表现出剂量依赖性抑制作用（P < 0.05）；而≤1 μM Src酪氨酸激酶抑制剂对H226、PC14PE6和RERFLCOK细胞增殖没有明显的抑制作用。Src酪氨酸激酶抑制剂对肺癌细胞体外侵袭浸润呈现明显的剂量依赖性抑制作用（P < 0.05）。结论Src蛋白的活化，而不是过度表达，在NSCLC细胞体外增殖和浸润中发挥着重要作用。

Regulation of PI3K/Akt signaling by N-desmethylclozapine through activation of δ-opioid receptor

We have previously reported that N-desmethylclozapine (NDMC), a major clozapine metabolite, acts as a δ-opioid receptor agonist. Here, we show that in different cellular systems NDMC regulates protein kinase B/Akt (Akt) signaling through the activation of δ-opioid receptors. In Chinese hamster ovary cells transfected with the human δ-opioid receptor (CHO/DOR), NDMC induced a time- and concentration-dependent phosphorylation of Akt at Thr308 and glycogen synthase kinase-3β (GSK-3β) at Ser9 and these effects were fully blocked by the δ-opioid receptor antagonist naltrindole. NDMC-induced Akt and GSK-3β phosphorylations were completely prevented by pertussis toxin, the Src tyrosine kinase inhibitor PP2 and the selective insulin-like growth factor-I (IGF-I) receptor tyrosine kinase inhibitor tyrphostin AG 1024. NDMC stimulated IGF-I receptor β subunit tyrosine phosphorylation and this effect was prevented by either naltrindole or PP2. Blockade of phosphatidylinositol 3-kinase (PI3K) α, but not PI3Kγ, suppressed NDMC-induced Akt and GSK-3β phosphorylation, whereas inhibition of Akt curtailed the stimulation of GSK-3β phosphorylation. In rat nucleus accumbens, NDMC induced Akt and GSK-3β phosphorylation either in vitro or in vivo and these effects were prevented by naltrindole. NDMC also regulated Akt and GSK-3β phosphorylation through δ-opioid receptors in NG108-15 cells. In these cells NDMC counteracted oxidative stress-induced apoptosis and the effect was lost following PI3K inhibition. These data demonstrate that in different cell systems NDMC can stimulate Akt signaling by activating Gi/Go-coupled δ-opioid receptors, which, at least in CHO/DOR cells, regulate PI3Kα through Src-dependent transactivation of the IGF-I receptor, and indicate that through this mechanism NDMC can exert neuroprotective effects.

The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy

Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.

Effect of Src tyrosine kinase inhibition on secretion of MMP-2 and MMP-9 by non-small cell lung cancer cells

背景与目的Src酪氨酸激酶和基质金属蛋白酶在肺癌的浸润和转移中发挥重要作用。本研究旨在探讨抑制Src酪氨酸激酶对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞分泌基质金属蛋白酶-2（matrix metalloproteinase 2, MMP-2）和基质金属蛋白酶-9（matrix metalloproteinase 9, MMP-9）以及NSCLC细胞侵袭浸润的影响。方法采用ELISA法检测NSCLC细胞（PC14PE6、H226、PC-9、A549）培养上清中MMP-2和MMP-9含量以及抑制Src酪氨酸激酶对NSCLC细胞分泌MMP-2和MMP-9的影响；Boyden chamber法检测抑制Src酪氨酸激酶对NSCLC细胞体外侵袭浸润的影响。结果NSCLC细胞中PC14PE6和H226中MMP-2和MMP-9的水平较高，A549细胞中MMP-9的水平较低，而MMP-2和MMP-9在PC-9细胞中检测不到。Src酪氨酸激酶抑制剂对PC14PE6中的MMP-2水平以及PC14PE6、H226和A549细胞中的MMP-9水平呈剂量依赖性抑制关系。10 µM Src酪氨酸激酶抑制剂使PC14PE6细胞中的MMP-2水平、H226细胞和A549细胞中的MMP-9水平降低50%以上。10 µM Src酪氨酸激酶抑制剂对H226细胞中的MMP-2无明显抑制作用。Src酪氨酸激酶抑制剂对4种NSCLC细胞体外侵袭浸润的抑制程度略有差异，但均呈现明显的剂量依赖性抑制作用。3 µM Src酪氨酸激酶抑制剂对PC14PE6、H226、A549和PC-9细胞体外侵袭浸润的抑制率分别为79.1%、68.09%、90.96%和96.98%（P < 0.001）。结论通过抑制NSCLC细胞分泌MMP-2和MMP-9，抑制Src酪氨酸激酶可降低细胞的体外侵袭浸润能力。

Targeting Tyrosine Kinases and Autophagy in Prostate Cancer

Tyrosine kinases play significant roles in tumor progression and therapy resistance. Inhibitors of tyrosine kinases are on the forefront of targeted therapy. For prostate cancer, tyrosine kinases play an additional role in the development of castration-resistant disease state, the most troubling aspect of prostate cancinogenesis which presently defies any effective treatment. Among the 30 or so tyrosine kinases expressed in a typical prostate cancer cell, nearly one third of them have been implicated in prostate carcinogenesis. Interestingly, most of them channel signals through a trio of non-receptor tyrosine kinases, Src/Etk/FAK, referred here as Src tyrosine kinase complex. This complex has been shown to play a significant role in the aberrant activation of androgen receptor (AR) mediated by growth factors (e.g., epidermal growth factor (EGF)), cytokines (interleukin (IL)-6), chemokines (IL-8), and neurokines (gastrin-releasing peptide). These factors are induced and released from the prostate cancer to the stromal cells upon androgen withdrawal. The Src kinase complex has the ability to phosphorylate androgen receptor, resulting in the nuclear translocation and stabilization of un-liganded androgen receptor. Indeed, tyrosine kinase inhibitors targeting Src can inhibit androgen-independent growth of prostate cancer cells in vitro and in preclinical xenograft model. While effective in inducing growth arrest and inhibiting metastasis of castration-resistant tumors, Src inhibitors rarely induce a significant level of apoptosis. This is also reflected by the general ineffectiveness of tyrosine kinase inhibitors as monotherapy in clinical trials. One of the underlying causes of apoptosis resistance is “autophagy,” which is induced by tyrosine kinase inhibitors and by androgen withdrawal. Autophagy is a self-digesting process to regenerate energy by removal of long-lived proteins and retired organelles to provide a survival mechanism to cells encountering stresses. Excessive autophagy, sometimes, could lead to type II programmed cell death. We demonstrated that autophagy blockade sensitizes prostate cancer cells toward Src tyrosine kinase inhibitor. Thus, a combination therapy based on Src tyrosine kinase inhibitor and autophagy modulator deserves further attention as a potential treatment for relapsed prostate cancer.

Gold nanoparticles inhibit vascular endothelial growth factor-induced angiogenesis and vascular permeability via Src dependent pathway in retinal endothelial cells

The purpose of this study was to investigate the effect of gold nanoparticles on the signaling cascade related to angiogenesis and vascular permeability induced by Vascular Endothelial Growth Factor (VEGF) in Bovine retinal endothelial cells (BRECs). The effect of VEGF and gold nanoparticles on cell viability, migration and tubule formation was assessed. PP2 (Src Tyrosine Kinase inhibitor) was used as the positive control and the inhibitor assay was performed to compare the effect of AuNPs on VEGF induced angiogenesis. The transient transfection assay was performed to study the VEGFR2/Src activity during experimental conditions and was confirmed using western blot analysis. Treatment of BRECs with VEGF significantly increased the cell proliferation, migration and tube formation. Furthermore, gold nanoparticles (500 nM) significantly inhibited the proliferation, migration and tube formation, in the presence of VEGF in BRECs. The gold nanoparticles also inhibited VEGF induced Src phosphorylation through which their mode of action in inhibiting angiogenic pathways is revealed. The fate of the gold nanoparticles within the cells is being analyzed using the TEM images obtained. The potential of AuNPs to inhibit the VEGF165-induced VEGFR-2 phosphorylation is also being confirmed through the receptor assay which elucidates one of the possible mechanism by which AuNPs inhibit VEGF induced angiogenesis. These results indicate that gold nanoparticles can block VEGF activation of important signaling pathways, specifically Src in BRECs and hence modulation of these pathways may contribute to gold nanoparticles ability to block VEGF-induced retinal neovascularization.

Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts.

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.

Inhibition of Src Impairs the Growth of Met-Addicted Gastric Tumors

We examined whether inhibition of Src tyrosine kinase, a downstream effector of the MET oncogene, can hinder the malignant properties of gastric tumors dependent on Met for growth and survival. Sensitivity to Src inhibition was determined in vitro by measuring clonogenic survival (anchorage-independent growth) and in vivo by establishing xenograft models. Four "Met-addicted" gastric carcinoma cell lines (GTL16, MKN45, HS746T, and SNU5) and three Met-independent gastric carcinoma cell lines (KATO III, AGS, and NCI-N87) were treated with the Src inhibitor saracatinib (AZD0530). In GTL16 and KATO III, Src neutralization was also achieved by dasatinib and RNA interference. The biochemical and transcriptional consequences of Src inhibition were explored using anti-phosphoprotein antibodies and oligonucleotide microarrays. Inhibition of Src in Met-addicted gastric carcinoma cell lines (a) decreased the phosphorylation/activation levels of signaling intermediates involved in cell proliferation and protection from apoptosis and down-modulated the expression of several cell cycle regulators; (b) reduced anchorage-independent growth; (c) enhanced impairment of cell viability produced by Met inhibition; and (d) delayed tumorigenesis in xenotransplantation models. Immunohistochemical analysis of tumor xenograft tissues following systemic treatment with saracatinib showed a reduction of tumor cell proliferation index, increased apoptosis, and diminished phospho-focal adhesion kinase and phospho-paxillin staining. Tumor stroma parameters such as angiogenesis or inflammatory infiltration were unaffected. In clonogenic survival assays, gastric carcinoma cells without addiction to Met were less sensitive than Met-addicted cells to Src inhibition. Src is as a key downstream transducer of Met-driven tumor growth. Targeting Src might provide therapeutic benefit in Met-addicted tumors.

Activities of SYK and PLCγ2 Predict Apoptotic Response of CLL Cells to SRC Tyrosine Kinase Inhibitor Dasatinib

B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking B-cell receptor signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. Fresh CLL B cells were treated with dasatinib, and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules, as well as with molecular and cytogenetic prognostic factors. Among 50 CLL cases, dasatinib treatment reduced cell viability by 2% to 90%, with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good, as well as poor, responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and phospholipase Cgamma2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with phospholipase Cgamma2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK, independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment.

Olmesartan Inhibits Angiotensin II–Induced Migration of Vascular Smooth Muscle Cells Through Src and Mitogen-Activated Protein Kinase Pathways

Clinical studies have shown that angiotensin-receptor blockers (ARBs) reduce the risk of cardiovascular diseases in hypertensive patients. It is assumed that the reduction of the risk by ARBs may be attributed in part to the inhibition of angiotensin II (AII)-induced vascular smooth muscle cell (VSMC) migration associated with atherosclerosis. However, the effect of ARBs on AII-induced changes in intracellular signaling and resultant cell migration has not been well established. Here, we investigated the effect of olmesartan, an ARB, on AII-induced extracellular signal–regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation and rat aortic smooth muscle cell (RASMC) migration. Olmesartan inhibited AII-induced ERK1/2 and JNK activation at lower concentrations (10 nM). On the other hand, PP2, a Src tyrosine kinase inhibitor, also inhibited AII-induced ERK1/2 and JNK activation, but its effect on ERK1/2 was less pronounced than that of olmesartan. Olmesartan, U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and PP2 potently inhibited AII-induced RASMC migration. From these findings, it was inferred that angiotensin-receptor blockade by olmesartan results in the inhibition of AII-induced activation of Src, ERK1/2, and JNK in RASMC. Olmesartan may be a potent inhibitor of AII-induced VSMC migration, which may be involved in the progression of atherosclerosis.

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG- modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.