Src inhibition induces mitotic arrest associated with chromosomal passenger complex.

The non-receptor tyrosine kinase Src plays a key role in cell division, migration, adhesion, and survival. Src is overactivated in several cancers, where it transmits signals that promote cell survival, mitosis, and other important cancer hallmarks. Src is therefore a promising target in cancer therapy, but the underlying mechanisms are still uncertain. Here we show that Src is highly conserved across different species. Src expression increases during mitosis and is localized to the chromosomal passenger complex. Knockdown or inhibition of Src induces multipolar spindle formation, resulting in abnormal expression of the Aurora B and INCENP components of the chromosomal passenger complex. Molecular mechanism studies have found that Src interacts with and phosphorylates INCENP. This then leads to incorrect chromosome arrangement and segregation, resulting in cell division failure. Herein, Src and chromosomal passenger complex co-localize and Src inhibition impedes mitotic progression by inducing multipolar spindle formation. These findings provide novel insights into the molecular basis for using Src inhibitors to treat cancer.

Toxoplasma gondii induces prolonged host epidermal growth factor receptor signalling to prevent parasite elimination by autophagy: Perspectives for in vivo control of the parasite.

Toxoplasma gondii causes retinitis and encephalitis. Avoiding targeting by autophagosomes is key for its survival because T. gondii cannot withstand lysosomal degradation. During invasion of host cells, T. gondii triggers epidermal growth factor receptor (EGFR) signalling enabling the parasite to avoid initial autophagic targeting. However, autophagy is a constitutive process indicating that the parasite may also use a strategy operative beyond invasion to maintain blockade of autophagic targeting. Finding that such a strategy exists would be important because it could lead to inhibition of host cell signalling as a novel approach to kill the parasite in previously infected cells and treat toxoplasmosis. We report that T. gondii induced prolonged EGFR autophosphorylation. This effect was mediated by PKCα/PKCß âž” Src because T. gondii caused prolonged activation of these molecules and their knockdown or incubation with inhibitors of PKCα/PKCß or Src after host cell invasion impaired sustained EGFR autophosphorylation. Addition of EGFR tyrosine kinase inhibitor (TKI) to previously infected cells led to parasite entrapment by LC3 and LAMP-1 and pathogen killing dependent on the autophagy proteins ULK1 and Beclin 1 as well as lysosomal enzymes. Administration of gefitinib (EGFR TKI) to mice with ocular and cerebral toxoplasmosis resulted in disease control that was dependent on Beclin 1. Thus, T. gondii promotes its survival through sustained EGFR signalling driven by PKCα/ß âž” Src, and inhibition of EGFR controls pre-established toxoplasmosis.

Quercetin Inhibits the Proliferation and Metastasis of Human Non-Small Cell Lung Cancer Cell Line: The Key Role of Src-Mediated Fibroblast Growth Factor-Inducible 14 (Fn14)/ Nuclear Factor kappa B (NF-κB) pathway.

BACKGROUND Quercetin (Que) is reported to induce apoptosis of lung cancer cells. Src is closely related to the progression of non-small cell lung cancer (NSCLC) and can be modulated by Que in macrophages. In the current study, the interaction between Que and Src signaling in NSCLC cells was explored to explain the anti-NSCLC function of Que. MATERIAL AND METHODS NSCLC cell line HCC827 was subjected to the administrations of Que at different concentrations. The effect of Que on tumor cell proliferation was detected using MTT and colony formation assays. Then the effect on the migration and invasion abilities was assessed using scratch and Transwell assays. At molecular level, the changes in Src/Fn14/NF-kappaB signaling were determined using western blotting assays. The role of Src in the function of Que was further explored by inducing the expression of Src gene in NSCLC cells before Que administration. The results of the in vitro assays were verified using a NSCLC mice model. RESULTS Que inhibited the proliferation and anchorage-independent growth of NSCLC cells. Additionally, Que delayed in the gap closure rate in scratch assays and decreased the membrane-penetrating cell number in Transwell assays. At a molecular level, Que suppressed the expression of Src, which subsequently inhibited Fn14/NF-kappaB signaling. In in vivo assays, Que inhibited the growth of solid tumors. After the overexpression of Src in NSCLC cells, the anti-NSCLC effect of Que was blocked by inducing NSCLC proliferation and metastasis, and by activating Fn14/NF-kappaB signaling. Moreover, the induced level of Src promoted the growth and metastasis potential of solid tumors in mice. CONCLUSIONS Que exerted the anti-NSCLC effect by inhibiting Src-mediated Fn14/NF-kappaB pathway both in vitro and in vivo.

Second-generation Src/Abl inhibitor bosutinib effectively induces apoptosis in human esophageal squamous cell carcinoma (ESCC) cells via inhibiting Src/Abl signaling.

Esophageal cancer is a prevalent type of cancer worldwide and is ranked sixth among cancer-associated mortalities. Aberrant activation of the non-receptor tyrosine kinase Src and c-Abl contribute to the progression of ESCC. Thus, targeting these kinases to treat ESCC is a promising strategy. In this paper, we report that the potent dual Src/Abl inhibitor bosutinib exerts anti-tumor effects on ESCC. Bosutinib inhibits ESCC cell proliferation in a dose-dependent manner. Furthermore, bosutinib suppresses the colony formation ability of ESCC cells. Mechanistically, bosutinib effectively inhibits c-Abl and Src and its downstream signaling pathways, PI3K/AKT/mTOR and JAK/STAT3. In addition, bosutinib enhances the cytotoxic effects of doxorubicin on ESCC cells. In summary, our results reveal that Src and Abl are potential therapeutic targets in ESCC and that the novel Src/Abl inhibitor bosutinib alone or in combination with other chemotherapeutic agents may be a viable option for treating ESCC patients.

Identification of inhibitors synergizing gemcitabine sensitivity in the squamous subtype of pancreatic ductal adenocarcinoma (PDAC).

Pancreatic adenocarcinoma (PDAC) is a highly aggressive cancer with a high chance of recurrence, limited treatment options, and poor prognosis. A recent study has classified pancreatic cancers into four molecular subtypes: (1) squamous, (2) immunogenic, (3) pancreatic progenitor and (4) aberrantly differentiated endocrine exocrine. Among all the subtypes, the squamous subtype has the worst prognosis. This study aims to utilize large scale genomic datasets and computational systems biology to identify potential drugs targeting the squamous subtype of PDAC through combination therapy. Using the transcriptomic data available from the International Cancer Genome Consortium, Cancer Cell Line Encyclopedia and Connectivity Map, we identified 26 small molecules that could target the squamous subtype of PDAC. Among them include inhibitors targeting the SRC proto-oncogene (SRC) and the mitogen-activated protein kinase kinase 1/2 (MEK1/2). Further analyses demonstrated that the SRC inhibitors (dasatinib and PP2) and MEK1/2 inhibitor (pimasertib) synergized gemcitabine sensitivity specifically in the squamous subtype of PDAC cells (SW1990 and BxPC3), but not in the PDAC progenitor cells (AsPC1). Further analysis revealed that the synergistic effects are dependent on SRC or MEK1/2 activities, as overexpression of SRC or MEK1/2 completely abrogated the synergistic effects SRC inhibitors (dasatinib and PP2) and MEK1/2 inhibitor (pimasertib). In contrast, no significant toxicity was observed in the MRC5 human lung fibroblast and ARPE-19 human retinal pigment epithelial cells. Together, our findings suggest that combinations of SRC or MEK inhibitors with gemcitabine possess synergistic effects on the squamous subtype of PDAC cells and warrant further investigation.

Bone-Targeted Therapies in Cancer-Induced Bone Disease.

Cancer-induced bone disease is a major source of morbidity and mortality in cancer patients. Thus, effective bone-targeted therapies are essential to improve disease-free, overall survival and quality of life of cancer patients with bone metastases. Depending of the cancer-type, bone metastases mainly involve the modulation of osteoclast and/or osteoblast activity by tumour cells. To inhibit metastatic bone disease effectively, it is imperative to understand its underlying mechanisms and identify the target cells for therapy. If the aim is to prevent bone metastasis, it is essential to target not only bone metastatic features in the tumour cells, but also tumour-nurturing bone microenvironment properties. The currently available bone-targeted agents mainly affect osteoclasts, inhibiting bone resorption (e.g. bisphosphonates, denosumab). Some agents targeting osteoblasts begin to emerge which target osteoblasts (e.g. romosozumab), activating bone formation. Moreover, certain drugs initially thought to target only osteoclasts are now known to have a dual action (activating osteoblasts and inhibiting osteoclasts, e.g. proteasome inhibitors). This review will focus on the evolution of bone-targeted therapies for the treatment of cancer-induced bone disease, summarizing preclinical and clinical findings obtained with anti-resorptive and bone anabolic therapies.

Suppression of IL-8-Src signalling axis by 17ß-estradiol inhibits human mesenchymal stem cells-mediated gastric cancer invasion.

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs-mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin-8 (IL-8) protein. Administration of IL-8 specific neutralizing antibody significantly inhibits HBMMSCs-mediated gastric cancer motility. Treatment of recombinant IL-8 soluble protein confirmed the role of IL-8 in mediating HBMMSCs-up-regulated cell motility. IL-8 up-regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17ß -estradiol inhibit HBMMSCS-induced cell motility via suppressing activation of IL8-Src signalling in human gastric cancer cells. 17ß-estradiol inhibits IL8-up-regulated Src downstream target proteins including p-Cas, p-paxillin, p-ERK1/2, p-JNK1/2, MMP9, tPA and uPA. These results suggest that 17ß-estradiol significantly inhibits HBMMSCS-induced invasive motility through suppressing IL8-Src signalling axis in human gastric cancer cells.

In Vitro and In Vivo Anti-Melanoma Effects of Pituranthos tortuosus Essential Oil Via Inhibition of FAK and Src Activities.

A large number of plants used in traditional medicines have been shown to possess antitumor activities. The aims of this study were to evaluate any anticancer effect of the essential oil (EO) extracted from P. tortuosus against B16F10 melanoma cancer cells in vitro as well as in vivo. In vitro, EO was shown to induce apoptosis and to inhibit migration and invasion processes. Further investigation revealed that EO decreased focal adhesion and invadopodia formation which was accompanied by a drastic downregulation of FAK, Src, ERK, p130Cas and paxillin. Moreover, EO treatment decreased the expression level of p190RhoGAP, and Grb2, which impair cell migration and actin assembly. Mice bearing the melanoma cells were used to confirm any in vivo effectiveness of the EO as an anti-tumor promoting agent. In mice dosed with 100 mg EO/kg/d (for 27 days), tumor weight was inhibited by 98% compared to that in mice that did not receive the product. In conclusion, these data suggested to us that an EO of P. tortuosus could evolve to be a potential medicinal resource for use in the treatment of cancers.

Oxidative stress contributes to the enhanced expression of Gqα/PLCß1 proteins and hypertrophy of VSMC from SHR: role of growth factor receptor transactivation.

We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCß1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCß1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCß1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCß1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCß1 proteins and resultant VSMC hypertrophy.

Explaining why Gleevec is a specific and potent inhibitor of Abl kinase.

Tyrosine kinases present attractive drug targets for specific types of cancers. Gleevec, a well-known therapeutic agent against chronic myelogenous leukemia, is an effective inhibitor of Abl tyrosine kinase. However, Gleevec fails to inhibit closely homologous tyrosine kinases, such as c-Src. Because many structural features of the binding site are conserved, the molecular determinants responsible for binding specificity are not immediately apparent. Some have attributed the difference in binding specificity of Gleevec to subtle variations in ligand-protein interactions (binding affinity control), whereas others have proposed that it is the conformation of the DFG motif, in which ligand binding is only accessible to Abl and not to c-Src (conformational selection control). To address this issue, the absolute binding free energy was computed using all-atom molecular dynamics simulations with explicit solvent. The results of the free energy simulations are in good agreement with experiments, thereby enabling a meaningful decomposition of the binding free energy to elucidate the factors controlling Gleevec's binding specificity. The latter is shown to be controlled by a conformational selection mechanism and also by differences in key van der Waals interactions responsible for the stabilization of Gleevec in the binding pocket of Abl.

Oestrogen receptor-alpha regulates non-canonical Hedgehog-signalling in the mammary gland.

Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-src(Act) mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty.

High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

BACKGROUND: Elevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, ß-Catenin or GSK3ß but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling. METHODS: Twelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level. RESULTS: High basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of ß-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased ß-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity. CONCLUSIONS: High basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.

Cigarette smoke activates the proto-oncogene c-src to promote airway inflammation and lung tissue destruction.

The diagnosis of chronic obstructive pulmonary disease (COPD) confers a 2-fold increased lung cancer risk even after adjusting for cigarette smoking, suggesting that common pathways are operative in both diseases. Although the role of the tyrosine kinase c-Src is established in lung cancer, less is known about its impact in other lung diseases, such as COPD. This study examined whether c-Src activation by cigarette smoke contributes to the pathogenesis of COPD. Cigarette smoke increased c-Src activity in human small airway epithelial (SAE) cells from healthy donors and in the lungs of exposed mice. Similarly, higher c-Src activation was measured in SAE cells from patients with COPD compared with healthy control subjects. In SAE cells, c-Src silencing or chemical inhibition prevented epidermal growth factor (EGF) receptor signaling in response to cigarette smoke but not EGF stimulation. Further studies showed that cigarette smoke acted through protein kinase C α to trigger c-Src to phosphorylate EGF receptor and thereby to induce mitogen-activated protein kinase responses in these cells. To further investigate the role of c-Src, A/J mice were orally administered the specific Src inhibitor AZD-0530 while they were exposed to cigarette smoke for 2 months. AZD-0530 treatment blocked c-Src activation, decreased macrophage influx, and prevented airspace enlargement in the lungs of cigarette smoke-exposed mice. Moreover, inhibiting Src deterred the cigarette smoke-mediated induction of matrix metalloproteinase-9 and -12 in alveolar macrophages and lung expression of cathepsin K, IL-17, TNF-α, MCP-1, and KC, all key factors in the pathogenesis of COPD. These results indicate that activation of the proto-oncogene c-Src by cigarette smoke promotes processes linked to the development of COPD.

Inhibition of Gαi activity by Gßγ is mediated by PI 3-kinase-γ- and cSrc-dependent tyrosine phosphorylation of Gαi and recruitment of RGS12.

Others and we have characterized several Gßγ-dependent effectors in smooth muscle, including G protein-coupled receptor kinase 2 (GRK2), PLCß3, and phosphatidylinositol (PI) 3-kinase-γ, and have identified various signaling targets downstream of PI 3-kinase-γ, including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gßγ acting via PI 3-kinase-γ and cSrc exerts an inhibitory influence on Gαi activity. The Gi2-coupled δ-opioid receptor agonist d-penicillamine (2,5)-enkephalin (DPDPE) activated cSrc, stimulated tyrosine phosphorylation of Gαi2, and induced regulator of G protein signaling 12 (RGS12) association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the COOH-terminal sequence of GRK2-(495-689), a Gßγ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002, and a selective PI 3-kinase-γ inhibitor, AS-605420. Expression of tyrosine-deficient (Y69F, Y231F, or Y321F) Gαi2 mutant or knockdown of RGS12 blocked Gαi2 phosphorylation and Gαi2-RGS12 association and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or ACh to activate, respectively, Gi1-coupled somatostatin (sstr3) receptors, and Gi3-coupled adenosine A1 or muscarinic m2 receptors elicited cSrc activation, Gαi1 or Gαi3 phosphorylation, Gαi1-RGS12 or Gαi3-RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and PP2. The results demonstrate that Gßγ-dependent tyrosine phosphorylation of Gαi1/2/3 by cSrc facilitated recruitment of RGS12, a Gαi-specific RGS protein with a unique phosphotyrosine-binding domain, resulting in rapid deactivation of Gαi and facilitation of smooth muscle relaxation.

Cbl negatively regulates erythropoietin-induced growth and survival signaling through the proteasomal degradation of Src kinase.

We examined the biological functions of the gene Cbl in erythropoietin (EPO) signaling using Cbl-deficient F-36P human erythroleukemia cells by the introduction of the Cbl siRNA expression vector. Knockdown of Cbl promoted EPO-dependent proliferation and survival of F-36P cells, especially at a low concentration of EPO (0.01U/mL), similar to serum concentrations of EPO in healthy volunteers (0.005-0.04U/mL). We found that Src was degraded mainly by the proteasomal pathway because the proteasome inhibitor MG-132 but not the lysosome inhibitor NH4Cl suppressed the EPO-induced degradation of Src in F-36P cells and that knockdown of Cbl inhibited EPO-induced ubiquitination and degradation of Src in F-36P cells. The experiments using the Src inhibitor PP1 and co-expression experiments further confirmed that Cbl and the kinase activity of Src are required for the EPO-induced ubiquitination of Src. In addition, the co-expression experiments and in vitro kinase assay demonstrated that the EPO-induced tyrosine phosphorylation and ubiquitination of Cbl were dependent on the kinase activity of Src but not Jak2. Thus, Cbl negatively regulates EPO signaling mainly through the proteasome-dependent degradation of Src, and the E3 ligase activity of Cbl and its tyrosine phosphorylation are regulated by Src but not Jak2.

Ligand-based pharmacophore modeling and Bayesian approaches to identify c-Src inhibitors.

Abstract Cellular Src (c-Src) kinases play a critical role in cell adhesion, proliferation, angiogenesis and cancer. Ligand-based pharmacophore models, used to identify the critical chemical features of c-Src inhibitors, were generated and validated by training, test and decoy sets, respectively. Best pharmacophore model, Hypo1, consists of four features such as HBA, HBD, Hy-Ar and RA. Hypo1 was used in virtual screening of the chemical databases such as Maybridge, Chembridge and NCI. The sorted compounds by Hypo1 were further reduced by applying drug-like properties and ADMET. Totally, 85 compounds which showed the good drug-like properties were selected from three databases and subjected to molecular docking for refinement of the retrieved hits by analysing the suitable orientation of the compounds in the active site of c-Src. Finally, 18 compounds were selected based on consensus scoring and hydrogen bond interactions with critical amino acids such as Met341, Thr338, Glu339 or Asp404. In addition, the Bayesian model was generated from the training set to find suitable fragments for inhibition of the c-Src function. Based on the above finding, we suggested that the Hypo1 and the good fragments from the Bayesian model will be helpful to select the compounds from various databases to identify the novel and potent c-Src inhibitor.

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways.

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors.

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.

False-negative MRI biomarkers of tumour response to targeted cancer therapeutics.

BACKGROUND: Non-invasive quantitative imaging biomarkers are essential for the evaluation of novel targeted therapeutics. Before deployment in clinical trials, such imaging biomarkers require qualification, typically through pre-clinical identification of imaging-pathology correlates. METHODS: First, in investigating imaging biomarkers of invasion, the response of orthotopic murine PC3 prostate xenografts to the Src inhibitor saracatinib was assessed using susceptibility contrast MRI. Second, the longitudinal response of chemically induced rat mammary adenocarcinomas to the VEGFR2 inhibitor vandetanib was monitored by intrinsic susceptibility MRI, to identify the time window of transient vascular normalisation. RESULTS: No significant differences in fractional blood volume (%), vessel calibre (µm), native T(1) (ms) or apparent water diffusion coefficient were determined, despite reduced expression of activated Fak and paxillin in the saracatinib cohort. Treatment with vandetanib elicited a 60% antitumour response (P<0.01), 80% inhibition in vessel density (P<0.05) and reduction in hypoxia (P<0.05). There was, however, no significant change in tumour baseline R(2)* (s(-1)) or carbogen-induced ΔR(2)* with treatment. CONCLUSION: Reporting negative imaging biomarker responses is important, to avoid the risk of clinical trials using the same biomarkers being undertaken with a false expectation of success, and the abandonment of promising new therapeutics based on a false-negative imaging biomarker response being mistaken for a true-negative.