The MEK1/2 inhibitor, selumetinib (AZD6244; ARRY-142886), enhances anti-tumour efficacy when combined with conventional chemotherapeutic agents in human tumour xenograft models.

BACKGROUND: The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy. METHODS: The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations. RESULTS: Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib. CONCLUSION: The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.

Metal organic chemical vapour deposition of vertically aligned ZnO nanowires using oxygen donor adducts.

Vertically aligned zinc oxide (ZnO) nanowires (NWs) have been grown by liquid injection Metal Organic Chemical Vapour Deposition, using oxygen donor adducts of Me2Zn. The growth and characterisation of the nanowires grown using [Me2Zn(L)] where L = monodentate ethers, tetrahydrofuran (C4H8O) (1), tetrahydropyran (C5H10O) (2), furan (C4H4O) (3) and the bidentate ethers, 1,2-dimethoxyethane (C4H12O2,) (4) 1,4-dioxane (C4H8O2) (5) and 1,4-thioxane (C4H8SO) (6) is discussed. Single crystal X-ray structures of (4), (5), (6) have been established and are included here. The ZnO NWs were deposited in the absence of a seed catalyst on Si(111) and F-doped SnO2/glass substrates over the temperature range 350-600 degrees C. X-ray diffraction (XRD) data shows that the nanowires grown from all adduct precursors were deposited in the wurtzitic phase.

Specific demonstration of drug-induced tumour cell apoptosis in human xenografts models using a plasma biomarker.

Pharmacodynamic (PD) assays should be used before advancing new drugs to clinical trials. Most PD assays measure the response to drugs in tissue, a procedure which requires tissue biopsies. The M30-Apoptosense ELISA is a PD biomarker assay for the quantitative determination of caspase-cleaved cytokeratin 18 (CK18) released from apoptotic carcinoma cells into blood. We here demonstrate that whereas the M30-Apoptosense ELISA assay detects human caspase-cleaved CK18, the mouse and rat CK18 caspase cleavage products are detected with low affinity. The M30-Apoptosense ELISA therefore facilitates the determination of drug-induced apoptosis in human tumour xenografts in rodents using plasma samples, largely independently from host toxicity. Increases of caspase-cleaved CK18 were observed in plasma from different carcinoma xenograft models in response to anticancer drugs. The appearance caspase-cleaved CK18 in plasma was found to reflect formation of the caspase-cleaved epitope in FaDu head-neck carcinomas and in cultured cells. The M30-Apoptosense assay allows determination of tumour response in blood from xenograft models and from patients, providing a powerful tool for translational studies of anticancer drugs.

Multiplex PCRs for identification of Escherichia coli virulence genes.

PCRs were developed to detect 11 Escherichia coli virulence genes. Primers amplified the respective genes without cross-reaction with other genes. Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions. These reactions successfully identified genes in E. coli from the feces of four dogs.

Evidence for a compensatory increase in synthesis of brush border enzymes in the jejunum of Irish Setters with gluten-sensitive enteropathy.

OBJECTIVE: To determine whether biosynthesis of aminopeptidase N (ApN), alkaline phosphatase (AP), and total microvillus membrane protein is altered in Irish Setters with gluten-sensitive enteropathy (GSE). ANIMALS: A litter of 6 Irish Setters with GSE and 3 healthy Greyhounds. PROCEDURES: Explants obtained from affected dogs at 4 and 12 months were maintained in vitro and were compared with material from healthy control dogs. Biosynthesis of ApN and AP was monitored by incorporation of [35S]methionine and immunoprecipitation of these enzymes. RESULTS: Jejunal explants from affected Irish Setters had significantly higher rates of biosynthesis of total protein, microvillus membrane protein, AP, and ApN, compared with control tissue. Two forms of ApN with apparent molecular mass of 155 and 135 kd and 4 forms of AP with apparent molecular mass of 210 to 260, 150, 130, and 105 kd were identified in total membrane fractions from control and affected dogs. CONCLUSIONS: Reduced activities of ApN and AP in dogs with GSE are not attributable to decreased synthesis of these proteins and document enhanced synthesis of microvillar membrane proteins, which may be a compensatory response to enterocyte damage. The 150-kd form of AP was most prominent in tissue from the most affected dogs, probably representing an early form of this enzyme. In contrast, the 105-kd form was most intense in tissue from controls and less intense in tissue of affected dogs.

Surface properties of diarrhoeagenic Escherichia coli isolates.

The surface properties of various Escherichia coli isolates associated with diarrhoeal illness were compared by aqueous partitioning between polyethylene glycol (PEG) and Dextran phases. Two well characterised strains of enteropathogenic E. coli (EPEC) were found to be very hydrophobic, based on the critical polymer concentration. EPEC strain E2348 cured of the EPEC adherence factor (EAF) plasmid had much reduced surface hydrophobicity. Partitioning of a series of diarrhoeagenic E. coli strains demonstrated that the majority of EAF+ EPEC strains were significantly more hydrophobic than EAF- EPEC strains. E. coli strains defined as enteroaggregative on the basis of hybridisation with a specific DNA probe showed much greater heterogeneity in their partitioning behaviour, possibly indicating that the AAF/I pili were not expressed in all strains. The E. coli K-12 strain used as a transformation host for adhesion studies had very low surface hydrophobicity but had a detectable negative charge. No alteration in these properties was observed when transformed with EPEC and recombinant plasmids known to specify adherence to tissue culture cells.

Growth and propagation of normal rat intestinal epithelial cells.

A combination of mild proteolytic digestion and selective growth stimulation has been used to isolate and propagate adult rat intestinal epithelial cells with a finite life span. Growth of these cells on a variety of matrices and on mesenchymal cells has resulted in the expression of brush border enzymes including sucrase-isomaltase, aminopeptidase N, and alkaline phosphatase. Examination of the cells at the electron microscopic level has revealed that although these cells express key brush border enzymes, they do not have a fully formed brush border. These findings suggest that the expression of brush border enzymes and structural proteins represent distinct stages of enterocyte differentiation that are under separate transcriptional and temporal control.

Effects of concanavalin A and pokeweed lectins on microvillar membrane proteins during the organ culture of rabbit intestinal mucosa.

The effects of pokeweed lectin (PWL) and concanavalin A (Con A) on microvillar membrane (MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli (EPEC). PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium. Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased. Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles. Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio. These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis.

Use of indirect immunofluorescence and western blotting to assess the role of circulating antimyocardial antibodies in dogs with dilated cardiomyopathy.

The presence of circulating anti-heart IgG and IgM autoantibodies was assessed by indirect immunofluorescence and by probing Western blot transfers of normal canine myocardial proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis with samples of sera from dogs with dilated cardiomyopathy (n = 27) and from healthy control dogs (n = 20). No difference was demonstrated between the two groups using either method. Sera taken from the affected dogs at three monthly intervals throughout the course of the disease showed no change at all in the pattern of reactivity. The results of this study suggest that indirect immunofluorescence and the probing of Western blot transfers of a crude preparation of normal canine myocardium with canine sera will demonstrate too many apparent autoantigens for the technique to identify unique, disease-associated autoantibodies.

Low molecular weight angiogenesis factors.

A number of substances have been proposed for the role of angiogenesis factors. Many of these are of protein origin and are therefore amenable to the tools of the molecular biologist. However a number of low molecular weight angiogenesis factors are emerging as important initiators and/or cofactors of neovascularization. Of these a number are known to stimulate angiogenesis indirectly, possibly through an inflammatory response. Some putative angiogenic factors stimulate microvessel endothelial cells nonspecifically, also causing migration and proliferation of large vessel cells. Others are specific for microvessel cells either for stimulating migration, proliferation or both. The nature and action of the low molecular weight factors in vivo and in vitro are reviewed.

A synergistic effect on microvessel cell proliferation between basic fibroblast growth factor (FGFb) and endothelial cell stimulating angiogenesis factor (ESAF).

Stimulation of microvessel endothelial cells grown on collagen gels by endothelial cell stimulating angiogenesis factor was confirmed (1). The potent endothelial cell growth stimulating activity of basic fibroblast growth factor has also been demonstrated for cells grown on collagen gels. However, the growth factor activity of basic fibroblast growth factor towards microvessel endothelial cells was almost eliminated when experiments were carried out in the presence of diafiltered foetal calf serum, but was partially restored by the addition of endothelial cell stimulating angiogenesis factor to medium containing the diafiltered serum. In contrast to these observations, foetal skin fibroblasts were stimulated by basic fibroblast growth factor when grown in medium with diafiltered serum, and this stimulation was not modified by the presence of endothelial cell stimulating angiogenesis factor.