Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of src activity is associated with the inability of src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and dormancy of angiogenesis. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signalling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF – dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms

Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis

The aims of the study were to meet the demand of new tubulin antagonists with fewer side effects by characterizing the antiangiogenic properties of the experimental compound spongistatin 1, and to elucidate nonmitotic mechanisms by which tubulin antagonists inhibit angiogenesis. Although tubulin-inhibiting drugs and their antiangiogenic properties have been investigated for a long time, surprisingly little is known about their underlying mechanisms of action. Antiangiogenic effects of spongistatin 1 were investigated in endothelial cells in vitro, including functional cell-based assays, live-cell imaging, and a kinome array, and in the mouse cornea pocket assay in vivo. Spongistatin 1 inhibited angiogenesis at nanomolar concentrations (IC50: cytotoxicity>50 nM, proliferation 100 pM, migration 1.0 nM, tube formation 1.0 nM, chemotaxis 1.0 nM, aortic ring sprouting 500 pM, neovascularization in vivo 10 μg/kg). Further, a kinome array and validating data showed that spongistatin 1 inhibits the phosphorylation activity of protein kinase Cα (PKCα), an essential kinase in angiogenesis, and its translocation to the membrane. Thus, we conclude that PKCα might be an important target for the antiangiogenic effects of tubulin antagonism. In addition, the data from the kinase array suggest that different tubulin antagonists might have individual intracellular actions.—Rothmeier, A. S., Ischenko, I., Joore, J., Garczarczyk, D., Fu¨rst, R., Bruns, C. J., Vollmar, A. M., Zahler, S. Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis

EFEMP1 binds the EGF receptor and activates MAPK and Akt pathways in pancreatic carcinoma cells

The EGF-related protein EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1) has been shown to promote tumor growth in human adenocarcinoma. To understand the mechanism of this action, the signal transduction activated upon treatment with this protein has been investigated. We show that EFEMP1 binds EGF receptor (EGFR) in a competitive manner relative to epidermal growth factor (EGF), implicating that EFEMP1 and EGF share the same or adjacent binding sites on the EGFR. Treatment of pancreatic carcinoma cells with purified EFEMP1 activates autophosphorylation of EGFR at the positions Tyr-992 and Tyr-1068, but not at the position Tyr-1048. This signal is further transduced to phosphorylation of Akt at position Thr-308 and p44/p42 MAPK (mitogen-activated protein kinase) at positions Thr-202 and Tyr-204. These downstream phosphorylation events can be inhibited by treatment with the EGFR kinase inhibitor PD 153035. The observed signal transduction upon treatment with EFEMP1 can contribute to the enhancement of tumor growth shown in pancreatic carcinoma cells overexpressing EFEMP1

EFEMP1 binds the EGF receptor and activates MAPK and Akt pathways in pancreatic carcinoma cells

The EGF-related protein EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1) has been shown to promote tumor growth in human adenocarcinoma. To understand the mechanism of this action, the signal transduction activated upon treatment with this protein has been investigated. We show that EFEMP1 binds EGF receptor (EGFR) in a competitive manner relative to epidermal growth factor (EGF), implicating that EFEMP1 and EGF share the same or adjacent binding sites on the EGFR. Treatment of pancreatic carcinoma cells with purified EFEMP1 activates autophosphorylation of EGFR at the positions Tyr-992 and Tyr-1068, but not at the position Tyr-1048. This signal is further transduced to phosphorylation of Akt at position Thr-308 and p44/p42 MAPK (mitogen-activated protein kinase) at positions Thr-202 and Tyr-204. These downstream phosphorylation events can be inhibited by treatment with the EGFR kinase inhibitor PD 153035. The observed signal transduction upon treatment with EFEMP1 can contribute to the enhancement of tumor growth shown in pancreatic carcinoma cells overexpressing EFEMP1

Large scale analysis of amino acid substitutions in bacterial proteomics

Background: Proteomics of bacterial pathogens is a developing field exploring microbial physiology, gene expression and the complex interactions between bacteria and their hosts. One of the complications in proteomic approach is micro- and macro-heterogeneity of bacterial species, which makes it impossible to build a comprehensive database of bacterial genomes for identification, while most of the existing algorithms rely largely on genomic data. Results: Here we present a large scale study of identification of single amino acid polymorphisms between bacterial strains. An ad hoc method was developed based on MS/MS spectra comparison without the support of a genomic database. Whole-genome sequencing was used to validate the accuracy of polymorphism detection. Several approaches presented earlier to the proteomics community as useful for polymorphism detection were tested on isolates of Helicobacter pylori, Neisseria gonorrhoeae and Escherichia coli. Conclusion: The developed method represents a perspective approach in the field of bacterial proteomics allowing to identify hundreds of peptides with novel SAPs from a single proteome

Additional file 1 of Large scale analysis of amino acid substitutions in bacterial proteomics

Supplementary materials with Figures S1-S12 and Tables S1-S5. (PDF 855 kb