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

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

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

Preventive activities of the National Police of Ukraine regarding violations of rights and freedoms of internally displaced persons

The purpose of the investigation is to make a theoretical generalization in terms of revealing the essence of the preventive activities of the National Police aimed at the exclusion of violations of the rights and freedoms of internally displaced persons. Main content. It should be noted that today Ukraine has been faced with an unknown problem - the need to solve problems related to forced migrants, which requires the development of effective mechanisms for the realization of the right to social protection of this category of the population, as part of the activities of the National Police. Methodology: The dialectical method of scientific knowledge is the methodological basis of the investigation. Conclusions. It has been established that the protection of the rights and freedoms of internally displaced persons within the activities of the preventive police forces is carried out by observing the following norms: the principle of supremacy of the law; preventive police forces guarantee the protection of the rights and freedoms of internally displaced persons regardless of their political beliefs and party affiliation.El propósito de la investigación consiste en realizar una generalización teórica en cuanto a develar la esencia de las actividades preventivas de la Policía Nacional dirigidas a la exclusión de violaciones a los derechos y libertades de los desplazados internos. Contenido principal. Cabe señalar que hoy Ucrania se ha enfrentado a un problema desconocido: la necesidad de resolver los problemas relacionados con los migrantes forzosos, lo que requiere el desarrollo de mecanismos efectivos para la realización del derecho a la protección social de esta categoría de población, como parte de las actividades de la Policía Nacional. Metodología: El método dialéctico del conocimiento científico es la base metodológica de la investigación. Conclusiones. Se ha establecido que la protección de los derechos y libertades de los desplazados internos dentro de las actividades de las fuerzas policiales preventivas se realiza mediante la observación de las siguientes normas: el principio de supremacía de la ley; las fuerzas policiales preventivas garantizan la protección de los derechos y libertades de los desplazados internos independientemente de sus creencias políticas y afiliación partidaria

Abstract 149: Antisense inhibition of microRNA-21 and -221 in tumor-initiating stem-like cells modulates biological functions of pancreatic cancer including tumorigenesis, metastasis, and chemoresistance

Abstract Introduction. Our preliminary studies identified a small population of pancreatic cancer cells with stem-like properties. These cells, called Side Population (SP) cells, were able to induce fast and aggressive tumor formation in nude mice. Cultured SP were able to differentiate into daughter SP or non-SP cells and were found to be highly chemoresistant. Furthermore, performed gene expression analysis showed a significant difference in the expression of more than 1300 genes in SP cells, among which the difference in microRNA expression between SP and non-SP cells was identified as the most interesting candidate for our further studies. Methods. Pancreatic cancer stem-like cells from highly metastatic cell line L3.6pl were identified and characterized by flow cytometry using Hoechst 33342 dye staining. The gene expression was assessed by Affymetrix and the results were further confirmed by quantitative RT-PCR. The antagomir transfection was performed using microRNA-21 and -221 oligonucleotide antisense. Tumor cell apoptosis, cell cycle progression, chemoresistance, and invasion were quantitated by propidium iodide staining and Boyden chamber assay, respectively. Results. In our study, some microRNAs, including miR-21, miR-221, miR-211, and miR-30c-2 were significantly upregulated in stem-like SP from L3.6pl cells. Interestingly, in these cells both miR-21 and miR-221 were involved in the modulation of expression of more than 200 genes, including RASSF6, RAB2B, TP63, TP53INP1, TP53INP2, TET1, MAPK10, MAP2K6, CDK6, TNFRSF11B, SOCS6, STK33, and SMAD7. The administration of antagomir-21 and -221 significantly reduced the SP fracture, decreased SP cell differentiation, positively affected L3.6pl cell proliferation, invasion, and chemoresistance against gemcitabine and 5-Fluorouracil. Conclusion. Our results demonstrate the significance of both microRNA-21 and -221 in tumor-initiating capability of stem-like tumor cells in pancreatic cancer. Both microRNAs contribute to the most important biological functions of pancreatic cancer including apoptosis, metastasis, and chemoresistance, and may further serve as a potential target for pancreatic cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 149. doi:10.1158/1538-7445.AM2011-149</jats:p

Abstract 3901: Sox9-associated overexpression of IFIT3 leads to pancreatic cancer progression by activation of “pseudoinflammatory” pathways

Abstract The understanding of invasion, angiogenesis and metastasis is essential for the development of new molecular therapy against cancer. Inflammation plays important role in tumour initiation and progression. Here we report the role of the transcription factor Sox9 for regulation of IFIT3 an inflammation-related and tumour-promoting protein in pancreatic cancer. For in vivo and in vitro experiments we utilized the following human pancreatic cancer cell lines: low metastatic FG, high metastatic L3.6pl, and the stable transfected cell line FG-IFIT3. To demonstrate effects on primary tumor growth and metastases in vivo we orthotopically injected the different cell lines in the pancreas of nude mice. To evaluate the VEGF depending angiogenic capacity of the different cell lines ELISA technology was used. By One STrEP technology we were able to identify IFIT3-binding partners. Chromosomal immunoprecipitation (ChIP) using anti-Sox9 antibody, followed by PCR amplifying the IFIT3-promoter was used to identify the interaction of the IFIT3 promoter with the transcription factor Sox9. To investigate Sox9-depending expression of IFIT3 (protein and RNA) we used stable transfected L3.6pl-Sox9-shRNA cells under control of the Tet-CMV promoter in presence or absence of tetracycline, respectively. Analysis of differential gene expression by gene array technology demonstrated that the IFIT3 gene is up-regulated in L3.6pl cells as compared to FG cells. Results of animal experiment and in vitro experiments clearly demonstrated tumor-promoting, pro-metastatic and pro-angiogenic features of IFIT3. RT-PCR has revealed that both treatment with IFna as well as NFkB led to up-regulation of IFIT3-RNA expression. One STrEP experiments identified JNK and STAT1 as binding partners of IFIT3. ChIP has demonstrated binding of the transcription factor Sox9 to the IFIT3 promoter. RT-PCR and immunoblot data demonstrated constitutive up-regulation of Sox9 expression in L3.6pl cells. By Western blotting and RT-PCR we could show that diminishing of Sox9 expression in stable transfected L3.6pl Sox9-shRNA cells leads to a significant down-regulation of IFIT3-epression on the RNA and protein level. The inflammation associated protein IFIT3 is up-regulated in metastatic L3.6pl human pancreatic cancer cells and is in part responsible for the aggressive primary pancreatic tumor growth in vivo. This gene is up-regulated by IFna and NFkB. Interestingly Sox9 binds to the IFIT3P and activates its expression. Since in L3.6pl cells Sox9 is constitutively over-expressed, IFIT3 is up-regulated independent on the presence of the cytokine IFna. Therefore, the pro-inflammatory IFna-signaling pathway is activated even without actual inflammation in absence pro-inflammatory cytokine. The activation of such a “pseudo-inflammatory pathway” seems to be in part responsible for pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3901.</jats:p