Dynein Function and Protein Clearance Changes in Tumor Cells Induced by a Kunitz-Type Molecule, Amblyomin-X

Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. the supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. the collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. the characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-kappa B blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. in investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Butantan Inst, Biochem & Biophys Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/05969-4FAPESP: CAT/CEPID 1998/14307-9FAPESP: CETICs 2013/07467-1Web of Scienc

Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patient’s tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types

Effects of Lopap on Human Endothelial Cells and Platelets

Severe consumption coagulopathy has been detected in rats after Lopap (a prothrombin activator from Lonomia obliqua caterpillar bristles) infusion and in humans after accidental contact with L. obliqua bristles. However, platelet count and antithrombin (AT) levels were only modestly affected, suggesting that a different form of blood coagulation activation may be involved in this hemorrhagic syndrome. Here we describe that Lopap had no effect on aggregation of washed human platelets induced by several agonists, suggesting that it might not impair platelet function in vivo. AT was able to inhibit the amidolytic activity of thrombin generated by incubation of Lopap with prothrombin in a purified system, which may be different from that generated by the prothrombinase complex in vivo. The surface expression of both ICAM-1 and E-selectin but not of VCAM-1 was upregulated by Lopap in cultured HUVEC, suggesting that it may behave differently from other mediators, such as thrombin and tumor necrosis factor-α.Fil: Chudzinski-Tavassi, A.M.. Butantan Institute; BrasilFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fritzen, M.. Butantan Institute; BrasilFil: Pozner, Roberto Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Reis, C. V.. Butantan Institute; BrasilFil: Lourenço, D.. Universidade Federal de Sao Paulo; BrasilFil: Lazzari, María Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Expression of two endoplasmic reticulum stress markers, GRP78 and GADD153, is involved in the mechanism of action of the Amblyomin-X.

Inst Butantan, Biochem & Biophys Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniv São Paulo, Fac Med, Dept Radiol & Oncol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilWeb of Scienc

Relationship between autophagy marker expression and dynein in the mechanism of action of Amblyomin-X.

<p>Representative western blots of whole-cell lysates of cultured cells treated with vehicle (PBS), 0.5 µM Ambly for 2 h, 4 h or 24 h, 5 µM MG-132 for 24 h or 0.2 µM rapamycin for 16 h. Images are representative of three independent experiments containing the autophagic markers (mTOR, AMBRA1, LC3-I and LC3-II) in <b>A</b>) SK-MEL-28 cells and (<b>B</b>) MIA PaCa-2 cells and (<b>C</b>) autophagic/apoptosis marker (Bim) in SK-MEL-28 cells and (<b>D</b>) MIA PaCa-2 cells. Confocal microscopy analysis of cultured cells treated with vehicle (PBS) or 0.5 µM Ambly for 24 h. The final overlay image represents five fields of three independent experiments in which (<b>E</b>) the red fluorescence represents HC1, while the green fluorescence represents mTOR and the merging of the two is in yellow; or (<b>F</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents AMBRA1 (originally, the yellow fluorescence was artificially colored by the microscope software) and the merging of the two is in yellow; or (<b>G</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents Bim and the merging of the two is in yellow.</p