Skip regulates TGF- β 1-induced extracellular matrix degrading proteases expression in human PC-3 prostate cancer cells

Purpose. To determine whether Ski-interacting protein (SKIP) regulates TGF-β1-stimulated expression of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and uPA Inhibitor (PAI-1) in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA) compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF-β1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF-β1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF-β1. The ectopic expression of SKIP inhibited both TGF-β1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF-β1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment. © 2013 Victor Villar et al

Spred2 inhibits TGF-beta 1-induced urokinase type plasminogen activator expression, cell motility and epithelial mesenchymal transition

TGF-beta 1 is a potent inductor of malignance in cancer cells. TGF-beta 1 stimulates the expression of extracellular matrix degrading proteases, cell migration and it is also involved in the epithelial-mesenchymal transition (EMT). In the present work, we analyzed the role of Spred2 in the urokinase-type plasminogen activator (uPA) stimulation, EMT and cell migration by TGF-beta 1. We found that both the expression of mRNA and the protein level of Spred2 were lower in transformed keratinocytes PDV compared with immortalized keratinocytes MCA-3D. The transient ectopic expression of Spred2 in PDV cells inhibited the TGF-beta 1-transactivated SRE-Luc reporter which is related with the ERK1,2 signal. The stable ectopic expression of Spred2 in PDV cells (SP cells) led to the loss of ERK 1,2 activation by TGF-beta 1, although Smad2 activation was not affected, and the knockdown of Spred2 enhanced the activation of ERK1,2 signal by TGF-beta 1. The increment of uPA expression induced by TGF-beta 1 was suppressed in SP cells. In contrast, the stimulus on PAI-1 expression was not affected and comparable to parental PDV cells. SP cells under TGF-beta 1 treatment were unable to display the EMT, since the overexpression of Spred2 abolished the TGF-beta 1-induced disruption of the E-cadherin cell to cell interactions, reorganization of the actin cytoskeleton and upregulation of the mesenchymal marker vimentin. Finally, SP cells could not respond to the TGF-beta 1 stimulus on cell migration. Taken together, the data in the present study suggests that Spred2 is a regulator of TGF-beta 1-induced malignance in transformed keratinocytes

Avaliação de marcadores de sequenciamento RAD para inferência filogenética de máxima verossimilhança no gênero Barbacenia (Velloziaceae)

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.Neste trabalho, testei marcadores obtidos a partir do sequenciamento RAD (Restricted siteassociated DNA sequencing) para a inferência de filogenias em um clado de plantas tropicais. Todas as análises foram feitas com a utilização de pipelines de montagem de genomas e de inferência filogenética de acesso livre. Para isso, amostras de sequenciamento RAD de 31 espécies do gênero Barbacenia, previamente coletadas e sequenciadas, foram analisadas. Os alinhamentos foram testados com parâmetros distintos e os diferentes datasets resultantes foram analisados para a reconstrução de uma árvore filogenética com método de inferência de máxima verosimilhança. As árvores filogenéticas resultantes de cada alinhamento foram comparadas para verificar os parâmetros como sustentação e comprimento dos ramos. As árvores inferidas somente com base nos marcadores RAD foram então comparadas com o conhecimento científico mais atual sobre a família, que baseia-se em quatro sequências intergênicas obtidas pelo método de sequenciamento Sanger. Com isso, foi possível identificar resultados discrepantes entre os marcadores, a partir dos quais o potencial de utilização de marcadores RAD foi avaliado, especialmente no que diz respeito à resolução das relações referentes aos nós mais profundos da filogenia em questão.I tested the precision of markers obtained by RAD (Restricted site-associated DNA) sequencing to infer phylogenies in a clade of tropical plants by the utilization of freely accessible pipelines. For that, samples of 31 species of the genus Barbacenia, previously collected and sequenced, were analyzed using assemblage pipelines. This process was tested with different parameters and the resulting datasets were analyzed to infer a phylogenetic tree by the method of maximum likelihood. The resulting trees were compared to verify parameters such as branch length and node support. The trees inferred using RAD markers only were then compared to the current knowledge of the group, which is based on 4 intergenic sequences obtained by the Sanger sequencing method. By evaluating the obtained trees and the combined tree it was possible to identify possible discrepancies within the methods. These analyses showed the potential of using RAD markers to elucidate phylogenetic relationships, especially regarding the most ancient nodes within this group

The Metabolic Features of Tumor-Associated Macrophages: Opportunities for Immunotherapy?

Besides transformed cells, the tumors are composed of various cell types that contribute to undesirable tumor progression. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in the tumor microenvironment (TME). Within the TME, TAMs exhibit high plasticity and undergo specific functional metabolic alterations according to the availability of tumor tissue oxygen and nutrients, thus further contributing to tumorigenesis and cancer progression. Here, we review the main functional TAM metabolic patterns influenced by TME, including glycolysis, amino acid, and fatty acid metabolism. Moreover, this review discusses antitumor immunotherapies that affect TAM functionality by inducing cell repolarizing and metabolic profiles towards an antitumoral phenotype. Also, new macrophage-based cell therapeutic technologies recently developed using chimeric antigen receptor bioengineering are exposed, which may overcome all solid tumor physical barriers impeding the current adoptive cell therapies and contribute to developing novel cancer immunotherapies

The metabolic features of tumor-associated macrophages : opportunities for immunotherapy?

Funding: This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia.Besides transformed cells, the tumors are composed of various cell types that contribute to undesirable tumor progression. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in the tumor microenvironment (TME). Within the TME, TAMs exhibit high plasticity and undergo specific functional metabolic alterations according to the availability of tumor tissue oxygen and nutrients, thus further contributing to tumorigenesis and cancer progression. Here, we review the main functional TAM metabolic patterns influenced by TME, including glycolysis, amino acid, and fatty acid metabolism. Moreover, this review discusses antitumor immunotherapies that affect TAM functionality by inducing cell repolarizing and metabolic profiles towards an antitumoral phenotype. Also, new macrophage-based cell therapeutic technologies recently developed using chimeric antigen receptor bioengineering are exposed, which may overcome all solid tumor physical barriers impeding the current adoptive cell therapies and contribute to developing novel cancer immunotherapies.Publisher PDFPeer reviewe

SKIP is required for TGF-beta 1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor-beta 1 (TGF-beta 1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-beta 1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-beta 1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-beta 1-induced EMT, and the cells were sensitized to the TGF-beta 1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-beta 1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-beta 1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-beta 1 in transformed cells