INTRODUCTION: Astrocytomas are common brain tumors. Increased expression levels of Interleukin-13 Receptor α2 (IL-13RA2) have been reported in astrocytomas. The Interleukin-13 signaling pathway may be associated with cell migration when binding to Interleukin-13 Receptor α1. OBJECTIVE: To investigate Interleukin-13 Receptor α1 (IL-13RA1) and IL13RA2 expression levels in human diffusely infiltrative astrocytomas and test the involvement of Interleukin-13 levels in cell migration in two glioblastoma cell lines. METHODS: IL13RA expression levels were accessed by quantitative real time PCR in 128 samples of astrocytomas and 18 samples of non-neoplastic brain tissues from temporal lobe epilepsy surgery. The impact of IL-13 levels (10 and 20 ng/mL) on cell migration was analyzed by the wound assay in U87MG and A172 cells. RESULTS: Glioblastoma presented higher IL13RA1 and IL13RA2 expression levels compared to lower grades astrocytomas and to non-neoplastic cases. U87MG and A172 cells presented higher expression levels of IL-13RA1 vs. IL-13RA2. A significant difference in migration rate was observed in A172 cells treated with 10 ng/mL of IL-13 vs. control: treated cells presented slower migration than non-treated cells. U87MG cells treated with IL-13 20ng/ mL presented slower migration than non-treated cells. This indicates that the IL13Rα1 signaling pathway was not activated, indeed inhibited by the decoy IL-13Rα2, slowing cell migration. This impact occurred with a lesser concentration of IL-13 on the A172 than on the U87MG cell line, because A172 cells have a higher IL-13RA2/A1 ratio. CONCLUSION: The present results suggest IL-13 receptors as possible targets to decrease tumor cell migration.
RESUMO: Este artigo refere-se ao Projeto de Extensão intitulado
Introduction: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in several cell processes, including the cell cycle, proliferation, apoptosis and mRNA processing. MELK has shown multiple features consistent with the potential utility of this gene as an anticancer target, including in astrocytomas, the most common brain tumor in adults. Nevertheless the pathway of MELK in tumorigenesis is not well known yet. Purpose: This study aim to analyze genes and proteins involved in the MELK pathway in the tumorigenesis of gliomas, enabling the discovery of new therapeutic targets. Material and Methods: Analysis of the expression profile of a panel of protein relevant to the process of tumorigenesis that MELK is involved was performed by Proteomic analysis by two dimensional electrophoresis and mass spectrometry for human glioma cell line U87MG transfected with siRNA for the MELK compared to U87MG transfected with non-target control (NTC) cells. Analysis and validation of gene expression was performed by Quantitative real time PCR (qRT-PCR) using SYBR Green method in a series of 154 astrocytomas of different malignant grade (23 AGI, 26 AGII, 18 AGIII and 87 GBM) relative to 22 non neoplastic (NN) brain samples. Results: Differentially expressed proteins in U87MG cell line transfected with siRNA for the MELK compared to NTC control cells were identified by proteomic analysis. One of the proteins with lower expression when MELK was silenced was Stathmin (STMN1). The analysis in astrocytoma samples showed lower significant STMN1 relative expression of astrocytoma samples when compared to NN. Spearman correlation for gene expression levels of MELK and STMN1 for GBM samples were statistically significant (r=0.66, p<0.003). We found significant differences of median survival times of two GBM groups: 39 with hypoexpression of both STMN1 and MELK (8 months), versus 9 with hypoexpression of STMN1 and hiperexpression of MELK (5 months), log rank=0.016. Conclusions: STMN1 plays a role in regulation of the cell cycle and is defined as an oncoprotein. Thus, our results showed that STMN1 is involved in the MELK pathway suggesting that this protein plays an important role in glioma tumorigenesis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B134. Citation Format: Miyuki Uno, Sueli Mieko Oba-Shinjo, Roseli Silva, Marcela Gimenez, Gisele Reis, Jose C. Rosa, Suely K. N. Marie. Stathmin is involved in the maternal embryonic leucine zipper kinase pathway in human astrocytomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B134.
Mitochondrial dysfunction plays a role in determining the phenotype through bioenergetic depletion and increased production of ROS in several diseases, including cancer. We have previously demonstrated a relevant reduction of mitochondrial DNA (mtDNA) copy number in astrocytoma of different grades of malignancy, predominantly in grade IV-glioblastoma (GBM). We observed a stepwise increase of TFAM in parallel to the increase of malignancy, and TFAM expression was higher in GBM patients with overall survival longer than 24 months than less than 12 months. TFAM is codified in the nucleus and transported into mitochondria, where is essential for mtDNA replication, repair and protection by forming nucleoid complexes. To further understand the role of TFAM in astrocytoma progression we performed TFAM immunohistochestry in human astrocytoma samples, confocal analysis in GBM cell lines, in vitro functional assays after silencing TFAM by siRNA in two GBM cell lines (U87MG, A172), and high throughput RNA-Seq in Illumina platform for network analysis by Metacore. TFAM protein was detected in cytoplasm in granular pattern of low grade astrocytomas, and also in nuclei of malignant atsrocytomas. Such localization was also confirmed by confocal analysis in two GBM cell lines. TFAM-siRNA transfection decreased TFAM expression level >80% in D2, which persisted up to D7 with evident decrease of mtDNA copy number after 4 days, and maintained until 7 days. Interestingly, a decrease in migration but not in proliferation was observed in both cell lines after TFAM knockdown. Comparison of RNA-Seq of TFAM-siRNA to NTC- siRNA, with 20 million of reads in each experiment performed in duplicate, resulted in 1000 genes differentially expressed and among them 315 were detected in both cell lines. These genes were in pathways related to cell adhesion (integrin-mediated: p = 5.79e-5, histamine H1 receptor signaling in the interruption of cell barrier integrity: p = 3.98e-5, chemokines-mediated: p = 1.22e-5) and ECM remodeling (p = 9.17e-5), supporting the results of in vitro migration assay. 82 genes were downregulated together with TFAM in both cell lines. These genes were in pathways related to immune response IL15 signaling via JAK-STAT cascade involving MyD88-independent toll-like receptor signaling pathway involving ADAM17 and STAT2 (p = 1.89e-5), regulation of cytoskeleton proteins (p = 1.65e-2), hypoxia-induced EMT in cancer and fibrosis (p = 3.37e-2), polyamine and arginine metabolism (p = 2.73e-2, p = 4.65e-2) and as expected, regulation of apoptotic process (p = 9.50e-21). The role of TFAM in cytoskeleton and ECM remodeling may worth additional studies. Also, TFAM in extracellular space, as released by necrotic cells, may potentiate mitochondrial N-formyl peptide-induced secretion of chemokines and activate inflammatory responses, which may open another strategy to further understand TFAM role in tumorigeniesis. Citation Format: Suely K. Marie, Roseli Silva, Antonio Lerario, Miyuki Uno, Sueli Mieko Oba-Shinjo. Mitochondrial DNA copy variation and TFAM expression in astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2015-3047
Toll-like receptors (TLRs) are the first receptors of the immune system that identify disturbances in homeostasis, capable of recognizing molecules associated to cell and tissue damage. TLRs signaling pathway activates inflammatory transcription factors, mainly Nuclear Factor-kappa B (NF-kB) and Interferon Regulatory Factors (IRF3/7). Due to their importance in inflammatory process, TLRs are research hot targets for therapy in several diseases, including cancer. This study aims to analyze TLRs (1, 2, 4 and 6) expressions in human diffusely infiltrative astrocytomas, WHO grades II to IV of malignancy (AGII-AGIV), and correlate their expressions among them and with the clinical outcome of AGIV (glioblastoma, GBM) patients. TLRs mRNA levels were evaluated by qRT-PCR in 143 astrocytoma samples and 22 non-neoplastic (NN) brain specimens from epilepsy surgery. Upregulated mRNA levels of all studied TLRs were observed in astrocytomas compared to NN cases (p<0.05), except for TLR2 in AGII. TLR1 and TLR2 median expression values increased according to astrocytoma malignancy. TLRs mRNA expressions presented positive correlation with significance (p<0.05) in all grades of astrocytoma, except for TLR2 and TLR4 in AGIII. Strong associations (r<0.7) were detected with TLR1-TLR2 mRNA expressions among AGII, AGIII and AGIV, TLR1-TLR4 in AGIII, and TLR6-TLR1 in AGII. In GBM patients, TLR2 upregulation was associated to better outcome with longer overall survival (p<0.007). The same was verified for concomitant upregulation of TLR1, 2 and 4 (p<0.05). Aiming to analyze the distribution of these receptors in different cells present in the tumor, we performed immunofluorescence staining of GBM cell lines (A172 and U87MG). All TLRs were detected in both cell lines, indicating that their expression were not limited to immune cells. The current literature has described controversial roles for the TLRs in cancer, either activating an anti-inflammatory condition leading to tumor progression or a pro-inflammatory state leading to tumor abrogation. TLRs distribution among different cells in the tumor environment might be relevant for the inflammatory process. Our findings of TLRs on tumor cells may suggest a tendency towards an anti-inflammatory state. The comprehension of TLRs role in each cell type, that compose the tumor, may help to determine the signaling pathways involved in diffusely infiltrative astrocytoma progression, and potential new therapeutic strategies. Citation Format: Isabele F. Moretti, Daiane Gil Franco, Roseli Silva, Thais F. Galatro, Sueli M. Oba-Shinjo, Suely K.N. Marie. Toll-like receptors 1, 2, 4 and 6 expression levels in diffusely infiltrative astrocytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1458.
ResumoO trabalho investiga a formação e dinâmica de redes temporárias, a partir dos princípios sociais de redes, e apresenta os dados de pesquisa do ramo de eventos móveis. A pesquisa bibliográfica revelou que são raros os trabalhos que investigam redes temporárias, isto é, grupos de empresas que se unem num trabalho coletivo, com tempo determinado. A proposição básica é que o prazo curto, como é o caso de eventos móveis, dificulta a rápida emergência de categorias sociais que formam as bases da rede, que são a confiança, o comprometimento, a governança e a solução de assimetrias. Foram realizadas entrevistas com representantes de empresas de eventos móveis. A análise sustentou a afirmativa em parte. A governança informal e a solução de assimetrias foram as categorias mais presentes, o que é coerente com as características técnicas e a necessidade de sincronismo do negócio. A rede dos entrevistados não apresenta sinais de confiança e comprometimento, mas uma parceria entre dois sujeitos apontou que essas categorias podem se desenvolver mesmo no curto período do evento. A presente pesquisa se justifica por trazer o benefício de se investigar as diferenças entre redes temporárias e redes perenes, quando o foco está nas relações sociais. Como benefício secundário, o artigo apresenta indicadores das categorias sociais, o que facilita a construção de instrumentos de coleta.Palavras-chave: Teoria Social de Redes. Redes Temporárias. Eventos Móveis.
INTRODUCTION: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in several cell processes, including the cell cycle, differentiation, proliferation, and apoptosis. MELK has also shown multiple features consistent with the potential utility as an anticancer target, including in astrocytomas. However, the detailed downstream signaling pathway of MELK in cancer cells is still not fully understood and the putative role of MELK in astrocytomas remains unclear. The discovery of proteins involved in the MELK pathway might be important to develop additional small molecular compound to improve effectiveness. PURPOSE: This study aim to analyze proteins and/or genes involved in the MELK pathway in the tumorigenesis of gliomas. MATERIAL AND METHODS: Analysis of the expression profile of a panel of protein relevant to the process of tumorigenesis that MELK is involved was performed by Proteomic analysis by two dimensional electrophoresis and mass spectrometry for human glioma cell line U87MG transfected with siRNA for the MELK compared to U87MG transfected with non-target control (NTC) cells. Analysis and validation of gene expression was performed by Quantitative real time PCR (qRT-PCR) using SYBR Green method in a series of 153 astrocytomas of different malignant grade (23 AGI, 26 AGII, 18 AGIII and 86 AGIV or glioblastoma - GBM) and 22 non neoplastic (NN) brain samples. RESULTS: We identified Stathmin/oncoprotein 18 (STMN1) involved in the cell cycle, as one of twelve proteins differentially expressed, with lower expression when MELK was silenced. A significant higher expression of STMN1 in malignant diffusely infiltrative astrocytomas when compared to non-invasive pilocytic astrocytoma was found (p<0.0001). Strong correlation for MELK and STMN1 associated expression was observed (r=0.741, p<0.0001) in GBM. STMN1 hyperexpression alone impacted at GBM outcome with an overall survival of 5.4 months in contrast to 13.4 months when both genes were hypoexpressed (log rank=0.002). CONCLUSIONS: Thus, our results showed that STMN1 is downstream in the MELK pathway and might play an important role in astrocytoma tumorigenesis. Citation Format: Miyuki Uno, Sueli Mieko Oba-Shinjo, Roseli Silva, Marcela Gimenez, Jose Cesar Rosa, Suely Kazue Nagahashi Marie. Stathmin is involved in the maternal embryonic leucine zipper kinase pathway and impacts in the outcome of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2014-4607
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