ABSTRACT
In the initial online version of the article, author F.M. Soriani was missing. The original article has been corrected.
ABSTRACT
INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal form of gliomas. New therapies are currently in development to tackle treatment limitations such as chemotherapy resistance. One mechanism of resistance may be the stress granules (SG) assembly, a stress-related cellular response that allows cells to recruit and protect mRNAs during stress. SG are composed of various proteins, being G3BP1 a core element that enucleates and results in SG assembly. Here, we aimed to evaluate the effects of inhibiting the G3PB1 expression in the chemotherapeutical-induced cell death of the U87 glioblastoma cell line. MATERIALS AND METHODS: G3BP1 mRNA and protein expression were modulated with short-interference RNA (siRNA). The viability of U87 cells after Bortezomib (BZM), a proteasome inhibitor, and Temozolomide (TMZ), an alkylating agent, was assessed by MTT assay. Apoptosis was evaluated by staining cells with Annexin-V/7-AAD and analyzing by flow cytometry. Caspase-3 activation was evaluated by immunoblotting. The chorioallantoic membrane in vivo assay was used to evaluate angiogenesis. RESULTS: When G3BP1 was knocked-down, the SG assembly was reduced and the BZM-treated cells, but not TMZ-treated cells, had a significant increase in the apoptotic response. Corroborating this data, we observed increased Caspase-3 activation in the BZM-treated and G3BP1-knocked-down cells when compared to vehicle-treated and scramble-transfected cells. Worth mentioning, the conditioned culture medium of G3BP1-knocked-down BZM-treated cells inhibited angiogenesis when compared to controls. CONCLUSION: Our data suggest G3BP1 knockdown diminishes SG formation and stimulates BZM-induced apoptosis of U87 cells in vitro, in addition to inhibiting glioblastoma-induced angiogenesis in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Bortezomib/pharmacology , Cytoplasmic Granules/drug effects , DNA Helicases/antagonists & inhibitors , Glioblastoma/drug therapy , Neovascularization, Pathologic/drug therapy , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , RNA Helicases/antagonists & inhibitors , RNA Recognition Motif Proteins/antagonists & inhibitors , Antineoplastic Agents, Alkylating/pharmacology , Cell Proliferation/drug effects , Cytoplasmic Granules/pathology , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Temozolomide/pharmacology , Tumor Cells, CulturedABSTRACT
BACKGROUND AND OBJECTIVE: The angiotensin type 1 (AT1) receptor has been implicated in the pathogenesis of inflammatory bone disorders. This study aimed to investigate the effect of an AT1 receptor antagonist in infection-induced and arthritis-associated alveolar bone loss in mice. MATERIAL AND METHODS: Mice were subjected to Aggregatibacter actinomycetemcomitans oral infection or antigen-induced arthritis and treated daily with 10 mg/kg of the prototype AT1 antagonist, losartan. Treatment was conducted for 30 d in the infectious condition and for 17 d and 11 d in the preventive or therapeutic regimens in the arthritic model, respectively. The mice were then killed, and the maxillae, serum and knee joints were collected for histomorphometric and immunoenzymatic assays. In vitro osteoclast assays were performed using RAW 264.7 cells stimulated with A. actinomycetemcomitans lipopolysacharide (LPS). RESULTS: Arthritis and A. actinomycetemcomitans infection triggered significant alveolar bone loss in mice and increased the levels of myeloperoxidase and of TRAP(+) osteoclasts in periodontal tissues. Losartan abolished such a phenotype, as well as the arthritis joint inflammation. Both arthritis and A. actinomycetemcomitans conditions were associated with the release of tumor necrosis factor alpha (TNF-α), interferon-gamma, interleukin-17 and chemokine (C-X-C motif) ligand 1 and an increased RANKL/osteoprotegerin ratio in periodontal tissues, but such expression decreased after losartan treatment, except for TNF-α. The therapeutic approach was as beneficial as the preventive one. In vitro, losartan prevented LPS-induced osteoclast differentiation and activity. CONCLUSION: The blockade of AT1 receptor exerts anti-inflammatory and anti-osteoclastic effects, thus protecting periodontal tissues in distinct pathophysiological conditions of alveolar bone loss.
Subject(s)
Alveolar Bone Loss/prevention & control , Anti-Inflammatory Agents/metabolism , Arthritis/complications , Losartan/metabolism , Pasteurellaceae Infections/complications , Receptor, Angiotensin, Type 1/metabolism , Aggregatibacter actinomycetemcomitans/pathogenicity , Animals , Arthritis/microbiology , Histocytochemistry , Knee Joint/pathology , Male , Maxilla/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Pasteurellaceae Infections/microbiology , RAW 264.7 Cells/drug effects , Serum/chemistryABSTRACT
Aspergillus fumigatus possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consumption activities. Mitochondrial reactive oxygen species mediate signaling for alternative oxidase (AOX) expression. A 1173 bp-long Afaox gene encoding a 40 kDa protein has been cloned and identified. Recombinant constructs containing the Afaox ORF were transformed into Escherichia coli and Saccharomyces cerevisiae for heterologous expression. In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress. Therefore, positive transformants showed a cyanide-resistant and salicylhydroxamic acid-sensitive respiration, whereas in control cells the oxygen uptake was completely inhibited after KCN addition.
Subject(s)
Aspergillus fumigatus/genetics , Fungal Proteins/genetics , Oxidative Stress , Oxidoreductases/genetics , Amino Acid Sequence , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/metabolism , Blotting, Western , Cloning, Molecular , Cyanides/pharmacology , Enzyme Activation/drug effects , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Proteins , Molecular Sequence Data , Oxidoreductases/metabolism , Oxygen Consumption/drug effects , Paraquat/pharmacology , Plant Proteins , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Salicylamides/pharmacology , Sequence Homology, Amino Acid , Vitamin K 3/pharmacologyABSTRACT
The understanding of the controlling factors of calcium homeostasis in Aspergillus fumigatus is very poor, although this ion is involved in several important events of these particular cells. We have cloned, identified and expressed for functional complementation a PMR1-like Ca(2+)-ATPase gene from A. fumigatus. The Afpmr1 gene encodes a protein of 1061 deduced amino acids, containing all the conserved subdomains found in other P-type ATPases: the phosphatase region, phosphorylation site, FITC labelling site, ATP binding domain; E(386), N871, D875 amino acid residues for calcium ion interaction and Q880, a residue that alters ion selectivity in PMR1. The expressed AfPMR1 in S. cerevisiae K616 strain functionally complemented the deficient growth in EGTA (5-20 mM)- and MnCl2 (4 mM)-containing medium. These results demonstrate the first evidence of a Ca(2+)-ATPase in A. fumigatus and strongly suggest a role for this enzyme in calcium and manganese homeostasis.
