Hemodynamic impairment induced by Crotoxin using in vivo and ex vivo approach in a rat model.

Crotalus durissus snakebite represent 10 % of snakebite cases in Brazil, which cardiovascular disorders are associated with severe cases. Considering crotoxin (CTX) as the major venom component, the present study aimed to evaluate the hemodynamic alterations induced by CTX using in vivo and ex vivo approaches in a rat model. In vivo cardiac function parameters were analyzed from anesthetized rats treated with CTX or saline only (Sham), along with serum creatine kinase MB (CK-MB) and lung myeloperoxidase. From the same animals, hearts were isolated and functional parameters evaluated in Langendorff method ex vivo. CTX binding to myoblast cell line in vitro were evaluated using confocal microscopy and flow cytometry. CTX was capable of reducing arterial and diastolic blood pressure, heart rate, along with left ventricle pressure development or decay during systole (LVdP/dtmax and LVdP/dtmin) in vivo, however no differences were found in the ex vivo approach, showing that intrinsic heart function was preserved. In vitro, CTX binding to myoblast cell line was mitigated by hexamethonium, a nicotinic acetylcholine receptor antagonist. The present study has shown that CTX induce hemodynamic failure in rats, which can help improve the clinical management of cardiovascular alterations during Crotalus durissus snakebite.

Crotoxin-Induced Mice Lung Impairment: Role of Nicotinic Acetylcholine Receptors and COX-Derived Prostanoids.

Respiratory compromise in Crotalus durissus terrificus (C.d.t.) snakebite is an important pathological condition. Considering that crotoxin (CTX), a phospholipase A2 from C.d.t. venom, is the main component of the venom, the present work investigated the toxin effects on respiratory failure. Lung mechanics, morphology and soluble markers were evaluated from Swiss male mice, and mechanism determined using drugs/inhibitors of eicosanoids biosynthesis pathway and autonomic nervous system. Acute respiratory failure was observed, with an early phase (within 2 h) characterized by enhanced presence of eicosanoids, including prostaglandin E2, that accounted for the increased vascular permeability in the lung. The alterations of early phase were inhibited by indomethacin. The late phase (peaked 12 h) was marked by neutrophil infiltration, presence of pro-inflammatory cytokines/chemokines, and morphological alterations characterized by alveolar septal thickening and bronchoconstriction. In addition, lung mechanical function was impaired, with decreased lung compliance and inspiratory capacity. Hexamethonium, a nicotinic acetylcholine receptor antagonist, hampered late phase damages indicating that CTX-induced lung impairment could be associated with cholinergic transmission. The findings reported herein highlight the impact of CTX on respiratory compromise, and introduce the use of nicotinic blockers and prostanoids biosynthesis inhibitors as possible symptomatic therapy to Crotalus durissus terrificus snakebite.

Cancer Stem Cells: Powerful Targets to Improve Current Anticancer Therapeutics.

A frequent observation in several malignancies is the development of resistance to therapy that results in frequent tumor relapse and metastasis. Much of the tumor resistance phenotype comes from its heterogeneity that halts the ability of therapeutic agents to eliminate all cancer cells effectively. Tumor heterogeneity is, in part, controlled by cancer stem cells (CSC). CSC may be considered the reservoir of cancer cells as they exhibit properties of self-renewal and plasticity and the capability of reestablishing a heterogeneous tumor cell population. The endowed resistance mechanisms of CSC are mainly attributed to several factors including cellular quiescence, accumulation of ABC transporters, disruption of apoptosis, epigenetic reprogramming, and metabolism. There is a current need to develop new therapeutic drugs capable of targeting CSC to overcome tumor resistance. Emerging in vitro and in vivo studies strongly support the potential benefits of combination therapies capable of targeting cancer stem cell-targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This review will address the main characteristics, therapeutic implications, and perspectives of targeting CSC to improve current anticancer therapeutics.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

Lymph node or perineural invasion is associated with low miR-15a, miR-34c and miR-199b levels in head and neck squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. METHODS: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. RESULTS: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. CONCLUSIONS: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. GENERAL SIGNIFICANCE: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario.

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer.

Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models.

BACKGROUND: SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. METHODS: Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. RESULTS: The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. CONCLUSIONS: SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.