A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system. (C) 2010 Elsevier Ltd. All rights reserved.

Cytotoxicity of Diepoxybutane and Epichlorohydrin in Relation to Stages of the Cell Cycle

Work conducted in the Millard Biochemistry Research Laboratory examines the dual nature of molecules as carcinogens and anti-tumor agents through the molecular mechanisms of duplex DNA damage by bifunctional alkylating agents. Diepoxybutane (DEB) and epichlorohydrin (ECH) are polar molecules that form covalent DNA interstrand lesions by cross-linking the N7 position of deoxyguanosine residues. A recent experiment indicated that ECH preferentially targets nuclear DNA over mitochondrial DNA, whereas DEB shows similar rates of lesion formation for both loci. It was concluded that preferential targeting of nuclear DNA results from relatively poor uptake of ECH across the mitochondrial membrane. The objective of my honors research was to determine if the cytotoxicities of DEB and ECH vary according to the presence of the nuclear envelope in 6C2 chicken erythro-progenitor cells. The cytotoxicity of DEB and ECH was compared between cells randomly distributed throughout the cell cycle (Go/G, and S » G2/M) and cells enriched in G2/M stages. Results indicated that ECH is more cytotoxic than DEB in both unsynchronized control 6C2 cells and synchronized 6C2 cells enriched in G2/M stages of the cell cycle. Treatment with either bifunctional alkylating agent induced greater cytotoxicity in 6C2 cells enriched in G2/M stages than in unsynchronized control 6C2 cells, suggesting that the presence of the nuclear envelope-or any plasma membrane-may inhibit the reactivity of DEB and ECH.

Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p ((d) under bar efective for (f) under bar ilamentous (g) under bar rowth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a beta-glucan linked protein that undergoes N -glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues. The P. brasiliensis dfg5 nucleotide and deduced protein, PbDfg5p, sequences reported in this paper had been submitted to the GenBank database under Accession Nos AY307855 (cDNA) and DQ534495 (genomic). Copyright (C) 2007 John Wiley & Sons, Ltd.

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of cell cycle regulatory proteins in epithelial components of dental follicles

Dental follicle is a component of tooth germs, which remain adjacent to the crown of unerupted or impacted teeth. Under the influence of pathologic changes, however, dental follicles that possess reduced epithelium can proliferate into stratified squamous epithelium as far as originate dental cysts. In order to clarify the role of apoptosis and cellular proliferation herein, expression of p53 and PCNA was examined in epithelial components of dental follicles associated with impacted third molars by means of immunohistochemistry. A total of 40 cases was included in this study being 22 cases with reduced epithelium and 18 cases with stratified epithelium. Expression of p53 expression was weak or not detected in dental follicles with reduced and stratified squamous epithelium. By contrast, PCNA positive cells were evidenced in basal and supra basal layers of the stratified squamous epithelium and in reduced epithelium of dental follicles, but without any significant statistically differences between them (P > 0.05). In conclusion, these data suggest that dental follicles possess proliferative activity as depicted by PCNA-positive nuclei in some epithelial cells. However, the biological behavior of dental follicles during the late stage of dental eruptive process may not be associated with deregulation of death and/or cell proliferation.

Toxicogenomic activity of gemcitabine in two TP53-mutated bladder cancer cell lines: special focus on cell cycle-related genes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD(A (R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Anticlastogenic activity of aqueous extract of Agaricus blazei in drug-metabolizing cells (HTCs) during cell cycle

The mushroom Agaricus blazei has been extensively investigated because of evidence of its antimutagenic, antitumor, and anticarcinogenic activities. This study investigated the clastogenic and/or anticlastogenic activity of aqueous extract of Agaricus blazei (10% w/v) in drug-metabolizing rat hepatoma tissue cells (HTCs), with continuous treatment and treatment during different phases of the cell cycle. DNA damage was induced utilizing two directacting agents-methyl methane sulfonate and ethyl methane sulfonate-and two indirect-acting agents-2-aminoanthracene and cyclophosphamide. The aqueous extract of A. blazei with either continuous treatment or treatment during different phases of the cell cycle showed clastogenic activity. The results with continuous treatment showed that A. blazei does not protect against DNA damage-inducing agents that are direct acting. Meanwhile, when combined with indirect-acting agents, a protective effect was demonstrated. A protective effect was also found during different phases of the cell cycle when cells were treated with indirect-acting agents. The protective effects against indirect-acting agents (continuous treatment and during the different phases of the cell cycle) suggest that A. blazei may provide some health benefits to the public when used as a functional food.