Silvio Sanches Veiga

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16‐F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16‐F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16‐F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16‐F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.

Identification of proteases in the extract of venom glands from brown spiders

In the present investigation, in order to dispute the rational criticism against the presence of proteolytic enzymes in the electrostimulated venom obtained from spiders of the genus Loxosceles, as a consequence of contamination with abdominal secretions, venoms of L. intermedia and L. laeta were directly collected from venom glands by microdissection and gentle homogenization. Gel electrophoresis stained by silver method carried out to compare L. intermedia electrostimulated venom and venom gland extract demonstrated no significant differences in protein profile. Zymogram analysis of L. intermedia venom gland extract detected a gelatinolytic activity in the 32-35 kDa region. The inhibitory effect of 1,10-phenanthroline on this proteolytic activity further supported its metalloprotease nature. In proteolytic digestion experiments L. intermedia venom gland extract was also able to cleave purified fibronectin and fibrinogen. The inhibitory effect of 1,10-phenanthroline on these degrading activities confirmed the presence of metalloproteases in the venom. In addition, when purified fibrinogen was incubated with L. intermedia abdominal extract, the fibrinogenolysis was completely different, generating low mass fragments that ran away from the gel, a proteolytic event not blocked by 1,10-phenanthroline. Zymogram experiments using L. laeta venom gland extracts further detected a gelatinolytic band at 32-35 kDa, also inhibited by 1,10-phenanthroline, confirming the presence of metalloproteases in both species.

Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins

The physiological functions of the cellular prion protein, PrPC, as a cell surface pleiotropic receptor are under debate. We report that PrPC interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrPC-vitronectin interaction were mapped to residues 105-119 of PrPC and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrPC antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrPC-null mice. Functional assays demonstrated that relative to wild-type cells, PrPC-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater αvβ3 activity. Our findings indicate that PrPC plays an important role in axonal growth, and this function may be rescued in PrPC-knockout animals by integrin compensatory mechanisms.

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.

Identification of high molecular weight serine-proteases in Loxosceles intermedia (brown spider) venom.

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowmans space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.

Extracellular matrix molecules as targets for brown spider venom toxins

Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spiders venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

Effect of brown spider venom on basement membrane structures.

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1,10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.

In vivo and in vitro cytotoxicity of brown spider venom for blood vessel endothelial cells.

The effect of brown spider (Loxosceles intermedia) venom on endothelial cells was investigated in vivo and in vitro. Morphological and ultrastructural observations by light microscopy and transmission electron microscopy showed that the venom acts in vivo upon vessel endothelial cells of rabbits that were intradermally injected, evoking vessel instability, cytoplasmic endothelial cell vacuolization, and blebs. Likewise, treatment of rabbit endothelial cells in culture with the venom led to loss of adhesion of the cells to the substrate. Endothelial cells in culture were metabolically radiolabeled with sodium [35S]-sulfate and the sulfated compounds (proteoglycans and sulfated proteins) from medium, cell surface, and extracellular matrix (ECM) were analyzed. Agarose gel electrophoresis and SDS-PAGE showed that the venom is active on the ECM and on cell surface proteoglycans, shedding these molecules into the culture medium. In addition, when purified heparan sulfate proteoglycan (HSPG) and purified laminin-entactin (LN/ET) complex were incubated with the venom we observed a partial degradation of the protein core of HSPG as well as the hydrolysis of entactin. The above results suggest that the L. intermedia venom has a deleterious effect on the endothelium of vessels both in vivo and in culture, removing important constituents such as HSPG and entactin that are involved in the adhesion of endothelial cells and of subendothelial ECM organization.