Seek and destroy: the use of natural compounds for targeting the molecular roots of cancer.

One of the major issues facing anticancer research relies on the intrinsic inability of tumor cells to undergo apoptosis. Additionally, the development of cancer resistance to standard therapy and the great heterogeneity associated with frequent mutations and epigenetic changes make an ever increasing challenge to achieve treatment success. Thus, novel therapeutic approaches to induce cancer demise must be explored. Compelling evidence has shown the ability of naturally-occurring compounds to modulate signal transduction pathways, apoptosis and cell cycle progression, supporting their relevance to anticancer drug discovery. Moreover, millions of years of biological selection have led to an unlimited repertoire of chemical structures unmatched by any synthetic combinatorial library and recent advances in the fields of chemistry and biology are uncovering this still underexplored source of new promising natural agents, opening novel perspectives for the development of alternative strategies to fight cancer. This review presents the current status of natural products in modern oncology, illustrating the importance of some old and new agents, such as antimitotics and apoptosis inducers, as candidates of pharmacological interest in drug development and/or as chemical tools for the elucidation, as well as targeting, of deregulated cancer signaling pathways. Finally, some aspects of chemical modifications done in natural products core aiming to improve their activity and/or effectiveness will be discussed.

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom.

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles).

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase-D (dermonecrotic toxin) from brown spider venom.

Brown spiders have world-wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase-D, causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose-dependent and time-dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP-phospholipase-D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin-treated erythrocytes reacted with annexin-V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase-D, which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine-protease inhibitor) that had no effect on hemolysis. By site-directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase-D triggers direct human blood cell hemolysis in a catalytic-dependent manner.

Adult bone marrow-derived mononuclear cells expressing chondroitinase AC transplanted into CNS injury sites promote local brain chondroitin sulphate degradation.

Injury to the CNS of vertebrates leads to the formation of a glial scar and production of inhibitory molecules, including chondroitin sulphate proteoglycans. Various studies suggest that the sugar component of the proteoglycan is responsible for the inhibitory role of these compounds in axonal regeneration. By degrading chondroitin sulphate chains with specific enzymes, denominated chondroitinases, the inhibitory capacity of these proteoglycans is decreased. Chondroitinase administration involves frequent injections of the enzyme at the lesion site which constitutes a rather invasive method. We have produced a vector containing the gene for Flavobacterium heparinum chondroitinase AC for expression in adult bone marrow-derived cells which were then transplanted into an injury site in the CNS. The expression and secretion of active chondroitinase AC was observed in vitro using transfected Chinese hamster ovarian and gliosarcoma cells and in vivo by immunohistochemistry analysis which showed degraded chondroitin sulphate coinciding with the location of transfected bone marrow-derived cells. Immunolabelling of the axonal growth-associated protein GAP-43 was observed in vivo and coincided with the location of degraded chondroitin sulphate. We propose that bone marrow-derived mononuclear cells, transfected with our construct and transplanted into CNS, could be a potential tool for studying an alternative chondroitinase AC delivery method.

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

The physiological functions of the cellular prion protein, PrP(C), as a cell surface pleiotropic receptor are under debate. We report that PrP(C) interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrP(C)-vitronectin interaction were mapped to residues 105-119 of PrP(C) 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-PrP(C) antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrP(C)-null mice. Functional assays demonstrated that relative to wild-type cells, PrP(C)-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alphavbeta3 activity. Our findings indicate that PrP(C) plays an important role in axonal growth, and this function may be rescued in PrP(C)-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 and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a beta-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than beta-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53 kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom.

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.