ConBr lectin modulates MAPKs and Akt pathways and triggers autophagic glioma cell death by a mechanism dependent upon caspase-8 activation

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.Fil: Wolin, Ingrid A. V.. Universidade Federal de Santa Catarina; BrasilFil: Heinrich, Isabella A.. Universidade Federal de Santa Catarina; BrasilFil: Nascimento, Ana Paula M.. Universidade Federal de Santa Catarina; BrasilFil: Welter, Priscilla G.. Universidade Federal de Santa Catarina; BrasilFil: Sosa, Liliana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: de Paul, Ana Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Zanotto Filho, Alfeu. Universidade Federal de Santa Catarina; BrasilFil: Nedel, Cláudia Beatriz. Universidade Federal de Santa Catarina; BrasilFil: Lima, Lara Dias. Universidade Estadual do Ceará; BrasilFil: Osterne, Vinicius Jose Silva. Universidade Estadual do Ceará; BrasilFil: Pinto Junior, Vanir Reis. Universidade Estadual do Ceará; BrasilFil: Nascimento, Kyria S.. Universidade Estadual do Ceará; BrasilFil: Cavada, Benildo S.. Universidade Estadual do Ceará; BrasilFil: Leal, Rodrigo B.. Universidade Federal de Santa Catarina; Brasi

Differential vasodilator effect of Dioclea rostrata lectin in conductance and resistance arteries : mechanisms and glycoconjugate binding relationships

Lectins are proteins that recognize specific carbohydrates, and the vasorelaxant effect of legume lectins has been previously reported, for example the Dioclea rostrata lectin (DRL). This study evaluated major pathways of DRL-induced relaxation in different artery segments and the possible molecular interactions involved. Rat thoracic aorta, coronary and mesenteric resistance arteries were tested "in vitro" with concentration-response curves to DRL (0.01-100 mu g/mL). L-NAME, indomethacin and high KCl were used to evaluate nitric oxide, cyclooxygenase and hyperpolarization-dependent effects. DRL promoted relaxation of all vessels throughout different mechanisms. L-NAME blunted DRL-induced effects only in the aorta and mesenteric resistance artery. By the use of depolarizing KCl solution, vasodilation was reduced in all arteries, while incubation with indomethacin indicated a role of cyclooxygenase-derived factors for DRL effects in mesenteric and coronary arteries, but not in the aorta. Molecular docking results suggested interactions between DRL and heparan sulphate, CD31 and other glycans present on the membrane surface. These data indicate that the mechanisms involved in DRL-mediated vasodilation vary between conductance and resistance arteries of different origins, and these effects may be related to the capacity of DRL to bind a diversity of glycans, especially heparan sulphate, a proposed mechanoreceptor for nitric oxide synthase and cyclooxygenase activation

Differential vasodilator effect of Dioclea rostrata

Lectins are proteins that recognize specific carbohydrates, and the vasorelaxant effect of legume lectins has been previously reported, for example the Dioclea rostrata lectin (DRL). This study evaluated major pathways of DRL-induced relaxation in different artery segments and the possible molecular interactions involved. Rat thoracic aorta, coronary and mesenteric resistance arteries were tested "in vitro" with concentration-response curves to DRL (0.01-100 mu g/mL). L-NAME, indomethacin and high KCl were used to evaluate nitric oxide, cyclooxygenase and hyperpolarization-dependent effects. DRL promoted relaxation of all vessels throughout different mechanisms. L-NAME blunted DRL-induced effects only in the aorta and mesenteric resistance artery. By the use of depolarizing KCl solution, vasodilation was reduced in all arteries, while incubation with indomethacin indicated a role of cyclooxygenase-derived factors for DRL effects in mesenteric and coronary arteries, but not in the aorta. Molecular docking results suggested interactions between DRL and heparan sulphate, CD31 and other glycans present on the membrane surface. These data indicate that the mechanisms involved in DRL-mediated vasodilation vary between conductance and resistance arteries of different origins, and these effects may be related to the capacity of DRL to bind a diversity of glycans, especially heparan sulphate, a proposed mechanoreceptor for nitric oxide synthase and cyclooxygenase activation

Antiproliferative activity of Dioclea violacea lectin in CaCO3 particles on cancer cells after controlled release

Cancer is a serious health problem mainly characterized by unregulated cell divisions. It is known that malign cells display cancer-specific glycans that can be recognized by lectins, proteins with capacity of specific binding to carbohydrates. One such protein is the Dioclea violacea lectin (DVL), a known antiproliferative lectin that has been applied in this work to create and optimize a methodology for a controlled-release system by adsorption of lectin into CaCO3 particles. The system was tested by evaluating the cytotoxic effect of lectin particles against cervical cancer cells (HeLa) and compared to the non-conjugated lectin. DVL was adsorbed into CaCO3 particles of two sizes (average of 3.6 tm and 0.7 vim), a low-cost, biocompatible and biodegradable material, and the effect of DVL/bioconjugates against HeLa cells has been investigated in vitro and in silico. The bioconjugates possessed adsorption and release compatible with the biological assays. Cells assays applying the bioconjugates demonstrated their cytotoxicity, with a stronger effect compared to the free lectin. In conclusion, the anticancer potential of DVL against HeLa cells has been increased by the construction and application of a controlled-release system based on the adsorption of the lectin on CaCO3 particles with a relatively simple protocol. Also, the present data demonstrate the potential of lectin-particle bioconjugates as agents in cancer research and suggest an easy and low-cost methodology that can be extended to other lectins /proteins

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds

Cells use glycans to encode information that modulates processes ranging from cell–cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin’s specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38(MAPK) and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death

Purification, characterization and partial sequence of a pro-inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single-step affinity chromatography in Sephadex (R) G-50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30kDa (-chain) and two minor components (-chain and -chain) of 16 and 13kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA-like lectin. In comparison with the average molecular mass of -chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D-glucose, D-mannose, and -methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60 degrees C, and it was pH-sensitive for 1h, exhibiting maximal activity at pH7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright (c) 2014 John Wiley & Sons, Ltd