RESUMO
BACKGROUND: The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. METHODS: Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. RESULTS: Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced the expression of TRADD, p53, Bax, Bim and Bad and down-regulated the Bcl-2 expression and ROS levels in SK-MEL-28 cells. Interestingly, these treatments impaired SK-MEL-28 cell adhesion, migration and invasion through modulating the function and expression of αvß3 integrin along with regulating E-cadherin, vimentin, ß-catenin, c-Src and RhoA expression. In silico study suggested that only the α chain of macrovipecetin interacts with a region overlapping the RGD motif binding site on this integrin. CONCLUSIONS: We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells. GENERAL SIGNIFICANCE: The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.
Assuntos
Antineoplásicos/farmacologia , Lectinas Tipo C/isolamento & purificação , Melanoma/patologia , Venenos de Víboras/química , Viperidae/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/efeitos dos fármacos , Lectinas Tipo C/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.
Assuntos
Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Lectina de Ligação a Manose/metabolismo , Properdina/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Proteínas de Ciclo Celular , Cromatografia Líquida/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-18/metabolismo , Lectina de Ligação a Manose/análise , Camundongos , Nanotecnologia/métodos , Properdina/análise , Análise Serial de Proteínas/métodos , Ligação Proteica , Espectrometria de Massas em Tandem/métodosRESUMO
Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.
Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Lectinas Tipo C , Lectinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Colágeno/metabolismo , Venenos de Crotalídeos/farmacologia , Venenos Elapídicos/isolamento & purificação , Elapidae , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Homologia de Sequência de AminoácidosRESUMO
Alboluxin, a potent platelet activator, was purified from Trimeresurus albolabris venom with a mass of 120 kDa non-reduced and, after reduction, subunits of 17 and 24 kDa. Alboluxin induced a tyrosine phosphorylation profile in platelets that resembles those produced by collagen and convulxin, involving the time dependent tyrosine phosphorylation of Fc receptor gamma chain (Fc gamma), phospholipase Cgamma2 (PLCgamma2), LAT and p72SYK. Antibodies against both GPIb and GPVI inhibited platelet aggregation induced by alboluxin, whereas antibodies against alpha2beta1 had no effect. Inhibition of alphaIIb beta3 reduced the aggregation response to alboluxin, as well as tyrosine phosphorylation of platelet proteins, showing that activation of alphaIIb beta3 and binding of fibrinogen are involved in alboluxin-induced platelet aggregation and it is not simply agglutination. N-terminal sequence data from the beta-subunit of alboluxin indicates that it belongs to the snake C-type lectin family. The C-type lectin subunits are larger than usual possibly due to post-translational modifications such as glycosylation. Alboluxin is a hexameric (alphabeta)3 snake C-type lectin which activates platelets via both GPIb and GPVI.