Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell.

During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca(2+)] i was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X.

Tick salivary gland as potential natural source for the discovery of promising antitumor drug candidates.

Nowadays, the relationship between cancer blood coagulation is well established. Regarding biodiversity and bioprospection, the tick biology has become quite attractive natural source for coagulation inhibitors, since its saliva has a very rich variety of bioactive molecules. For instance, a Kunitz-type FXa inhibitor, named Amblyomin-X, was found through transcriptome of the salivary gland of the Amblyomma cajennense. tick. This TFPI-like inhibitor, after obtained as recombinant protein, has presented anticoagulant, antigionenic, and antitumor properties. Although its effects on blood coagulation could be relevant for antitumor effect, Amblyomin-X acts by non-hemostatic mechanisms, such as proteasome inhibition and autophagy inhibition. Notably, cytotoxicity was not observed on non-tumor cells treated with this protein, suggesting some selectivity for tumor cells. Considering the current efforts in order to develop effective anticancer therapies, the findings presented in this review strongly suggest Amblyomin-X as a promising novel antitumor drug candidate.

Estudo do papel da dineína no mecanismo de ação antitumoral do Amblyomin-X em células de melanoma e adenocarcinoma de pâncreas / Study of the role of dynein in the antitumor mechanism of action of Amblyomin X in melanoma and pancreas adenocarcinoma cells

Hematophagous animals are sources of natural anticoagulants, since, for feeding, they need to maintain fluid blood in their digestive tract and therefore may contain a series of physiologically active molecules. From the construction of a cDNA library from the salivary glands of the Amblyomma cajennense tick, it was identified a transcript that encodes a serine protease inhibitor with a Kunitz-type domain similar to the endogenous TFPI inhibitor. The protein showed an inhibitory effect on the coagulation factor FXa and was isolated and produced in recombinant form, being named Amblyomin-X. Furthermore, the recombinant protein exerted pro-apoptotic effects in tumor cells as well as anti-angiogenic activity, tumor regression and decreased metastasis. Until now, it was not identified cytotoxic effects of AmblyominX in non-malignant or normal cells, such as fibroblasts. Among the activities described in this new molecule, we emphasize its ability to inhibit the proteasome system and to induce an increase in gene expression of the dynein cytoplasmic 1 light-intermediate chain 2 in microarray analysis in melanoma (SK-MEL-28) and pancreatic adenocarcinoma (MIA PaCa-2) human cells. The dynein is a molecular motor that plays an important role in all eukaryotic cells in transport of proteins, organelles and vesicles, and also the positioning of the nucleus, and the mitotic spindle pole and microtubules. This study is proposed to investigate the role of dynein in the antitumor mechanism of action of Amblyomin-X, from its uptake by the cell towards to its target as well as the biological effects triggered by this novel molecule linked to the proteasome inhibition.

Animais hematófogos são fontes de anticoagulantes naturais, visto que, para se alimentarem, necessitam manter o sangue fluido no seu trato digestivo e assim,podem conter uma série de moléculasfisiologicamente ativas. A partir da construçãode uma biblioteca de cDNA da glândula salivar do carrapato-estrela Amblyomma cajennense, foi identificado um transcrito que codifica um inibidor de serino protease com um domínio tipo Kunitz semelhante ao inibidor endógeno TFPI. A proteína apresentou efeito inibidor sobre o fator FXa da coagulação e foi isolada e produzida na sua forma recombinante, sendo denominada Amblyomin-X. Além disso, a proteína recombinante exerceu efeitos pró-apoptóticos em células tumorais e também atividade anti-angiogênica, regressão da massa tumoral e diminuição de metástases. Até o momento, não foi identificado efeitos citotóxicos do Amblyomin-X em linhagens celulares não-tumorais ou normais, como por exemplo, fibroblastos. Dentre as atividades descritas desta nova molécula, destaca-se sua capacidade de inibição do sistema proteassomo e indução do aumento de expressão gênica da cadeia leve-intermediária 2 da dineína citoplasmática 1 em análise de microarray em células de melanoma (SK-MEL-28) e adenocarcinoma de pâncreas (MIA PaCa-2) humano. A dineína é um motor molecular que desempenha um papel importante em todas as células eucarióticas no transporte de proteínas, vesículas e organelas e também no posicionamento do núcleo, do fuso mitótico e dos microtúbulos. O presente trabalho é proposto para investigar o papel da dineína no mecanismo de ação antitumoral do Amblyomin-X, desde sua entrada na célula até o seu alvo e os efeitos biológicos desencadeados por esta nova molécula relacionados à inibição proteassomal.