Efeito da crotoxina sobre a reprogramação fenotípica dos macrófagos estimulados pelo microambiente tumoral

Abstract

Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has an antitumor effect both in experimental models and in clinical trials. Our group has been demonstrating that the antitumor activity of the toxin is also associated with the CTX modulatory actions on the secretory activity of inflammatory mediators (cytokines, reactive oxygen and nitrogen species) and on the energetic metabolism (glycolytic and glutaminolytic pathways) of macrophages. Despite this evidence, it has not been demonstrated to date whether the metabolic alterations induced by CTX establish a phenotypic profile of the resident macrophages and influence the reprogramming of macrophages induced by the tumor microenvironment. Therefore, the present study investigated the effect of CTX on the phenotypic reprogramming of macrophages in the tumor microenvironment. Balb/c mice received administration of different CTX concentrations (0.9 or 5 μg/animal subcutaneously in sterile saline) at the 1st, 6th or 1st and 6th day of inoculation of tumor cells (1x107 /0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT). After 6 or 13 days of EAT induction, we analyzed: a) immunophenotyping of macrophages; b) total volume of ascitic fluid; total and differential count of cells (tumor and leukocytes) in the ascitic fluid; determination of nitric oxide (NO) production and secretion of cytokines IL-1β, IL-10 and release of TNF-. Functional and phenotypic parameters were also established in macrophages obtained from the peritoneal cavity of healthy animals to evaluate the action per se of the toxin. The different doses of CTX, administered on day 1, modulated the macrophages phenotype (M1), accompanied by increased NO production. In healthy animals, the phenotypic profile of macrophages was dependent on the administered dose (0.9 μg/animal: M1 and 5 μg/animal: M2), accompanied by increased NO production when administered at a dose of 0.9 μg/animal. When the parameters were evaluated on the 6th day after the treatment concomitant to the induction of EAT, there was a significant decrease in ascitic volume, accompanied by the decrease of tumor cells and inhibition of IL-10 production. In healthy animals, both doses induced increased leukocytes in the peritoneal cavity. When the animals were treated with CTX (particularly at the dose of 0.9 μg/animal), on the 6th day of EAT induction, a decrease in the number of tumor cells and inhibition of TNF-α release was again observed. In addition, treatment with CTX induced an increase in the number of leukocytes in the peritoneal cavity of healthy animals. In another treatment protocol, where the animals were treated with two doses given on the 1st and 6th days of EAT induction, the ascitic volume decreased again, accompanied by a significant increase in TNF-α release. Therefore, the data obtained in the present study indicate that CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of these cells, influencing events involved with tumor progression.