Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells

Abstract

Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

Crotoxin-Treated Macrophages Stimulate ROS Production and Killing Activity in Co-Cultured Neutrophils

“…Three peaks (p1, p2 and p3) were obtained of which p2 corresponded to the pure CTX fraction (about 60% of the crude venom), and peaks 1 and 3 included the other CdtV toxins. Prior to pooling, the fractions containing CTX were tested for homogeneity by non-reducing SDS-PAGE (12.5%) (50) and the phospholipase A2 activity was assessed by a colorimetric assay using a synthetic chromogenic substrate, as previously described by our group (13,19,44,51).…”

“…Both in vivo and in vitro studies have shown the dual effects of CTX on macrophage functions, such as inhibition of macrophage spreading and phagocytosis, and stimulation of H2O2 and NO production and release (13,19). These changes are accompanied by increased activity of the key glycolytic enzymes and higher consumption of glucose and glutamine, which demonstrates the metabolic effects of this toxin (20).…”

“…These changes are accompanied by increased activity of the key glycolytic enzymes and higher consumption of glucose and glutamine, which demonstrates the metabolic effects of this toxin (20). CTX also enhances the production of ROS, RNS, and cytokines in macrophages, thereby inducing the M1 or inflammatory phenotype, and enhancing their antitumor and anti-leishmanial properties (13,14). In contrast, CTX inhibits both migration and phagocytosis of neutrophils induced by flogistic agents such as carrageenan or FMLP, and down-regulates the Syk-GTPase pathway (15,21).…”

Caracterização do efeito da crotoxina sobre a funcionalidade dos neutrófilos da medula óssea

“…Dentre essas, a lipoxina inibe a quimiotaxia, a adesão e a transmigração dos neutrófilos, bem como a expressão do receptor CR3 nessas células (Maderna & Godson, 2009;Ryan & Godson, 2010). Corrobora para essa hipótese, o fato de a CTX induzir a liberação de lipoxina por macrófagos em cultura (Costa et al, 2013) e por neutrófilos no tecido subcutâneo após administração de carragenina (Nunes et al, 2015, em publicação). Adicionalmente, recentemente foi demonstrado que a CTX induz a secreção de lipoxina no tecido do cólon, após a indução da inflamação intestinal (Almeida et al, 2015).…”

Efeito da crotoxina na ação moduladora dos macrófagos sobre eventos de neovascularização Ensaios <i>in vitro</i>.

“…Ainda, neste mesmo estudo foi observado aumento da produção de mediadores lipídicos Lipoxina A 4 e análogo mais estável 15-epi-LXA 4, que resultou no decréscimo da proliferação das células tumorais (COSTA et al, 2013).…”