Crotoxin promotes macrophage reprogramming towards an antiangiogenic phenotype

Abstract

Crotoxin (CTX) is the primary toxin of South American rattlesnake Crotalus durissus terrificus venom. CTX reduces tumour mass, and tumour cell proliferation and these effects seem to involve the formation of new vessels. Angiogenesis has a key role in tumour growth and progression and is regulated by macrophage secretory activity. Herein, the effect of CTX on macrophage secretory activity associated with angiogenesis was investigated in vitro. Thymic endothelial cells (EC) were incubated in the presence of macrophages treated with CTX (12.5 nM) or supernatants of CTX-treated macrophages and endothelial cell proliferation, migration and adhesion activities, and the capillary-like tube formation in the matrigel-3D matrix was measured. Angiogenic mediators (MMP-2, VEGF and TNF-α) were measured in the cell culture medium. Macrophages pre-treated with CTX and supernatant of CTX-treated macrophages inhibited EC proliferation, adhesion to its natural ligands, and migration (as evaluated in a wound-healing model and Time Lapse assay) activities. Decreased capillary-like tube formation and MMP-2, VEGF and TNF-α levels in the supernatant of macrophages treated with CTX was also described. CTX promotes macrophage reprogramming towards an antiangiogenic phenotype.

Crotoxin Inhibits Endothelial Cell Functions in Two- and Three-dimensional Tumor Microenvironment

“…Purification and phospholipase activity of the CTX was performed as described in previous studies ( Fraenkel-Conrat and Singer, 1956 ; Laemmli, 1970 ; Lobo de Araujo and Radvanyi, 1987 ; Faure and Bon, 1988 ; Sampaio et al, 2003 ; Sampaio et al, 2006a ; Sampaio et al, 2006b ; Brigatte et al, 2016 ; de Araujo Pimenta et al, 2019 ). Briefly, crude venom solution (CdtV) was subjected to anion-exchange chromatography, using a Mono-Q HR 5/5 column in an FPLC system (Pharmacia, Uppsala, Sweden).…”

“…For counting the migrating cells in the field induced by the probe, the images were inserted into the rules on the left and right edges of the field, based on the image obtained in the T0 coverslip. After insertion of dashed lines, the count of migrating cells in the field was performed ( de Araujo Pimenta et al, 2019 ).…”

“…The ImageJ plugin manual tracking was employed to track cell nuclei. Hence, the velocity of the cells was analyzed; the net distances per hour were calculated and summed up to determine the total cell path length ( Hauff et al, 2015 ; de Araujo Pimenta et al, 2019 ). Since the trajectory of individual cells was monitored, directionality was evaluated by calculating the D/T ratio (0–1), which is the ratio between the smallest distance between the initial and final position of the cell (D) by the total distance traveled (T).…”

“…On the other hand, in vivo experimental models have indicated that the prolonged inhibitory action of CTX promotes the reduction of solid tumors ( Faiad, 2012 ; Brigatte et al, 2016 ) and ascites tumor ( Nunes et al, 2010 ; Neves et al, 2019 ). The significance of CTX immunomodulatory activity on macrophages in the tumor microenvironment has been demonstrated as the most marked inhibitory effect on solid tumor development and progression ( de Araujo Pimenta et al, 2019 ). The inhibitory effect of CTX on neovascularization induced by the tumor could significantly contribute to the antitumor action described for this toxin.…”

Venom of the Red-Bellied Black Snake Pseudechis porphyriacus Shows Immunosuppressive Potential

“…C. durissus terrificus causes significant mortality across South America, yet components of its venom have been extensively researched for potential therapeutic applications [9][10][11][12]. In vitro studies have shown that the venom's principal neurotoxin, crotoxin (CTX), and its isolated basic phospholipase A 2 (PLA 2 ) subunit, downmodulate the expression of surface molecules with crucial stimulatory function on immune cells.…”