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

Abstract

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25–200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

Crotoxin Modulates Events Involved in Epithelial–Mesenchymal Transition in 3D Spheroid Model

“…Taken together, our data suggest that CTX significantly inhibits expression of N-cadherin, α-SMA, and integrin αv in MRC-5/A549 spheroids, which correlates with the reduced invasion area in the collagen gel. It also suggests an involvement of CTX with actin polymerization via integrin-dependent signaling pathway with subsequent impairment of migratory ability, a finding that was also observed in endothelial cells in the tumor microenvironment [33]. These findings are in agreement with a similar study conducted with PLA2 (BthTX-II) extracted from the venom of Bothrops jararacussu.…”

“…It has already been reported that the TGF-β pathway is the dominant mediator of crosstalk to initiate the process of CAF activation [37]. Previous studies have demonstrated the inhibitory effect of CTX on the functions of stromal cells such as macrophages [29] and endothelial cells [33] when co-cultivated with tumor-conditioned media. Thus, CTX impairs tumor progression.…”

“…Many studies have shown the inhibitory effect of other PLA2s on tumor cells, however the findings did not correlate the effect with the enzymatic activity of the molecule. CTX acts on focal adhesion kinases (FAK), a crucial component of integrin-mediated cell signaling in endothelial cells [33], and inhibits tyrosine phosphorylation, consequently inhibiting the activity of proteins involved in the intracellular signaling pathway of macrophages [44].…”

“…However, it has low antitumor activity against HL-60 leukemia cells [32]. Recent studies have demonstrated that CTX not only inhibits tumor growth but also modulates stromal cells in the tumor microenvironment, such as the reprogramming of endothelial cells and macrophages, thus exhibiting an antiangiogenic phenotype [26,28,29,33].…”

Snake venom toxins: Potential anticancer therapeutics

“…Crotoxin isolated from snake venom of the South American Crotalus durissus terrificus showed an antineoplastic effect, reduction of cell migration and inhibition of tumour development in oral squamous cell carcinoma (OSCC) cell lines and in mice that were chemically induced with cancer (da Rocha et al, 2022). Crotoxin-induced anti-angiogenesis was achieved by its ability to decrease endothelial cell adhesion to their respective matrix components, which can interfere with effector proteins that play a key role in the regulation of cell proliferation, migration and invasion (Kato et al, 2021). A phase I clinical trial whereby crotoxin was administered intramuscularly for 30 days in 23 patients with different treatment-resistant cancers at 0.21 mg/m 2 maximum tolerated dose showed a report of no death and partial response in cancer patients (Cura et al, 2002).…”