Antineoplastic properties and pharmacological applications of Crotalus durissus terrificus snake venom

ABSTRACT Snake toxins are widely studied owing to their importance in snakebite accidents, a serious public health issue in tropical countries, and their broad therapeutic potential. Isolated fractions from venom produced by snakes of the genus Crotalus sp. present a wide variety of pharmacological uses such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Given the direct effect of this venom on tumor cells, isolation of its compounds is important for the characterization of its anticarcinogenic actions. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies that are efficient against different tumor lines and cellular targets. This review highlights the venom toxins of this species, with a focus on their antineoplastic properties.


INTRODUCTION
Currently, approximately 11,341 reptile species are recognized worldwide 1 , with 1,116 species found in Australia, 974 in Mexico, and 830 in Brazil. Crotalus comprises of the venomous Viperidae snakes 2-7 from the subfamily Crotalinae, also known as rattlesnakes. These are distributed across South America, mainly from Colombia to Argentina 5,[7][8][9] , with the following six subspecies found in Brazil: Crotalus durissus cascavella, C. d. collilineatus, C. d. terrificus, C. d. marajoensis, C. d. ruruima, and C. d. durissus 4,6,9 . These snakes are primarily nocturnal 5 and solenoglyphic dentition 5,10 presents loreal pits, a thermoreceptor organ of viperid species, visible as openings between the eye and the nostril of the animal head, which are of great importance for the detection of temperature variations, particularly of prey and predators 5 . The most striking characteristic of Crotalus snakes is the presence of a rattle at the end of their tails (Figure 1) 5,6 .
This neurotoxic action is mainly attributed to the inhibitory mechanism of acetylcholine release in presynaptic neurons 48,52,54 . Desensitization of postsynaptic nicotinic receptors is another mechanism that reduces the response to acetylcholine 48,52 . Thus, crotoxin acts by blocking potassium channels and prolonging the action potential at neuromuscular junctions, thereby increasing calcium influx into the channels, mainly due to the presence and high activity of PLA 2 in its composition 8,48,52 .
In vitro and in vivo studies have described activating mechanisms of cell apoptosis in different cancer cell lines 19,[47][48][49][50][51]53,55 induced by cellular autophagy mechanisms 47,53 . Both cell death pathways activated by crotoxin (apoptosis and autophagy) can occur simultaneously or sequentially through mechanisms such as changes in mitochondrial membrane potential and release of intracellular cytochrome C. Another important factor related to the mechanism of action of crotoxin is its apparent selectivity for cells with high expression of epidermal growth factor receptors (EGFR) 19,21,47,50,56 .
The cytotoxic action on glioblastoma and benign pituitary adenoma cells was partially attributed to crotoxin, which is also cytotoxic to human mammary duct carcinoma and human lung adenocarcinoma cell lines 4,19,[47][48][49][50][51]55,57 . The application of portions of the toxin to murine erythroleukemia cells demonstrated the potential to reduce the viability of the strain 38 . To observe cytotoxicity, the B subunit of crotoxin was separated from PLA 2 and used alone 38 .
The isolated crotoxin is cytotoxic to different cell lines, with different cell response 53 . The mechanisms evaluated involved changes in the mitochondrial membrane potential, release of cytochrome C, and activation of caspase-3, a protease essential for the process of cell apoptosis [47][48][49][50]52,53,55 . Furthermore, it was possible to conclude that the toxin did not interfere with the viability of keratinocytes, which are highly affected by current antineoplastic therapies 53 .
Crotoxin provokes possible damage to the cellular DNA of PANC-1 cells, associated with pancreatic tumors, by upregulating protein expression 53 . DNA damage has also been observed in glioma cell lines, leading to an increase in the percentage of cells undergoing apoptosis. Some in vitro studies have also reported a higher percentage of apoptosis among SK-MES-1 cells, a lung cancer cell line, in addition to damage such as nuclear condensation and fragmentation 50,57 .
When associated with tyrosine kinase inhibitors, crotoxin potentiates the antitumor effect of the drug against lung tumor cell lines 50,53,57 . In a dose-dependent manner, the toxin prevents DNA synthesis and interrupts the cell cycle in the S phase, suppressing the proliferation of SK-MES-1 cells both in vitro and in vivo 52,57 . One of the mechanisms identified was the increased expression and cleavage of caspase-3, which is responsible for inducing cell apoptosis 50,57 . Another mechanism observed was the induction of cytochrome C release, which increased the occurrence of cellular autophagy, a mechanism also observed in MCF-7 breast cancer lines 47,49,53 .
Crotoxin also induces the release of LXA4, pro-inflammatory eicosanoid lipoxin, and its analogs through the induction of its synthesis in macrophages 36, 44,46,48,51 . In vivo studies of Walker 256 carcinoma cells concluded that this mechanism is responsible for the antineoplastic action of crotoxin on the lineage, and the concentration of lipoxin was 74% higher in the plasma of animals treated with crotoxin than in those treated with saline solution 51 . Lipoxins have been shown to be antineoplastic owing to their ability to inhibit tumor growth by inhibiting endothelial cell proliferation and reducing the production of angiogenic growth factors 46,51 .
Macrophages cultured in vitro in the presence of crotoxin secreted 47% less angiogenesis mediators than macrophages from a control group 46 , confirming the role of the toxin in reducing tumor blood vessel neoformation.
The efficacy of crotoxin in dose-dependent inhibition of human esophageal carcinoma tumor growth (Eca-109 cells) was demonstrated in vivo 55,57 . The toxin causes cellular damage to the lineage, such as formation of pyknotic cell nuclei, cell lysis, and DNA damage 55 . Exposure of tumor cells to crotoxin also resulted in an increase in the number of stagnant cells in the G1 phase of cell division 53,55,57 . Increased expression of caspase 3, p17, and p15 proteins and reduced production of Bcl-25 protein can be envolved 55 .
In vivo studies on the HL-60 leukemia cell line showed lower tumor growth inhibitory activity, suggesting that it acts preferentially on solid tumors 21,47,48,50 . The treatment of patients with solid tumors refractory to conventional antineoplastic therapies with the administration of different doses of crotoxin has demonstrated efficacy in reducing different types of carcinomas 21 . Mechanisms of mitochondrial collapse, cytochrome C release, and caspase 3 activation induced cell death in the human leukemiaassociated K562 cell line, with the induction of apoptosis and autophagy observed 50,57 .
Crotoxin has been shown to be more cytotoxic than standard chemotherapeutic agents for the treatment of glioma, pancreatic cancer, esophageal cancer, and cervical cancer. Therefore, novel antineoplastic therapies are of great interest, particularly against leukemia, lung cancer, colon cancer, renal cancer, ovarian cancer, esophageal carcinoma, breast carcinoma, melanoma, and brain tumors, whose proliferation is already known to be preventable by the toxin 19,53,57 . New drugs derived from the toxin, such as VRCTC-310 and CB24, have already been studied in murine and human cell lines 16,17,21,41,48 .
These enzymes represent the largest family of proteins contained in the venom 23,58 , accounting for up to 80% of total proteins 24 . PLA 2 induces processes such as edema, blockage of neuromuscular junctions, platelet aggregation, and muscle necrosis 21,59 . It has a substantial pharmacological interest owing to a wide range of biological actions 60 . Some enzymes have anticoagulant activity through mechanisms of hydrolysis of procoagulant phospholipids, antagonistic effects with coagulant proteins, and interaction with factor X 25 . Cotrim et al. (2011) suggested that PLA 2 activity is attributable to its actions at different pharmacological sites, which are responsible for platelet aggregation, myotoxicity, and antibacterial activity, as well as anti-inflammatory and neurotoxic effects 58 . PLA 2 has shown anticancer properties by acting on epithelial growth factor receptors (EGFR), reducing the production of tumor necrosis factor, and inhibiting neoplastic growth in lung carcinoma, human breast carcinoma, and leukemia.
The Cdt crotoxin and Naja naja atra cardiotoxin association has been conducted to develop "VRCTC-310-Onco," which aims to interfere with the signaling of EGRFs, reduce the production of tumor necrosis factor, and exert cytotoxic action on tumor cells 16,48 . The development of EGFR receptor inhibitor drugs represents a new type of therapy against epithelial neoplasms 61,62 given that the receptors act in the signaling responsible for the formation of epithelial cell tumors 61 .
Reduction in tumor necrosis factor production is also an important mechanism of anticancer action, since the presence of necrosis stimulates tumor phosphorescence mediators, favoring angiogenesis and tumor metastasis.

GYROXIN
Gyroxin, a member of the serinoprotease family, is a neurotoxic enzyme with coagulant action 4,6,25,45,63 , including thrombin-like action 4,37,45,63,64 , and represents the second most commonly found family of venoms 37 . Montoni et al. (2020) demonstrated that the toxin also has the ability to cross the blood-brain barrier 35 .
In vitro studies have revealed that the enzyme generates clotting in human plasma samples with citrate, with the speed of clot formation being directly proportional to the amount of gyroxin 25 , causing the breakdown of fibrinogen into fibrinopeptide A 25 . Gyroxin is the enzyme responsible for the coagulant activity of Cdt venom as it rapidly consumes circulating fibrinogen, making the blood incoagulable.
Brazilian researchers have used this activity to develop a biopolymer (Heterologous Fibrin Sealant, HFS), which consists of a fibrinogen-rich cryoprecipitate extracted from buffalo blood and a thrombin-like enzyme (gyroxin) purified from Crotalus durissus terrificus snake venom 27,63-65 . They successfully evaluated the safety and immunogenicity of HFS for the first time, estimated the optimum dose, and assessed its preliminary efficacy in the treatment of chronic venous ulcers (CVU) in a phase 2 clinical trial 27,63-65 .
As gyroxin can cross the blood-brain barrier, it can be an important tool for studies of tumors of the brain and central nervous system.

CONVULXIN
Convulxin is a high-molecular-mass glycoprotein of the C-type lectin family, which has potent platelet activating and aggregating action 4,6,66,67 , with high affinity for platelets 66 . However, its effect on human peripheral blood mononuclear cells (PBMCs) and the immune system remains unclear.
The mechanism of action of convulxin involves the activation of phospholipase C and its rapid phosphorylation, which is similar to the mechanism induced by collagens in mediating platelet aggregation 66 .
In in vitro studies utilizing citrated human plasma samples, the protein generated clot formation without interfering with factors of the coagulation cascade 25 .
This toxin induces skeletal muscle contraction through its action on sodium channels, interfering with ion permeability in the sarcolemma and reducing the resting potential of the membranes 18,28,69 . The changes in permeability cause a greater influx of sodium and calcium ions, which are responsible for depolarization, muscle contraction, vacuolization of sarcoplasmic reticulum, rupture of actin and myosin muscle filaments, and muscle necrosis 18,28,69,71 .
Translocation across cell membranes occurs by binding between crotamine and cell surface heparan sulfate proteoglycans, endocytosis, and accumulation of the toxin in intracellular vesicles 18,28,[69][70][71][72]75,76 . To reach the cytoplasm, crotamine induces changes in the permeability of vesicles, causing it to be released and dispersed in the cell 18,69,72,76 . In the cytoplasm, it can bind to centrosomes in the G1 phase of cell proliferation and enables the diagnosis of cell division phases by functioning as a molecular marker 18,68-71 .
The antitumor and antimicrobial properties of crotamine are due to its ability to bind to surfaces and acidic cellular compartments such as lysosomes 28, [74][75][76] . In tumor cells, the prevalence of negatively charged surface molecules, such as phospholipids and mucins, allows preferential binding with the toxin compared to that in healthy cells with electrically neutral surfaces 70 .
To develop new drugs, synthetic analogs of crotamine were produced, composed of peptides with smaller chains, and were used to study their functions in comparison to natural crotamine, revealing the possibility of producing crotamine derivatives with important antimicrobial and antineoplastic functions 18,74 .
Crotamine possesses preferential selectivity for proliferating cells and for certain phases of the cell cycle 18 The role of crotamine against murine melanoma cells, human melanoma cells, and primary human pancreatic adenocarcinoma cells has been extensively studied 26, [68][69][70][71] . Although it is cytotoxic to normal cells when administered at high doses, it is non-toxic at low doses 18 .
Crotamine administered via the intraperitoneal route at a concentration of one microgram per animal per day for 21 days demonstrated efficacy in reducing tumors in rats with subcutaneous melanoma [68][69][70][71][72] .
Crotamine's action mechanisms to induce cell apoptosis include the activation of caspases, the reduction of mitochondrial membrane potential, and consequent alteration of organelle membrane permeability, inducing the release of intracellular calcium ions and the influx of extracellular calcium 28, 68,70,71,73,76 . The activation of caspases is one of the mechanisms responsible for cell apoptosis signaling 78 . Its activation can occur by alterations in mitochondrial membrane permeability, which generates cytochrome C release that amplifies apoptosis signals 69 in HL-60 cells from human leukemia and urinary bladder tumors 69 .
Owing to the ability of the toxin to penetrate cells, it is a potential delivery mechanism for other drugs and antitumor agents 18,28,68,69,[71][72][73][74] . In addition to representing a possible antineoplastic or adjuvant therapy, crotamine can be used as a diagnostic marker for cancer 70,73,76 . Crotamine can be used as a diagnostic marker in human epithelial carcinoma (HeLa), human pancreatic adenocarcinoma (BxPC-3), human breast carcinoma (BT-474), and human colorectal carcinoma (Caco2) cells.
These enzymes induce apoptosis in human leukemia cells. Their toxicity is mainly attributed to the formation of hydrogen peroxide during oxidative reactions, among other mechanisms 16,21,30,79,80 . Although cytotoxic to tumor cells, LAAOs do not affect healthy cells 21,80 .
The species-specific cytotoxicity of LAAOcdt was evaluated using nine human cancer cell lines, including pancreatic, esophageal, cervical, and glioblastoma tumors 80 .
Purified LAAOs can act on different targets of cellular mechanisms such as DNA fragmentation, chromatin condensation, and nuclear fragmentation. Another mechanism is the induction of P53 protein expression, which is synthesized from a tumor suppressor gene that is functionally deficient in more than half of the human tumors 78,79,81 . Moreover, the induction of protein expression would be relevant to stimulate the monitoring of genome integrity, allowing the identification of damage and repair, resulting in the reduced proliferation of cells with genetic mutations.

LECTINS
Lectins belong to a family of proteins and glycoproteins that generate platelet aggregation 10,16,20,67 . C-type lectins are non-enzymatic calcium-dependent proteins that affect cell adhesion, endocytosis, and neutralization of pathogens 67 . These proteins may also interfere with tumor proliferation, which has been observed using lectins from venom of other species, offering potential for antineoplastic therapy 16 .

METALLOPROTEASES
Metalloproteases present hemorrhagic action and induce coagulation alterations in the prey 16,21,82 . These proteins are copious in crotalid venom 82 but are present in low quantities in Cdt venom, thus conferring low proteolytic and hemorrhagic activity 33 .
This class of enzymes is composed of endopeptidases that degrade extracellular matrix proteins, blood components, and endothelial cells 21 . In addition, metalloproteases play a fibrinolytic role and act as prothrombin activators, blood coagulation factor X activators, apoptosis inducers, platelet aggregation inhibitors, pro-inflammatory agents, and inactivators of serinoprotease inhibitors 82 . Different groups of metalloproteases found in viperid and crotalid venoms are involved in tumor proliferation and angiogenesis processes 16 . However, specific studies on Cdt venom metalloproteinases have not yet been conducted.

DISINTEGRINS
Disintegrins are also important for the inhibition of tumor cells, together with metalloproteases, by acting against angiogenesis and metastasis 16 . This group of non-enzymatic proteins of low molecular mass can interact with integrins expressed by different cells 16,17,20,83 , important cell surface receptors that are involved in interactions between cells and between cells and the extracellular matrix 16,17,20,21,83 .
Integrins, one of the most important targets of antineoplastic action, are cell surface adhesion molecules that function as receptors and transmitters of cellular signals for migration, invasion and cell proliferation 16,83 . Inhibition of integrins is important because it affects cell proliferation, angiogenesis, and metastasis and is a widely studied antineoplastic treatment option 16,83 .
Disintegrins isolated from Cdt venom inhibit the interaction between tumor cells, impairing their motility, and preventing the invasion of other tissues 21 . One of the mechanisms involved is the deposition of fibrin around the tumor, which limits its growth.

PHOSPHODIESTERASES
These enzymes are less abundant in the venom, representing only approximately 2% of its total 33 . Despite being present in the venom in low quantities, it is responsible for inducing important clinical signs of intoxication 33 , and its antitumor activity has not yet been evaluated.

CLINICAL TRIALS
Crotoxin was administered to patients with solid tumors that were refractory to standard therapy in a phase 1 clinical trial that observed a partial response of more than 50% reduction in tumor mass and a complete response in three of the 23 evaluated patients 21,48,77 . The authors concluded that crotoxin is a new class of anticancer agents that acts through a novel mechanism of action and thought that neurotoxicity could be the principal toxic effect and appears to be manageable. They recommended 0.18 mg/m 2 a therapeutic dose for Phase II studies 77 .
The same research team proposed an innovative design for a phase 1 trial with intra-patient dose escalation to study crotoxin 85 .
As recorded on the clinical trial platform ClinicalTrial.gov, 18 patients were recruited for this study between 2015 and 2018. The researchers stated that the results would be published shortly 86 .

CONCLUSIONS
After elucidating the various mechanisms of action of the C. d. terrificus venom, it may be stated that this venom is a potential candidate for the development of new antineoplastic therapies that are efficient against different tumor lines and act on different cellular targets.
Considering the selective cytotoxicity of venom components for tumor cells to the detriment of healthy cells, the development of innovative therapies against cancer, based on the bioactive compounds of the rattlesnake, may present greater benefits compared to current therapeutic protocols, such as chemotherapy and radiotherapy, which are known to cause alterations in the normal cells of cancer patients.
The therapeutic use of compounds from Crotalus durissus terrificus snake venom also represents an alternative for the treatment of tumors resistant to drugs currently available on the market.
Therefore, one can conclude that the improvement of studies of the different fractions of ophidian venom is of great pharmacological interest, with potential for immense impact on the future of therapeutic medicine.