Biochemical Society Transactions
Portland Press Ltd.
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Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase
Volume: 48, Issue: 2
DOI 10.1042/BST20200110
Abstract

The phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are two major enzymes found in the venoms from most snake species. These enzymes have been structurally and functionally characterised for their pharmacological activities. Both PLA2 and LAAO from different venoms demonstrate considerable cytotoxic effects on cancer cells via induction of apoptosis, cell cycle arrest and suppression of proliferation. These enzymes produce more pronounced cytotoxic effects in cancer cells than normal cells, thus they can be potential sources as chemotherapeutic agents. It is proposed that PLA2 and LAAO contribute to an elevated oxidative stress due to their catalytic actions, for instance, the ability of PLA2 to produce reactive oxygen species during lipolysis and formation of H2O2 from LAAO catalytic activity which consequently lead to cell death. Nonetheless, the cell-death signalling pathways associated with exposure to these enzymatic toxins are not fully elucidated yet. Here in this review, we will discuss the cytotoxic effects of PLA2 and LAAO in relationship to their catalytic mechanisms and the underlying mechanisms of cytotoxic actions.

Keywords
Hiu and Yap: Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase

Introduction

Snake venom is a complex mixture of proteins and polypeptides with a diverse array of pharmacological activities. The proteins and polypeptides constitute ∼95% of the dry weight of the venom [1]. Significant differences in venom composition have been reported between closely related species or even between the same species from different geographical origins [2,3]. Among all the venom toxins, the enzymatic toxins phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are ubiquitously found in Elapidae and Viperidae whereby PLA2 exists as the most abundant enzymatic toxins, as revealed by venom proteome (Figure 1).

Distribution of different venom toxins from Elapidae and Viperidae
Figure 1.
The venom toxins are coloured according to their respective pharmacological activities, whereby colour intensity indicates the dose-dependent pharmacological actions. On the other hand, different colour distributions within the same toxins correspond to the multiple biological effects exerted by the toxins [90,91]. Of all venom enzymatic toxins, the enzymes LAAO and PLA2 exhibit cytotoxicity (represented by a yellow colour). Abbreviations: LAAO, l-amino acid oxidase; SVMP, snake venom metalloproteinase; PLA2, phospholipase A2; SVSP, snake venoms serine protease; 3FTX, three-finger toxin; DTX, dendrotoxin; CTL, C-type lectin; CRISP, cysteine-rich secretory protein; MTX, myotoxin.Distribution of different venom toxins from Elapidae and Viperidae

PLA2 is one of the most extensively studied enzymatic toxins in snake venoms [4]. Snake venoms are the major source of Group 1 and Group II secretory PLA2. Generally, the venom PLA2 is a small protein with the molecular mass of ∼13–15 kDa. The enzyme catalyses the hydrolysis of phospholipids at sn -2 positions to produce lysophospholipids and free fatty acids [5]. It requires Ca2+ for their catalytic actions [6]. The venom PLA2 possesses presynaptic or postsynaptic neurotoxicity [7,8], systemic or local myotoxicity [9,10], cardiotoxicity [11], platelet aggregation inhibition [12], anticoagulant [13] and oedema inducing activities [14]. The venom-induced neurotoxicity has been suggested to be attributed to the β-neurotoxin, a PLA2 enzyme in nature that inhibits pre-synaptic neuromuscular transmission [15]. Although the molecular mechanism is not well characterised, studies have shown that the neurotoxic effects exerted by venom PLA2 are presumably due to the influx of cytosolic calcium ions when binding to the voltage-gated ion channels on the neuronal membrane [16,17]. Besides, the PLA2 can cause mitochondrial membrane disruption in the respiratory muscle as a result of phospholipid hydrolysis [18,19]. These events further lead to acute neuromuscular weakness, followed by flaccid paralysis [20]. In general, PLA2 from Elapidae venom exists as a monomeric enzyme and possesses neurotoxicity while Viperidae venom PLA2 can exist in both monomer and dimer forms. The Viperidae monomeric PLA2 exhibits cytotoxic effects, whereas dimeric PLA2 possesses cytotoxic effects at a lower dose and neurotoxicity at a higher dose ([21], Figure 1).

LAAO is a flavoenzyme that catalyses the oxidative deamination of l-amino acid to α-keto acid and produces hydrogen peroxide (H2O2 ). Snake venom LAAOs display various pharmacological activities. Some enzyme LAAOs exhibit potent platelet inhibitory actions [22] while other LAAO isoforms induce platelet aggregation [23]. The antiplatelet mechanism of LAAO is attributed to the elevated production of H2O2 , ammonia, and α-keto acid [24]. The liberated H2O2 affects ADP-induced platelet formation and distorts the interactions between blood coagulation factors [25,26]. In addition, LAAO also possesses antimicrobial actions [27], oedema [28], haemolysis [29] and haemorrhage [30].

Although both enzymatic toxins demonstrate various pharmacological effects, they share a similar feature whereby the products from their catalytic actions pose potent cytotoxic agents. For example, venom PLA2 alters plasma membrane integrity in muscle cells to cause myonecrosis [31]. The membrane perturbation by PLA2 is a secondary process to its catalytic actions on membrane phospholipids [32], indicating that venom PLA2 exhibits remarkable cytotoxicity. On the other hand, venom LAAO has also been demonstrated to induce cell death due to the generated H2O2 [33–35]. Cancer is characterised by an uncontrolled cells proliferation, the ability to escape apoptosis and evading growth suppressors with active metastasis. Cancer cells differ from normal cells not only in the cellular metabolism but the lipid compositions on plasma membranes. Cancer cells have asymmetry in their membrane lipid compositions such as extracellular accumulation of phosphatidylserine [36] and higher lipid concentrations than normal cells [37]. Both enzymatic toxins exert their effects on the plasma membrane, it is thus suggested that cancer cells are more susceptible to toxins’ actions.

In this review, we outline our current understanding of the structural properties and catalytic actions of both PLA2 and LAAO. In addition, we also discuss and summarise the cytotoxic effects exerted by PLA2 and LAAO against different cancer cells with a specific focus on the underlying mechanisms.

Phospholipase A2

PLA2 (EC 3.1.1.4) is an enzyme belongs to a family of lipolytic enzyme esterase which specifically catalyses the hydrolysis of the ester linkages in glycerophospholipids at the sn-2 position. The hydrolysis of glycerophospholipids liberates free fatty acid, such as arachidonate and the release of lysophosphatidic which are the mediators in various biological processes.

The Ca2+ is a crucial cofactor for catalysis, thus the Ca2+ binding loop structure is highly conserved in most of the venom PLA2. The structure of PLA2 has three major α-helices and two antiparallel β-sheets cross-linked by disulfide bonds [38]. The disulfide-linked α-helices (residues 37–54 and residues 90–109) form a hydrophobic channel catalytic site which facilitates the binding of phospholipid substrates [31]. The four key residues in the active site involves in the coordination of the Ca2+ , are His48, Asp49, Tyr52 and Asp99 via hydrogen bond formation and coupling interaction [6]. The venom PLA2 can be classified into two major groups, namely Group I PLA2 (GIPLA2) and Group II PLA2 (GIIPLA2 ) according to the location of disulfide bonds [6,39].

Group I PLA2 (GIPLA2)

The venom GIPLA2 consists of 115–125 residues with a molecular mass of 13–15 kDa [40]. The GIPLA2 has a single polypeptide chain containing 6–8 disulfide bridges [6]. It contains ∼50% of α-helices and 10% of β-sheets [40]. The venom GIPLA2 has an elapid loop (residues 57–59) that links the α-helices and the β-sheets [41], thus, GIPLA2 is found ubiquitously in elapids venoms. The venom GIPLA2 is different from mammalian pancreatic PLA2 , which the latter enzyme has a pancreatic loop with an additional five amino acid residues at position 62–67 [42]. The GIPLA2 is further divided into Group IA and Group IB for snake venom PLA2 and mammalian pancreatic PLA2, respectively. Despite so, Group IB PLA2 enzymes have also been identified in the venoms from Oxyuranus scutellatus, Micrurus frontalis frontalis, Notechis scutatus and Ophiophagus hannah due to the presence of the α-helix that is identical with mammalian pancreatic PLA2 [43].

Group II PLA2s (GIIPLA2)

The venom GIIPLA2 is found exclusively in Viperidae venoms. It contains 120–125 amino acid residues and seven disulfide bonds [6]. Unlike GIPLA2, neither the pancreatic nor elapid loops are present in GIIPLA2 enzymes. However, it possesses a C-terminal extension with a different organisation of disulfide bonds, which clearly distinguishes GIIPLA2 from GIPLA2 [44]. In GIIPLA2, the D49 is conserved and contributes to Ca2+ -dependent catalytic activity [45]. Thus, GIIPLA2 is also recognised as D49 acidic PLA2 [46].

Mechanism of cytotoxicity

PLA2 catalyses the cleavage of the ester bond of phospholipids at the sn -2 site by nucleophilic attack [47]. Calcium ion, on the other hand, stabilises the negatively charged transition state by coordinating the phosphate oxygen and a carbonyl group during the catalysis [48]. Most of the biological membranes are composed of phospholipids, it is believed that PLA2 alters the membrane fluidity and causes membrane permeabilisation, which ultimately leads to cell death. The cytotoxic effects of PLA2 on a different cell are summarised in Table 1.

Table 1.
The cytotoxicity of different PLA2 from different snake species on various cell types. The IC50 indicates the concentration of venom PLA2 to kill 50% of the cell populations
SpeciesTypes of PLA2Cell typesIC50References
Bothrops asperbasic PLA2Mouse adrenal tumour cellsn.d.[92]
Bothrops braziliacidic PLA2Jurkat human acute T-cell leukaemia cells100.0 μg/ml[53]
Bothrops jararacaacidic PLA2peripheral blood mononuclear cells (PBMC)n.d.[93]
HL60 human leukaemia cellsn.d.
Bothrops jararacussuBth TX-1Jurkat human acute T-cell leukaemia cellsn.d.[54,57,94,95]
Erlich ascitic tumour cellsn.d.
SK-BR-3 human breast cancer cells81.2 μg/ml
MCF-7 human breast cancer cells104.35 μg/ml
MDAMB231 human breast cancer cells>409 μg/ml
PC-12 rat adrenal medulla pheochromocytoman.d.
C2C212 murine muscle cellsn.d.
B16F10 mouse melanoma cellsn.d.
S180 murine sarcoma cellsn.d.
Bothrops moojeniacidic PLA2Jurkat human acute T-cell leukaemia cellsn.d.[96]
K562-S human immortalised myelogenous leukaemia cells257 μg/ml[55]
K562-R human immortalised myelogenous leukaemia cells191 μg/ml
Crotalus durissus terrificusHeterodimeric basic PLA2Murine erythroleukemia cells3.0–5.0 μg/ml[97]
SK-LU-1 human lung cancer cells∼4.0 μg/ml[98]
Hs578T human breast cancer cells∼5.3 μg/ml
KYSE 30 oesophageal cancer cells1.0 μg/ml[99]
GAMG human glioblastoma cells<0.5 μg/ml
HCB151 glioma cells4.1 μg/ml
PSN-1 human pancreatic cancer cells0.7 μg/ml
PANC-1 pancreatic cancer cells<0.5 μg/ml
HeLa cervical cancer cells2.4 μg/ml
KYSE 270 oesophageal cancer cells8.7 μg/ml
U373 glioma cells30.2 μg/ml
SiHa cervical cells>30.0 μg/ml
Daboia siamensisdssPLA2SK-MEL-28 human skin melanoma cellsn.d.[60]
Daboia russelii siamensisdrsPLA2SK-MEL-28 human skin melanoma cells0.90 μg/ml[62]
Echis carinatus sochurekiSer49 PLA2A549 human adenocarcinoma cells8.5 μM[49]
HUVEC human umbilical vein cells12.2 μM
Echis coloratusSer49 PLA2A549 human adenocarcinoma cells3.5 μM
HUVEC human umbilical vein cells4.9 μM
Echis ocellatusSer49 PLA2A549 human adenocarcinoma cells5.2 μM
HUVEC human umbilical vein cells5.0 μM
Echis pyramidum leakeyiSer49 PLA2A549 human adenocarcinoma cells2.9 μM
HUVEC human umbilical vein cells2.5 μM
Micrurus lemniscatusMyotoxic group I PLA2 (lemnitoxin)Rat myocytesn.d.[100]
Naja atraPLA2SK-N-SH human neuroblastoma cellsn.d.[101]
Naja najaacidic PLA2Erlich ascitic tumour cellsn.d.[102]
partially differentiated L6 rat myoblastsn.d.[103]
platelets from citrated goat bloodn.d.
rat pheochromocytoma PC-12 cellsn.d.
Naja nigricollisNigexine (basic PLA2)Epithelial FL cells1.6 mM[104]
C-13 T neuroblastoma cells2.9 mM
HL60 human leukaemia cells3.1 mM
Vipera ammodytes ammodytesneurotoxic secretory PLA2Motoneuronal NSC34 cellsn.d.[7]

In general, venom PLA2 variants can be classified into D49 acidic PLA2 (Asp-49), K49 basic PLA2 (presence of Lys-49 instead of Asp-49) and S49 PLA2 (presence of Ser-49). The basic PLA2 homologues, K49 and S49 PLA2s are responsible for many Ca2+ independent biological activities and thus they are catalytically inactive [45]. The D49 acidic PLA2 is less cytotoxic than K49 basic PLA2, whereby acidic PLA2 possesses higher IC50 than basic PLA2 (Table 1). On the other hand, S49 PLA2 variants have been isolated from the venoms of saw-scaled vipers Echis sp. [49] which also exhibit Ca2+ independent biological activities with potent cytotoxic effects than K49 PLA2 (IC50 = 2.5–12.2 μM). Despite so, S49 PLA2 demonstrates weaker lipolytic activity compared with K49 PLA2 [50]. The basic PLA2 homologues display more pronounced cytotoxic effects in cancer cells.

The C-terminal region of the PLA2 is believed to be responsible for compromised membrane integrity and interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) [51,52]. The C-terminal region of the enzyme could also bind to VEGFR-2 to inhibit angiogenesis, an essential process in cancer metastasis. Therefore, the cytotoxicity of PLA2 is probably mediated by the interaction between the C-terminal region and the plasma membrane [53–55]. Besides, the PLA2 -induced cytotoxicity might involve the liberated reactive oxygen species (ROS) during its phospholipid metabolism, further increases intracellular oxidative stress. Elevated oxidative stress leads to the activation of cell death pathways. Although there is no establishment of the exact pathways, it might involve the down-regulation of anti-apoptotic proteins such as Bcl2, Bcl-XL and c-FLIP [56]. There is also an increase in pro-apoptotic BAD expression and the activation of caspase 3 [56]. Moreover, PLA2 alters the distribution of different phases in the cell cycle to cause apoptosis [57]. PLA2 also exerts genotoxic effects to induce cytotoxicity in human lymphocytes [58]. In addition, PLA2 induces cytotoxicity through DNA damage and the formation of micronuclei [58]. The PLA2 also significantly ameliorates the expression of proto-oncogene NOTCH1 and BRAF V600E genes in SK-MEL-28 cells [59]. As revealed by Annexin V-Propidium iodide double-staining flow cytometry, apoptosis remains as the predominant cell death mechanism in PLA2 -associated cytotoxicity [60]. It is noteworthy that, the venom PLA2 exhibits time-dependent and dose-dependent cytotoxicity in cancer cells without any effects on normal cells [61]. Besides, the venom PLA2 has been reported for its in vivo antitumour properties. The PLA2 from Bothrops jararacussu , BthTX-1 could reduce the S180tumour size by 79% in BALB/c mice [54]. In addition, Drs-PLA2 from Daboia russelii siamensis has also been found to reduce tumour nodules by 65% in BALB/c mice [62]. So far, only crotoxin, a PLA2 from Crotalus durissus terrificus venom undergoes phase I clinical trials which shows the objective partial response in cancer patients [63]. The cytotoxicity of PLA2 is described in a schematic diagram (Figure 2).

Summary of the cytotoxic effects of venom phospholipase A2 in cancer cells
Figure 2.
An example of the three-dimensional structure of a K49 basic PLA2 from Bothrops flavoviridis venom is shown [Protein Data Bank accession (PDB) ID: 6AL3]. The C-terminal of PLA2 interacts directly with the cell membrane to produce membrane perturbating effects. Accumulation of reactive oxygen species (ROS) occurs due to catalytic actions of PLA2 on membrane phospholipids which causes cell death. The venom PLA2 reduce the expression of anti-apoptotic proteins, for example, Bcl2, Bcl-XP, c-FLIP and proto-oncogene such as NOTCH1 and BRAF V600E. On the contrary, venom PLA2 increases the expression of pro-apoptotic proteins BAD and caspase-3. At the same time, venom PLA2 triggers cell cycle arrest in cancer cells. Altogether, the findings imply that apoptosis is the predominant cell death mode in PLA2-induced cytotoxicity.Summary of the cytotoxic effects of venom phospholipase A2 in cancer cells

l-amino acid oxidase

LAAO (EC. 1.4.3.2) is a homodimeric flavoenzyme with covalently linked-flavin adenine dinucleotides (FADs) contributes to a yellow appearance in snake venom. Each subunit in LAAO possesses a molecular mass of 50–70 kDa. The enzyme has a molecular mass of 110–159 kDa under a native state [26,64]. The LAAO consists of three major domains, which are a substrate-binding domain, a FAD-binding domain and a helical domain ([65], Figure 3a). The substrate-binding domain is characterised by seven strands of mixed β-pleated sheet forming a pocket for substrate binding.

The structural and cytotoxic properties of venom l-amino acid oxidase (LAAO)
Figure 3.
A ribbon representation of LAAO (PDD ID: 5Z2G, Naja atra venom) is illustrated in (a). The LAAO is a homodimeric flavoenzyme containing a substrate-binding domain (yellow colour of mixed β-pleated sheet), a flavin adenine nucleotide (FAD) binding site and a helical domain (red colour). The enzyme catalyses the oxidative deamination of l-amino acid and produces H2O2 as the main mediator for its cytotoxicity, as illustrated in (b). LAAO exerts apoptosis in cancer cells through extrinsic and intrinsic pathways. It is noteworthy that there is an up-regulation of CYP450 gene families to further enhance the oxidative by producing excessive ROS. On the contrary, the cell cycle arrest gene CDKN2B is down-regulated after exposure to LAAO. The CDKN2B is the main cell cycle regulator that inhibits G1 progression. It explains the role of LAAO in cell cycle arrest at the Go–G1 phase. Abbreviations: DISC-FADD, death-inducing signalling complex and Fas-associated death domain; MMP, mitochondrial membrane potential; BMP, Bcl2 modifying factor; IGFBP3, Insulin-like growth binding protein 3; PLEKHF1, Pleckstrin homology domain containing family F member 1; HSPD1, heat shock 60 kDa protein 1; SQSTM1, Sequestosome 1; MLF1, myeloid leukaemia factor 1; KLF10, Kruppel-like factor 10; CDKN2B, Cyclin-dependent kinase inhibitor 2B.The structural and cytotoxic properties of venom l-amino acid oxidase (LAAO)

The FAD-binding domain is composed of two conserved motifs, including the FAD-binding motif and the GG motif, with a consensus sequence of three glycine residues (Gly) residues [66]. The first Gly is highly conserved and contributes to the positioning of the second Gly. The second Gly allows a proximity of the main chain to the negatively charged pyrophosphate of the FAD. The second Gly residue of the GG motif plays an important role in interacting with the ribose of the FAD molecule Whereas, the third Gly promotes the close packing of α-helix and β-sheets of the motifs [67]. In brief, these interactions stabilise the tight binding of the FAD cofactor to the LAAO [68].

The helical domain forms a funnel-shaped entrance protruding into the protein core near the flavin cofactor, where the active site is located. This funnel-shaped helical domain facilitates the entry orientation of amino acid substrates through electrostatic interaction with the carboxylic groups (–COOH) of the substrates [65]. It appears that the key residues involved in the interaction with substrates are Arg90 and Gly 464 [65,69]. Besides, there are also two residues, His223 and Arg 322 which present at the active site to involve in the catalytic mechanisms of LAAO [69]. The LAAO exhibits high stereospecificity and enantioselectivity towards the oxidative deamination of l -amino acids due to the presence of a helical domain specifically in LAAO [70].

A catalytic reaction of LAAO comprises a reductive half reaction and the oxidation half reaction (Figure 3b). During the first half of the reduction reaction, FAD plays an important role as a cofactor. The reductive half reaction involves the abstraction of a proton from the amino group of the l -amino acid substrate by a basic His223 residue [65]. Concomitantly, an imino intermediate is formed when a hydride is transferred from α carbon of the substrate to the N5 of the FAD isoalloxazine ring. The cofactor FADH2 is produced in this reaction. The imino acid is further hydrolysed non-enzymatically into α-keto acid and ammonia [71]. The second oxidative half reaction involves the oxidation of the FADH2 into FAD and at the same time, generating H2O2 [72]. This reaction completes the LAAO catalytic cycle as the FAD cofactor is regenerated for subsequent cycles [73].

Mechanism of cytotoxicity

Extensive studies have demonstrated that snake venom LAAOs induce cytotoxic effects, particularly on cancer cell lines (Table 2). However, the actual cytotoxic mechanism is poorly understood. Most of the hypotheses are based on the accumulated H2O2 generated during the LAAO catalytic activity, which leads to oxidative stress [22,74,75]. This theory is further supported by a few studies which have demonstrated a reduction in the cytotoxic effect of LAAO upon exposure to glutathione (GSH) or catalase, which inhibit the H2O2 activity [34,75,76].

Table 2.
The cytotoxicity of different LAAO from different snake species on various cell types. The IC50 indicates the concentration of venomous LAAO to kill 50% of the cell populations
SpeciesName of LAAOCell typeIC50References
Agkistrodon acutusACTX-6A549 human lung cancer cells20 μg/ml[84]
ACTX-8HeLa cervical cancer cells[75]
Agkistrodon contortrix laticinctusACL LAOHL60 human leukaemia cellsn.d.[30]
Agkistrodon halysAhLAAOL1210 mouse lymphocytic leukaemian.d.[80]
MOLT-4 human lymphoblastic leukaemia cells
HL60 human leukaemia cells
RPMI 1788 human peripheral blood
A549 human lung cancer cellsn.d.[105]
Bothrops atroxBatroxLAAOHL60 human leukaemia cells50 μg/ml[78,83]
B16F10 mouse skin melanoma25 μg/ml
PC-12 rat adrenal medulla pheochromocytoma
Jurkat human acute T-cell leukaemia cells
Normal human keratinocytes5.1 μg/ml[33]
Bothrops insularisBiLAOTubular[106]
Bothrops jararacaBjarLAAO-IEhrlich ascites tumour cellsn.d.[107]
Bothrops leucurusBI-LAAOMKN-45 gastric cancer cellsn.d.[34]
HuTu human duodenocarcinoma
RKO human colorectal cells
LL-24 human fibroblast cells
Bothrops moojeniBmooLAAO-IEAT cells[23]
HL60 human leukaemia cells
Bothrops pirajaiBpirLAAO-IHL60 human leukaemia cells
BCR-ABL human leukaemia cells
n.d.[79]
HL60 human leukaemia cells
Jurkat human acute T-cell leukaemia cellsn.d.[76]
SKBR-3 human breast cancer cells
S180 murine sarcoma
Ehrlich ascites tumour cell
Bungarus fasciatusBF-LAAOA549 human lung cancer cellsn.d.[28]
Calloselasma rhadostomaCR-LAAOJurkat human acute T-cell leukaemia cellsn.d.[85]
Crotalus atroxApoxin IHL60 human leukaemia cellsn.d.[81]
A2780 human ovarian cancer cells
293T human embryonic kidney cells
KN-3 odontoblast cells
Eristocophis macmahoniLNV-LAOMM6 human monocytic cells[64]
Lachesis mutaLmlAAOAGS gastric adenocarcinoma22.7 μg/ml[108]
MCF-7 human breast cells1.41 μg/ml
VERO normal epithelial monkey kidney0.83 μg/ml[35]
EA. hy926 human umbilical vein
HeLa cervical cancer cells
MGSO-3 human breast cancer tissue
normal human keratinocyte
Ophiophagus hannahOH-LAAOB16F10 murine melanoma0.17 μg/ml[109]
HT-1080 human fibrosarcoma0.6 μg/ml
CHO Chinese hamster ovary cells0.3 μg/ml
murine epithelial cells Balb/3T30.45 μg/ml
PC3 human prostate cancer cells0.05 μg/ml[87]
MCF-7 human breast cancer cells0.04 μg/ml[110]
A549 human lung cancer cells0.05 μg/ml[110]
Trimeresurus flavoviridisOHAP-1rat C6 glioma cells RBR 17Tn.d.[111]
human glioma U251
Trimeresurus stejnegeriTSV-LAOC8166 human T cell leukaemia24 nM[112]
Vipera berus berusVB-LAAOHeLa cervical cancer cellsn.d.[22]
K562 human leukaemia cells

The liberated H2O2 accumulates as ROS to cause direct deterioration of the cell membranes. The oxidative stress by H2O2 could also lead to the dissipation of MMP to induce translocation of cytochrome c to cytosol [77]. Cytochrome c then activates caspase-9, an initiator caspase presence in the intrinsic mitochondrial-mediated apoptosis. The p53 apoptotic proteins are found to be substantially expressed in the presence of LAAO, followed by translocation of the cytoplasmic Bax protein to mitochondria to activate the downstream apoptotic pathways [75]. Furthermore, LAAO has been reported to activate another initiator caspase-8 in the extrinsic death-receptor apoptosis before downstream activation of caspase-3 (the executioner phase of apoptosis) [78,79]. Extrinsic apoptosis requires ligands–death receptor interactions to form DISC-FADD, followed by cleavage of pro-caspase 8 to active caspase-8. However, it is uncertain if LAAO interacts with the death receptors for the occurrence of the extrinsic pathway. On the other hand, caspase-3 is responsible for the endpoint apoptotic features such as chromatin condensation (karyorrhexis) and DNA fragmentation. The findings thus conclude that LAAO exerts apoptosis through extrinsic and intrinsic pathways.

Besides, LAAO from Agkistrodon halys venom displays cytotoxicity on murine lymphoblastic leukaemia cells (L1210) with prominent apoptotic features such as DNA fragmentation [80]. Similarly, apoxin 1, a type of LAAO from Crotalux atrox venom also induces DNA fragmentation in human umbilical endothelial cells, HL-60 (human leukaemia) A2780 (human ovarian carcinoma) and NK-3 (rat endothelial cells) due to elevated H2O2 levels [81] The ACL-LAAO isolated from Agkistrodon cntortrix venom has also been demonstrated to cause DNA fragmentation in HL60 cells [30]. On the other hand, LAAO from Ophiophagus hannah venom was found to alter several apoptotic, autophagic and cell cycle-related genes, as a result of accumulated H2O2 released from the enzyme action [82]. Furthermore, the LAAO also significantly up-regulates cytochrome P450 genes to further increase intracellular ROS levels [82]. Similar to PLA2, venom LAAO also induces cell cycle arrest in cancer cell lines. In a study on Bothrops atrox snake venom LAAO treated HL-60 cells, the BatroxLAAO exerts an arrest in the Go/G1 phase with a decrease in S and G2/M phases [83]. Another LAAO from Agkistrodon acutus venom, namely ACTX-6 also elicits cell cycle arrest in A549 cells [84]. Collectively, these findings suggest that venom LAAO activates both intrinsic and extrinsic apoptotic pathways (Figure 3).

In addition to apoptosis, the venom LAAO exhibits a dose-dependent transition of apoptosis to necrosis when its concentration increases [22,33,80,83]. This is presumably related to the levels of H2O2 produced by the enzyme, as the treatment with catalase significantly reduced the number of necrotic cells [85].

Although apoptosis remains as the predominant cell death mode in LAAO-induced cytotoxicity in cancer cells, the venom LAAO is able to cause autophagy in normal human keratinocyte [33]. Autophagy refers to a self-degenerative cell death process in which cellular components are degraded in autophagic vacuoles of dying cells [86]. The LAAO-induced cytotoxic effects are dose dependent and follow a sequential manner of cells undergoing autophagy, apoptosis to necrosis within 24 h [33,35]. On the other hand, preclinical trials of LAAO from Ophiophagus hannah revealed that LAAO suppresses PC-3 Solid Tumour Growth in a tumour xenograft mouse model [87]. The venom LAAO exhibits selectivity towards cancer cells and relatively non-toxic to normal cells [79,87–89].

Conclusion

The enzymatic toxins, PLA2 and LAAO from snake venoms, exhibit pronounced cytotoxic effects mainly on cancer cells. They suppress cancer cells proliferation, induce apoptosis and cell cycle arrest, although necrosis and autophagy cell death are also observed. The C-terminal region of PLA2 is suggested to contribute to its cytotoxicity upon interaction with the cell membranes. On the other hand, LAAO is known to produce notable levels of H2O2 through its enzymatic reaction. Therefore, the enzymes are known to cause the accumulation of ROS which eventually leads to cell death. Besides cytotoxicity, PLA2 and LAAO also possess anticoagulant activity which could be promising candidates in cancer research as venous thromboembolism is often observed in cancer. The exact modes cell death elicited by the enzymes, especially the potential agonistic actions on the death receptors, are not well established. Therefore, elucidation of the possible enzymes–receptors interactions is required in future studies. While considering the potential anticancer effects of both enzymes, we must not forget to ascertain the selectivity of the enzymes towards cancer cells only. Since non-cancer cells are less susceptible to both enzymes, it is most likely that the cytotoxic actions of PLA2 and LAAO are selective to cancer cells only. Nevertheless, before these enzymatic toxins can be developed into chemotherapeutic agent, their efficacy, potency and safety need to be established while considering new approaches for targeted delivery, these include formulation into nanoparticles or conjugation with ligands or monoclonal antibodies which recognises targeted cancer cells.

Perspectives

    Importance of the field: Although both enzymatic toxins exhibit various pharmacological actions, we should not neglect their cytotoxic properties on cancer cells. Both PLA2 and LAAO produce oxidative stress and trigger cell cycle arrest and apoptosis in cancer cells, thereby suggesting their potential applications as anticancer lead molecules.
    Current status: Despite well documented structural and catalytic properties of both enzymes, their cytotoxic actions remain superficial without in-depth analysis on the specific cell-death signalling pathways. It remains ambiguous if both PLA2 and LAAO interact directly with the surface cell death receptors to induce cytotoxicity.
    Future direction: The potential target actions of PLA2 and LAAO on cell surface death receptors remain poorly understood. Cancer cells possess abnormalities in cell surface death receptors, for instance, down-regulation of TRAIL receptor DR4, mutated DR5 as well as overexpression of TRAIL decoy and Fas decoy. Thus, investigation of enzymes–death receptor interaction will distinguish the selectivity of the enzymes targeting cancer cells. This is attainable via in-silico docking analysis and chemical cross-link mass spectrometry to detect enzyme–receptor interactomes which enables the annotation of signalling pathways targeted by enzymes PLA2 and LAAO during cytotoxicity.

Open Access

Open access for this article was enabled by the participation of Monash University in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

J.J.H. and M.K.K.Y. wrote the manuscript draft, M.K.K.Y. edited the manuscript. All authors approved the final article.

Abbreviations

FADs

flavin adenine dinucleotides

LAAO

L-amino acid oxidase

PLA2

phospholipase A2

ROS

reactive oxygen species

References

1 

Tu A.T. (1996) Overview of snake venom chemistry In Natural Toxins 2 Advances in Experimental Medicine and Biology (Singh B.R., Tu A.T., eds), vol. 391, pp. , pp.37–62, Boston, MA, Springer

2 

Queiroz G.P., Pessoa L.A., Portaro F.C.V., MdFD F. and Tambourgi D.V. (2008) . Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus. Toxicon52, , pp.842–851, doi: 10.1016/j.toxicon.2008.10.002

3 

Salazar A.M., Guerrero B., Cantu B., Cantu E., Rodríguez-Acosta A., Pérez J.C.et al. (2009) . Venom variation in hemostasis of the southern Pacific rattlesnake (Crotalus oreganus helleri): isolation of hellerase. Comp. Biochem. Physiol. C Toxicol. Pharmacol.149, , pp.307–316, doi: 10.1016/j.cbpc.2008.08.007

4 

Mackessy S.P. (2002) . Biochemistry and pharmacology of colubrid snake venoms. J Toxicol. Toxin Rev.21, , pp.43–83, doi: 10.1081/TXR-120004741

5 

Kini R.M. (1997) Venom Phospholipase A2 Enzymes: Structure, Functions and Mechanisms, John Wiley & Son Limited, Hoboken, NJ

6 

Doley R., Zhou X. and Kini M. (2010) Snake venom phospholipase A2 enzymes In Handbook of Venoms and Toxins of Reptiles (Mackessy S.P., ed.), pp. , pp.174–195, CRC Press, Boca Raton

7 

Praznikar, Z.J, Petan, T. and Pungercar, J. A neurotoxic secretory phospholipase A2 induces apoptosis in motoneuron-like cells. Ann NY Acad Sci.2009; 1152:, pp.215–224. , doi: 10.1111/j.1749-6632.2008.03999.x

8 

Rouault M., Rash L.D., Escoubas P., Boilard E., Bollinger J., Lomonte B.et al. (2006) . Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity. Biochemistry45, , pp.5800–5816, doi: 10.1021/bi060217r

9 

Andrião-Escarso S.H., Soares A.M., Rodrigues V.M., Angulo Y., Díaz C., Lomonte B.et al. (2000) . Myotoxic phospholipases A2 in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie82, , pp.755–763, doi: 10.1016/S0300-9084(00)01150-0

10 

Gutiérrez J.M., Ponce-Soto L.A., Marangoni S. and Lomonte B. (2008) . Systemic and local myotoxicity induced by snake venom group II phospholipases A2: comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue. Toxicon51, , pp.80–92, doi: 10.1016/j.toxicon.2007.08.007

11 

Zhang H.L., Xu S.J., Wang Q.Y., Song S.Y., Shu Y.Y. and Lin Z.J. (2002) . Structure of a cardiotoxic phospholipase A2 from Ophiophagus hannah with the “pancreatic loop”. J. Struct. Biol.138, , pp.207–215, doi: 10.1016/S1047-8477(02)00022-9

12 

Satish S., Tejaswini J., Krishnakantha T.P. and Gowda T.V. (2004) . Purification of a class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja naja) venom. Biochimie86, , pp.203–210, doi: 10.1016/j.biochi.2004.02.003

13 

Zhao K., Zhou Y. and Lin Z. (2000) . Structure of basic phospholipase A2 from Agkistrodon halys pallas: implications for its association, hemolytic and anticoagulant activities. Toxicon38, , pp.901–916, doi: 10.1016/S0041-0101(99)00193-2

14 

Yamaguchi Y., Shimohigashi Y., Chijiwa T., Nakai M., Ogawa T., Hattori S.et al. (2001) . Characterization, amino acid sequence and evolution of edema-inducing, basic phospholipase A2 from Trimeresurus flavoviridis venom. Toxicon39, , pp.1069–1076, doi: 10.1016/S0041-0101(00)00250-6

15 

Šribar J., Oberčkal J. and I K. (2014) . Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2: an update. Toxicon89, , pp.9–16, doi: 10.1016/j.toxicon.2014.06.019

16 

Rigoni M., Pizzo P., Schiavo G., Weston A.E., Zatti G., Caccin P.et al. (2007) . Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products. J. Biochem.282, , pp.11238–11245

17 

Vulfius C.A., Kasheverov I.E.Kryukova E.V., Spirova E.N., Shelukhina I.V., Starkov V.G.et al. (2017) . Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors. PLoS One12, , pp.e0186206, doi: 10.1371/journal.pone.0186206

18 

Rigoni M., Paoli M., Milanesi E., Caccin P., Rasola A., Bernardi P.et al. (2008) . Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores. J. Biochem.283, , pp.34013–34020

19 

Paoli M., Rigoni M., Koster G., Rossetto O., Montecucco C. and Postle A.D. (2009) . Mass spectrometry analysis of the phospholipase A2 activity of snake pre-synaptic neurotoxins in cultured neurons. J. Neurochem.111, , pp.737–744, doi: 10.1111/j.1471-4159.2009.06365.x

20 

Ranawaka U.K., Lalloo D.G. and de Silva H.J. (2013) . Neurotoxicity in snakebite–the limits of our knowledge. PLoS Negl. Trop. Dis.7, , pp.e2302, doi: 10.1371/journal.pntd.0002302

21 

Gutiérrez J.M., Calvete J.J., Habib A.G., Harrison R.A., Williams D.J. and Warrell D.A. (2017) . Snakebite envenoming. Nat. Rev. Dis. Primers3, , pp.17079, doi: 10.1038/nrdp.2017.79

22 

Samel M., Vija H., Rönnholm G., Siigur J., Kalkkinen N. and Siigur E. (2006) . Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom. Biochim. Biophys. Acta1764, , pp.707–714, doi: 10.1016/j.bbapap.2006.01.021

23 

Stábeli R.G., Sant'Ana C.D., Ribeiro P.H., Costa T.R., Ticli F.K., Pires M.G.et al. (2007) . Cytotoxic L-amino acid oxidase from Bothrops moojeni: biochemical and functional characterization. Int. J. Biol. Macromol.41, , pp.132–140, doi: 10.1016/j.ijbiomac.2007.01.006

24 

de Queiroz M.R., de Sousa B.B., da Cunha Pereira D.F., Mamede C.C.N., Matias M.S., de Morais N.C.G.et al. (2017) . The role of platelets in homeostasis and the effects of snake venom toxins on platelet function. Toxicon133, , pp.33–47, doi: 10.1016/j.toxicon.2017.04.013

25 

Belisario M.A., Tafuri S., Di Domenico C., Squillacioti C., Della Morte R., Lucisano A.et al. (2000) . H2o2 activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation. Biochim. Biophys. Acta1495, , pp.183–193, doi: 10.1016/S0167-4889(99)00160-3

26 

Du X.Y. and Clemetson K.J. (2002) . Snake venom L-amino acid oxidases. Toxicon40, , pp.659–665, doi: 10.1016/S0041-0101(02)00102-2

27 

Rodrigues R.S., da Silva J.F., Boldrini França J., Fonseca F.P.P., Otaviano A.R., Henrique Silva F.et al. (2009) . Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom. Biochimie91, , pp.490–501, doi: 10.1016/j.biochi.2008.12.004

28 

Wei J.F., Yang H.W., Wei X.L., Qiao L.Y., Wang W.Y. and He S.H. (2009) . Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom. Toxicon54, , pp.262–271, doi: 10.1016/j.toxicon.2009.04.017

29 

Ciscotto P., de Avila RA M., Coelho E.A.F., Oliveira J., Diniz C.G., Farías L.M.et al. (2009) . Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom. Toxicon53, , pp.330–341, doi: 10.1016/j.toxicon.2008.12.004

30 

Souza D.H., Eugenio L.M., Fletcher J.E., Jiang M.S., Garratt R.C., Oliva G.et al.) . Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data. Arch. Biochem. Biophys.1999368, , pp.285–290

31 

Montecucco C., Gutiérrez J.M. and Lomonte B. (2008) . Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell. Mol. Life Sci.65, , pp.2897–2912, doi: 10.1007/s00018-008-8113-3

32 

Gutiérrez J.M. and Ownby C.L. (2003) . Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon42, , pp.915–931, doi: 10.1016/j.toxicon.2003.11.005

33 

Costal-Oliveira F., Stransky S., Guerra-Duarte C., de Souza DL N., Vivas-Ruiz D.E., Yarlequé A.et al. (2019) . L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes. Sci. Rep.9, , pp.781, doi: 10.1038/s41598-018-37435-4

34 

Naumann G.B., Silva L.F., Silva L., Faria G., Richardson M., Evangelista K.et al. (2011) . Cytotoxicity and inhibition of platelet aggregation caused by an L-amino acid oxidase from Bothrops leucurus venom. Biochim. Biophys. Acta1810, , pp.683–694, doi: 10.1016/j.bbagen.2011.04.003

35 

Stransky S., Costal-Oliveira F., Lopes-de-Souza L., Guerra-Duarte C., Chávez-Olórtegui C. and Braga V.M.M. (2018) . In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom. PLoS Negl. Trop. Dis.12, , pp.e0006427, doi: 10.1371/journal.pntd.0006427

36 

Tan L.T.H., Chan K.G., Pusparajah P., Lee W.L., Chuah L.H., Khan T.M.et al. (2017) . Targeting membrane lipid a potential cancer cure?Front. Pharmacol.8, , pp.12, doi: 10.3389/fphar.2017.00012

37 

Costa T.R., Burin S.M., Menaldo D.L., de Castro F.A. and Sampaio S.V. (2014) . Snake venom L-amino acid oxidases: an overview on their antitumor effects. J. Venom. Anim. Toxins Incl. Trop. Dis.20, , pp.23, doi: 10.1186/1678-9199-20-23

38 

Scott D. (1997). Phospholipase A2: structure and catalytic properties In Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism (Kini R.M., ed.), pp. , pp.97–128, John Wiley & Son Limited, Chichester

39 

Six D.A. and Dennis E.A. (2000) . The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim. Biophys. Acta1488, , pp.1–19, doi: 10.1016/S1388-1981(00)00105-0

40 

Harris J.B. and Scott-Davey T. (2013) . Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry. Toxins5, , pp.2533–2571, doi: 10.3390/toxins5122533

41 

Carredano E., Westerlund B., Persson B., Saarinen M., Ramaswamy S., Eaker D.et al. (1998) . The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2. Toxicon36, , pp.75–92, doi: 10.1016/S0041-0101(97)00051-2

42 

Davidson F.F. and Dennis E.A. (1990) . Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms. J. Mol. Evol.31, , pp.228–238, doi: 10.1007/BF02109500

43 

Huang M.Z., Gopalakrishnakone P., Chung M.C. and Kini R.M. (1997) . Complete amino acid sequence of an acidic, cardiotoxic phospholipase A2 from the venom of Ophiophagus hannah (King cobra): a novel cobra venom enzyme with “pancreatic loop”. Arch. Biochem. Biophys.338, , pp.150–156, doi: 10.1006/abbi.1996.9814

44 

Xiao H., Pan H., Liao K., Yang M. and Huang C. (2017) . Snake Venom PLA2, a promising target for broad-spectrum antivenom drug development. Biomed Res Int.2017, , pp.6592820, doi: 10.1155/2017/6592820

45 

Ward R.J., Chioato L., de Oliveira A.H.C., Ruller R. and  J.M. (2002) . Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis. Biochem. J.362(Pt 1), , pp.89–96, doi: 10.1042/bj3620089

46 

Matsui T., Kamata S., Ishii K., Maruno T., Ghanem N., Uchiyama S.et al. (2019) . SDS-induced oligomerization of Lys49-phospholipase A PLA2 from snake venom. Sci. Rep.9, , pp.2330, doi: 10.1038/s41598-019-38861-8

47 

Kang T.S., Georgieva D., Genov N., Murakami M.T., Sinha M., Kumar R.P.et al. (2011) . Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J.278, , pp.4544–4576, doi: 10.1111/j.1742-4658.2011.08115.x

48 

Scott D.L., White S.P., Otwinowski Z., Yuan W., Gelb M.H. and Sigler P.B. (1990) . Interfacial catalysis: the mechanism of phospholipase A2. Science250, , pp.1541–1546, doi: 10.1126/science.2274785

49 

Conlon J.M., Attoub S., Arafat H., Mechkarska M., Casewell N.R., Harrison R.A.et al. (2013) . Cytotoxic activities of [Ser49] phospholipase A2 from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus. Toxicon71, , pp.96–104, doi: 10.1016/j.toxicon.2013.05.017

50 

Petan T., Krizaj I. and Pungercar J. (2007) . Restoration of enzymatic activity in a Ser-49 phospholipase A2 homologue decreases its Ca2+-independent membrane-damaging activity and increases its toxicity. Biochemistry46, , pp.12795–12809, doi: 10.1021/bi701304e

51 

Lomonte B., Angulo Y. and Calderón L. (2003) . An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon42, , pp.885–901, doi: 10.1016/j.toxicon.2003.11.008

52 

Fujisawa D., Yamazaki Y., Lomonte B. and Morita T. (2008) . Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region. Biochem J.411, , pp.515–522, doi: 10.1042/BJ20080078

53 

Costa T.R., Menaldo D.L., Oliveira C.Z., Santos-Filho N.A., Teixeira S.S., Nomizo A.et al. (2008) . Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: cytotoxic effect on microorganism and tumour cells. Peptides29, , pp.1645–1656, doi: 10.1016/j.peptides.2008.05.021

54 

Gebrim L.C., Marcussi S., Menaldo D.L., de Menezes C.S.R., Nomizo A., Hamaguchi A.et al. (2009) . Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region. Biologicals37, , pp.222–229, doi: 10.1016/j.biologicals.2009.01.010

55 

Lomonte B., Angulo Y. and Moreno E. (2010) . Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from viperidae snake venoms: biomimetic activities and potential applications. Curr. Pharm. Des.16, , pp.3224–3230, doi: 10.2174/138161210793292456

56 

Benati R.B., Costa T.R., MdC C., Sampaio S.V., de Castro F.A. and Burin S.M. (2018) . Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojenisnake venom, towards leukemic cells. J. Venom. Anim. Toxins Incl. Trop. Dis.24, , pp.40, doi: 10.1186/s40409-018-0180-9

57 

da Silva C P., Costa T.R., Paiva R.M.A., Cintra A.C.O., Menaldo D.L., Antunes L.M.G.et al. (2015) . Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumour cell lines. J. Venom. Anim. Toxins21, , pp.44, doi: 10.1186/s40409-015-0044-5

58 

Marcussi S., Santos P.R.S., Menaldo D.L., Silveira L.B., Santos-Filho N.A., Mazzi M.V.et al. (2011) . Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes. Mutat. Res.724, , pp.59–63, doi: 10.1016/j.mrgentox.2011.06.004

59 

Khunsap S., Khow O., Buranapraditkun S., Suntrarachun S., Puthong S. and Boonchang S. (2016) . Anticancer properties of phospholipase A2 from Daboia siamensis venom on human skin melanoma cells. J. Venom. Anim. Toxins Incl. Trop. Dis.22, , pp.7, doi: 10.1186/s40409-016-0061-z

60 

Marcussi S., Stábeli R.G., Santos-Filho N.A., Menaldo D.L., Silva Pereira L.L., Zuliani J.P.et al. (2013) . Genotoxic effect of Bothrops snake venoms and isolated toxins on human lymphocyte DNA. Toxicon65, , pp.9–14, doi: 10.1016/j.toxicon.2012.12.020

61 

Tran T.V., Siniavin A.E., Hoang A.N., Le M.T.T., Pham C.D., Phung T.V.et al. (2019) . Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro. PeerJ7, , pp.e8055, doi: 10.7717/peerj.8055

62 

Khunsap S., Pakmanee N., Khow O., Chanhome L., Sitprija V., Suntravat M.et al. (2011) . Purification of a phospholipase A2 from Daboia russelii siamensis venom with anticancer effects. J. Venom. Res.2, , pp.42–51

63 

Cura J.E., Blanzaco D.P., Brisson C., Cura M.A., Cabrol R., Larrateguy L.et al. (2002) . Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA2, NSC-624244) in patients with advanced cancer. Clin. Cancer Res.8, , pp.1033–1041 PMID:

64 

Ali S.A., Stoeva S., Abbasi A., Alam J.M., Kayed R., Faigle M.et al. (2000) . Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom. Arch. Biochem. Biophys.384, , pp.216–226, doi: 10.1006/abbi.2000.2130

65 

Pawelek P.D., Cheah J., Coulombe R., Macheroux P., Ghisla S. and Vrielink A. (2000) . The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. EMBO J.19, , pp.4204–4215, doi: 10.1093/emboj/19.16.4204

66 

Hanukoglu I. (2015) . Proteopedia: Rossmann fold: A beta-alpha-beta fold at dinucleotide binding sites. Biochem. Mol. Biol. Edu.43, , pp.206–209, doi: 10.1002/bmb.20849

67 

Dym O. and Eisenberg D. (2001) . Sequence-structure analysis of FAD-containing proteins. Protein Sci.10, , pp.1712–1728, doi: 10.1110/ps.12801

68 

Suwannapan W., Chumnanpuen P. and E-Kobon T. (2018) . Amplification and bioinformatics analysis of conserved FAD-binding region of L-amino acid oxidase LAAO genes in gastropods compared to other organisms. Comput. Struct. Biotech16, , pp.98–107, doi: 10.1016/j.csbj.2018.02.008

69 

Moustafa I.M., Foster S., Lyubimov A.Y. and Vrielink A. (2006) . Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism. J. Mol. Biol.364, , pp.991–1002, doi: 10.1016/j.jmb.2006.09.032

70 

Umhau S., Diederichs K., Welte W., Ghisla S., Pollegioni L., Molla G.et al. (1999) Very high resolution crystal structure of d-amino acid oxidase. Insights into the reaction mechanisms and mode of ligand binding. In Flavins and Flavoproteins (Ghisla S., Kroneck P., Macheroux P., Sund H., eds.), pp. , pp.567–570, Ruldolf Weber, Berlin

71 

Gaweska H. and Fitzpatrick P.F. (2011) . Structures and mechanism of the monoamine oxidase family. Biomol. Concepts2, , pp.365–377, doi: 10.1515/BMC.2011.030

72 

Macheroux P., Seth O., Bollschweiler C., Schwarz M., Kurfürst M., Au L.C.et al. (2001) . L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme. Eur. J. Biochem.268, , pp.1679–1686, doi: 10.1046/j.1432-1327.2001.02042.x

73 

Sun M.Z., Guo C., Tian Y., Chen D., Greenaway F.T. and Liu S. (2010) . Biochemical, functional and structural characterization of akbu-LAAO: a novel snake venom L-amino acid oxidase from Agkistrodon blomhoffii ussurensis. Biochimie92, , pp.343–349, doi: 10.1016/j.biochi.2010.01.013

74 

Ribeiro P.H., Zuliani J.P., Fernandes C.F.C., Calderon L.A., Stábeli R.G., Nomizo A.et al. (2016) . Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom. Int. J. Biol. Macromol.92, , pp.329–337, doi: 10.1016/j.ijbiomac.2016.07.022

75 

Zhang L. and Wei L.-J. (2007) . ACTX-8, a cytotoxic L-amino acid oxidase isolated from Agkistrodon acutus snake venom, induces apoptosis in hela cervical cancer cells. Life Sci.80, , pp.1189–1197, doi: 10.1016/j.lfs.2006.12.024

76 

Izidoro L.F.M., Ribeiro M.C., Souza G.R.L., Sant'Ana C.D., Hamaguchi A., Homsi-Brandeburgo M.I.et al. (2006) . Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom. Bioorg. Med. Chem.14, , pp.7034–7043, doi: 10.1016/j.bmc.2006.06.025

77 

Singh M., Sharma H. and Singh N. (2007) . Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway. Mitochondrion7, , pp.367–373, doi: 10.1016/j.mito.2007.07.003

78 

Alves R.M., Antonucci G.A., Paiva H.H., Cintra A.C.O., Franco J.J., Mendonça-Franqueiro E.P.et al. (2008) . Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. Comp. Biochem. Physiol. A Mol. Integr. Physiol.151, , pp.542–550, doi: 10.1016/j.cbpa.2008.07.007

79 

Burin S.M., Ayres L.R., Neves R.P., Ambrósio L., de Morais F.R., Dias-Baruffi M.et al. (2013) . L-amino acid oxidase isolated from Bothrops pirajai induces apoptosis in BCR-ABL-positive cells and potentiates imatinib mesylate effect. Basic Clin. Pharmacol. Toxicol.113, , pp.103–112, doi: 10.1111/bcpt.12073

80 

Suhr S.M. and Kim D.S. (1996) . Identification of the snake venom substance that induces apoptosis. Biochem. Biophys. Res. Commun.224, , pp.134–139, doi: 10.1006/bbrc.1996.0996

81 

Torii S., Naito M. and Tsuruo T. (1997) . Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom. J. Biol. Chem.272, , pp.9539–9542, doi: 10.1074/jbc.272.14.9539

82 

Fung S.Y., Lee M.L. and Tan N.H. (2015) . Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase. Toxicon96, , pp.38–45, doi: 10.1016/j.toxicon.2015.01.012

83 

de Melo Alves Paiva R., de Freitas Figueiredo R., Antonucci G.A., Paiva H.H., de Lourdes Pires Bianchi M., Rodrigues K.C.et al. (2011) . Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom. Biochimie93, , pp.941–947, doi: 10.1016/j.biochi.2011.01.009

84 

Zhang L. and Wu W.T. (2008) . Isolation and characterization of ACTX-6: a cytotoxic L-amino acid oxidase from Agkistrodon acutus snake venom. Nat. Prod. Res.22, , pp.554–563, doi: 10.1080/14786410701592679

85 

Ande S.R., Kommoju P.R., Draxl S., Murkovic M., Macheroux P., Ghisla S.et al. (2006) . Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom. Apoptosis11, , pp.1439–1451, doi: 10.1007/s10495-006-7959-9

86 

Yu L., Chen Y. and Tooze S.A. (2018) . Autophagy pathway: cellular and molecular mechanisms. Autophagy14, , pp.207–215, doi: 10.1080/15548627.2017.1378838

87 

Lee M.L., Fung S.Y., Chung I., Pailoor J., Cheah S.H. and Tan N.H. (2014) . King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumour growth in a tumour xenograft mouse model. Int. J. Med. Sci.11, , pp.593–601, doi: 10.7150/ijms.8096

88 

Lu W., Hu L., Yang J., Sun X., Yan H., Liu J.et al. (2018) . Isolation and pharmacological characterization of a new cytotoxic L-amino acid oxidase from Bungarus multicinctus snake venom. J. Ethnopharmacol.213, , pp.311–320, doi: 10.1016/j.jep.2017.11.026

89 

Abidin SA Z., Rajadurai P., Hoque Chowdhury M.E., Othman I. and Naidu R. (2018) . Cytotoxic, anti-proliferative and apoptosis activity of L-amino acid oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) venom on human colon cancer cells. Molecules23, , pp.E1388, doi: 10.3390/molecules23061388

90 

Calvete J.J. (2017) . Venomics: integrative venom proteomics and beyond. Biochem J.474, , pp.611–634, doi: 10.1042/BCJ20160577

91 

Warrell D.A. (2010) . Snake bite. Lancet375, , pp.77–88, doi: 10.1016/S0140-6736(09)61754-2

92 

Butrón E., Ghelestam M. and Gutiérrez J.M. (1993) . Effects on cultured mammalian cells of myotoxin III, a phospholipase A2 isolated from Bothrops asper (terciopelo) venom. Biochim. Biophys. Acta1179, , pp.253–259, doi: 10.1016/0167-4889(93)90080-9

93 

Cedro R.C.A., Menaldo D.L., Costa T.R., Zoccal K.F., Sartim M.A., Santos-Filho N.A.et al. (2018) . Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom. J. Venom. Anim. Toxins Inc. Trop. Dis.24, , pp.33, doi: 10.1186/s40409-018-0170-y

94 

Bezerra P.H.A., Ferreira I.M., Franceschi B.T., Bianchini F., Ambrósio L., Cintra A.C.O.et al. (2019) . BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation. J. Venom. Anim. Toxins Inc. Trop. Dis.25, , pp.e20190010, doi: 10.1590/1678-9199-jvatitd-2019-0010

95 

Chioato L., Aragão E.A., Ferreira T L., Medeiros A.I., Faccioli L.H. and Ward R.J. (2007) . Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis. Biochim. Biophys Acta1768, , pp.1247–1257, doi: 10.1016/j.bbamem.2007.01.023

96 

Stábeli R.G., Amui S.F., Sant'Ana C.D., Pires M.G., Nomizo A., Monteiro M.C.et al. (2006) . Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins. Comp. Biochem. Physiol. C Toxicol. Pharmacol.142, , pp.371–381, doi: 10.1016/j.cbpc.2005.11.020

97 

Corin R.E., Viskatis L.J., Vidal J.C. and Etcheverry M.A. (1993) . Cytotoxicity of crotoxin on murine erythroleukemia cells in vitro. Invest New Drugs.11, , pp.11–15, doi: 10.1007/BF00873905

98 

Rudd C.J., Viskatis L.J., Vidal J.C. and Etcheverry M.A. (1994) . In vitro comparison of cytotoxic effects of crotoxin against three human tumours and a normal human epidermal keratinocyte cell line. Invest. New Drugs.12, , pp.183–184, doi: 10.1007/BF00873958

99 

Muller S.P., Silva V.A.O., Silvestrini A.V.P., de Macedo L.H., Caetano G.F., Reis R.M.et al. (2018) . Crotoxin from Crotalus durissus terrificus venom: In vitro cytotoxic activity of a heterodimeric phospholipase A2 on human cancer-derived cell lines. Toxicon156, , pp.13–22, doi: 10.1016/j.toxicon.2018.10.306

100 

Casais E Silva L.L., Teixeira C.F.P., Lebrun I., Lomonte B., Alape-Girón A. and Gutiérrez J.M. (2016) . Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2. Toxicol Lett.257, , pp.60–71, doi: 10.1016/j.toxlet.2016.06.005

101 

Chen K.C., Kao P.H., Lin S.R. and Chang L.S. (2009) . Upregulation of Fas and FasL in Taiwan cobra phospholipase A2 treated human neuroblastoma SK-N-SH cells through ROS- and Ca2+ - mediated p38 MAPK activation. J. Cell. Biochem.106, , pp.93–102, doi: 10.1002/jcb.21979

102 

Rudrammaji L.M. and Gowda T.V. (1998) . Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom. Toxicon36, , pp.921–932, doi: 10.1016/S0041-0101(97)00097-4

103 

Dutta S., Sinha A., Dasgupta S. and Mukherjee A.K. (2019) . Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity. Biochim. Biophys. Acta Biomembr.1861, , pp.958–977, doi: 10.1016/j.bbamem.2019.02.002

104 

Chwetzoff S., Tsunasawa S., Sakiyama F. and Ménez A. (1989) . Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties. J. Biol. Chem.264, , pp.13289–13297 PMID:

105 

Liu J.W., Chai M.Q., Du X.Y., Song J.G. and Zhou Y.C.) . Purification and characterization of L-amino acid oxidase from Agkistrodon halys pallas venom. Acta Biochim. Biophys. Sin.34, , pp.305–310 PMID:

106 

Braga M.D.M., Martins A.M.C., Amora D.N., de Menezes D.B., Toyama M.H., Toyama D.O.et al. (2008) . Purification and biological effects of L-amino acid oxidase isolated from Bothrops insularis venom. Toxicon51, , pp.199–207, doi: 10.1016/j.toxicon.2007.09.003

107 

de Vieira Santos M.M., Sant'Ana C.D., Giglio J.R., da Silva R.J., Sampaio S.V., Soares A.M.et al. (2008) . Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom. Basic Clin. Pharmacol. Toxicol.102, , pp.533–542, doi: 10.1111/j.1742-7843.2008.00229.x

108 

Bregge-Silva C., Nonato M.C., de Albuquerque S., Ho P.L., de Azevedo ILM J., Diniz MR V.et al. (2012) . Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom. Toxicon60, , pp.1263–1276, doi: 10.1016/j.toxicon.2012.08.008

109 

Ahn M.Y., Lee B.M. and Kim Y.S. (1997) . Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah). Int. J. Biochem. Cell Biol.29, , pp.911–919, doi: 10.1016/S1357-2725(97)00024-1

110 

Lee M.L., Chung I., Fung S.Y., Kanthimathi M.S. and Tan N.H. (2014) . Antiproliferative activity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase. Basic Clin. Pharmacol. Toxicol.114, , pp.336–343, doi: 10.1111/bcpt.12155

111 

Sun L.-K., Yoshii Y., Hyodo A., Tsurushima H., Saito A., Harakuni T.et al. (2003) . Apoptotic effect in the glioma cells induced by specific protein extracted from Okinawa Habu (Trimeresurus flavoviridis) venom in relation to oxidative stress. Toxicol In Vitro17, , pp.169–177, doi: 10.1016/S0887-2333(03)00010-9

112 

Zhang Y.J., Wang J.H., Lee W.H., Wang Q., Liu H., Zheng Y.T.et al. (2003) . Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity. Biochem. Biophys. Res. Commun.309, , pp.598–604, doi: 10.1016/j.bbrc.2003.08.044

https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1042/BST20200110&title=Cytotoxicity of snake venom enzymatic toxins: phospholipase A<sub>2</sub> and <span style="font-variant: all-small-caps">l</span>-amino acid oxidase&author=Jia Jin Hiu,Michelle Khai Khun Yap,&keyword=apoptosis,cancer,cytotoxicity,l-amino acid oxidase,phospholipase A2,reactive oxygen species,&subject=Pharmacology & Toxicology,Enzymology,Cell Death & Injury,Review Articles,