USE OF ANIMAL VENOM PEPTIDES/TOXINS IN CANCER THERAPEUTICS

Animal venoms possess variety of toxins/proteins/peptides which act as ionic channel inhibitors and target vital physiological processes. Toxin peptides isolated from honey bee, wasp and scorpion show membrane binding, growth inhibition and strong cytolytic properties. These also inhibit angiogenesis and induce caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the experimental animals against tumor development. These toxin peptides exert cytotoxic effects on human oral cancer cells by inducting apoptosis via a p53-dependent intrinsic apoptotic pathway. These can be used as cancer therapeutic agents by loading them in liposomes. This lead to increase in their target specificity against cancer cells, shorten cell survival, and produce higher reactive oxygen species (ROS), and does enhancement in the number of apoptotic cells. In addition, toxins enhance G0/G1 enrichment upon treatment of cancer cells. Further, encapsulated honey bee, wasp and scorpion venoms exert much potent anti-cancer effect on many cancer cells lines. Upon slight chemical modifications animal toxins could gear up higher selectivity, show much improved target specificity and lesser toxic effects because of charge optimization. After their improvement they show superiority over the conventional chemical drugs as an increase in net charge of the peptide, its hydrophobicity and anionicity and fluidity of the target cell membranes. It primarily imparts effect through biophysical interaction with the target cell membrane. Toxin peptides are best candidate as they selectively target malignant gliomas and play significant role in GBM immunotherapy. Venom toxins/peptides are good natural sources of compounds/molecules which could act as prototype or template which can be used for development of new therapeutic agents and best tools for diagnosis of not only for cancer but also for other diseases. WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES SJIF Impact Factor 7.421 Volume 7, Issue 11, 596-652 Review Article ISSN 2278 – 4357 Article Received on 19 September 2018, Revised on 09 October 2018, Accepted on 30 Oct. 2018, DOI: 10.20959/wjpps201818-12612 *Corresponding Author

Moreover, venom factors also cause acute and chronic inflammatory responses in laboratory animals. Histamine inhibits vaso-dilation in mast cells of lungs, liver and gastric mucosa with allergic hyper-sensitivity and inflammation. More specifically citrate present in arthropod venom inhibits phospholipase A2 activity. [3] After few seconds of envenomation toxins cause sudden inflammation in body cells with a severe pain and do massive inhibition of axonal transmission in neurons. Toxins also affect activity of ATP driven Na + -K + ATPase pump, which plays a key role in maintaining cell volume and intra cellular ionic composition specially Na + and K + gradients. This pump actively transports ions across the cell membrane and also helps in excitation of nerves, and does phosphorylation and de-phosphorylation in muscle cells. In this mechanism some transmembrane proteins/ enzymes utilize the energy stored in molecules of ATP to move K + into the neuron. Na + -K + pump helps the neurons to maintain resting potential for which pump allows interior negative charge and exterior positive charge on neurons by pumping Na + outside the cell and K + inside the cell (Figure 4). Both Na + and K + channels are competitively blocked by these toxins and induce the release of transmitters and cause repetitive firing of the axons (Table 2). Toxins also change the orientation and affinity of ion binding sites change ion permeability mediated by the nicotinic Ach receptors. Snake, neurotoxins such as α-bungarotoxin and cobra toxin block neuromuscular transmission, affect sodium and calcium exchanges and block ion channels forming a tight ring. [4] The toxicity of venom toxins depend upon the sequence of the amino acid residues present in the active site regions, topological folding, hydrophobic pockets and binding affinity.
Basically modifications like site-specific mutations and rearrangements occurred in the active site region in response to gradual environmental changes in due course of time have resulted in structural and functional diversifications of toxins. Therefore, different biologically active toxins have been evolved even within a single animal species. However, most of the toxins are identified as neurotoxic, which block various types of ion channels. Different animal groups produce different toxins but each of them shows significant evolutionary relationship in structure especially in their active site regions.
Tetrodotoxin is a highly potent poison that is isolated from puffer fish. It (TTX) blocks the conduction of nerve impulses along axons and in excitable membrane of nerve fibres, which lead to respiratory paralysis. Tetrodotoxins are insensitive (TTX-I) to voltage dependent Na + channels and are critical for initial rapid upstroke of the cardiac action potential and are responsible for most of the Na + current that occurs in mammalian heart. Similarly protein kinase C acts as inhibitor of tetrodotoxin-resistant Na+ channels in small dorsal root ganglion and sensory neurons in rats. [13] Besides this, sea anemone toxins interact with large variety of excitable membranes including myelinated and non-myelinated axons. There exist four sea anemone toxins namely ASVI, AX I, AX II and AS II. Among which AS II is one of the most abundant sea anemone toxin that works on mammalian Na + channels. Sea anemones produce cardio and neurotoxins, which mainly cause inactivation of Na + channels, slow down sodium permeability and do prolongation of action potential. In nerve, cardiac and skeletal muscle cell in culture animal toxins effectively inhibit transmission and do make failure of contraction and excitation. Besides these, Bg II and Bg III toxins have been purified and sequenced from the Sea anemone Bunodosoma granulifera. BgII specially affect the insect Na + channel (Table 1). Both Bg II and Bg III cause the inactivation of voltage gated Na + channels. [14] ATXII from Anemonia sulcata and Ap B from Anthopleura xanthogrammica inhibit the inactivation process of Na + channel but shows cardiotoxic and neurotoxic effects.
Similarly Jingzhaotoxin-I a novel spider neurotoxin preferentially inhibits cardiac function in rat, [15] target voltage gated sodium channels and inactivate them. [16] Besides this, few paralyzing peptides have been isolated from sea anemone Bunodosoma caissaum [17] and Phoneutria nigreventer. These are identified as neuronal sodium, channel inhibitor and interact with micro-conotoxin binding sites. [18] Similarly a neurotoxic lipopeptide kalkitoxin interacts with voltage sensitive sodium channels in cerebral granule neurons. [19] Similarly an alpha like scorpion neurotoxin BmK I play an important role in voltage gated Na+ channels in excitation-contraction coupling of heart rate. [20] Besides this, hainantoxin IV binds to sodium channels, block and inactivate those. [21] (Table 2).
Similarly delta-atracotoxin target the voltage gated sodium channels. [22] This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the Na + channels of insects. It also modulates the binding of alpha and beta type anti -mammal scorpion toxin to the sodium channels. [23] This toxin shows biological activity only in insects and cause contractional paralysis in them.
Electrophysiological and binding studies showed that pharmacological target of anti mammal toxin are the voltage dependent Na + channels of excitable cell. [24] Similarly, effects are also noted after inflction of saxitoxin (STX), produced by a marine dinoflagellate. Physiologically TTX (tetrodotoxin), STX (Saxitoxin), and μ-conotoxin block Na + channels from different tissues with vastly different affinities( Table 2). In contrast brain and skeletal muscle channels show a different magnitude of binding and are more sensitive to TTX and STX than are heart channels found in heart muscles. [25]

K + Channels Inhibitors
Potassium channels occur in almost all living organisms and form potassium selective pores which span cell membranes. [26] Due to their high ionic interaction they control a wide variety of cell functions. There is a family of K + channel inhibitors that includes charybdotoxin, noxiustoxins, kaliotoxins and iberiotoxins. Among these some are voltage-gated channels and some are pure ionic channels. All these channel inhibitors are short peptides of 28-35 amino acids in length and show different toxicity in different animals. Few scorpion toxins are K + channel inhibitors, which bind to a receptor site at the cell membrane or extra cellular vestibule and prevent conduction of cellular fluid and ion across the membrane. These toxins also variably affect the activity of ventricular myocytes in rat. Iberiotoxin is a small globular polypeptide isolated from Buthus tamulus, which shows high potential when added to extracellular fluid. [27,28] The same activity has also shown by TSTX -K isolated from Tityus serrulatus. [29] Besides these, there are some low conductance Ca + activated K + channels, which show mild toxicity. In this category PO5, a polypeptide and apamin, a bee toxin shows similar physiological properties and block K + channels. Bmpo2 is a 28 amino acid residues peptide purified from the venom of the Chinese scorpion Buthus mortensi Karsh, which shows very low inhibition of apamine sensitive calcium activated potassium channel. [30] Bgk toxin isolated from sea anemone blocks membrane potential and suppresses K + current in rat neurons [31] (Table 2B). Another toxin Shk from sea anemone inhibits K + channels in Tlymphocytes in very low concentration [32] (Table 2). Charybdotoxin (ChTX) and naxiustoxin bind to an extra cellular receptor site and prevent ion conduction by occluding the pore. Both toxin bind by a pore binding mechanism established high conductance of Ca + activated K + channels. It is formed due to presence of similar amino acid sequences especially in active site region. [33] Single amino acid modifications in toxin binding region can either increase or decrease the affinity of a given toxin for a specific  [34] and induce confirmation changes in potassium channels [35] (Lange, et al 2006) ( Table 2).  (Table 2C). Besides this, hemolysins secreted by pathogenic bacteria are also seem to function like chloride channel blockers which form pores in the plasma membrane of erythrocytes and through these pores pathogens enter inside the erythrocytes and affect ionic permeability and hemoglobin concentration. Another category of extra cellular toxins is leukocidins, which interrupt the phagocytosis of bacteria by leukocytes. Other proteins are streptomycin O and streptomycin S, which attack the cell membrane of many cells ( Table 2).

Action of animal toxins on cancer cells
Animal venom toxins display profound anticancer effects and are potential therapeutic agents. Toxins purified from snake; bee and scorpion venoms effect cancer cell proliferation, Hemilipin a heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom displays anti-angiogenesis both in vitro and in vivo. [43] This property remains intact even chemical treatment with p-bromophenacyl bromide that abolishes its enzymatic activity.

Melittin: an anticancer peptide
Bee venom is a mixture of proteins, polypeptides and low molecular weight aromatic and aliphatic constituents in variable amounts. It also contains some important enzymes i.e. phospholipase A, hyaluronidase, acid phosphatase and D-glucosidase which are highly antigenic. Honey bee wasp venom contain many toxic substances such as melittin, adiapin, apamine, bradykinin, cardiopep, mast cell degranulating peptide, mastoparan, phospholipase A2 and secapin. These toxin components have wider therapeutic applications. [ These also show antimicrobial, [46,5] anti-cancer [47] and antitumor activity both in vitro and in vivo. [48] Animal toxins act as antibiotics against microbial infections. [49,50] (Table 1).
Honey bee venom inhibits significantly nonenzymatic lipid peroxidation and displays a considerable hydroxyl radical scavenging activity. [51] Melittin causes apoptosis, necrosis and lysis of tumor cells and finally inhibits the tumor growth. [48] It also affects functions of several cell types such renal, lung, liver, prostate, and bladder, breast, and leukemia cancer cells. When melittin is treated with natural detergent it form tetramer aggregates on membranes, it leads to disorders in the structure of phospholipid bilayers. It causes changes in membrane potential, and does aggregation of membrane proteins, and does induction of hormone secretion. [52] It causes membrane disruption and interact with enzymatic systems, such as G-protein, [53] protein kinase C, [54] adenylate cyclase, [55] and phospholipase A. [56] Melittin inhibits calmodulin, a calcium-binding protein that plays a crucial role in cell proliferation. [57] Tumoral cells contain anionic phospholipids, mainly phosphatidylserine on the external leaflet of the plasma membrane. [58] To these leaflets melittin binds preferentially as it possesses cationic charges more than normal cells. It displays anti-proliferative [59,60] and antiangiogenic activity against cancer cells in vivo. [61][62][63] Conjugation of melittin to an antibody enhances its target specificity and minimizes cellular effects on normal cells. Further, for minimizing the cellular effects pro-cytolytic melittin systems are developed by associating melittin to transporting carriers. However, to enhance action of melittin on cancer cells melittin-based recombinant immunotoxins are prepared by fusion of genes that encode an antibody fragment derived from the murine monoclonal antibody K121 with an oligonucleotide encoding melittin. [64] Recombinant immunotoxins of melittin are also prepared and an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (Ca) is attached to them. It increases target specificity and ASGPR-specific cytotoxicity to hepatocellular carcinoma cells. [65] MMP2 cleavable melittin/avidin conjugates are also (pro-cytolytic) prepared by conjugation with tumor matrix metalloproteinase 2 which usually found over-expressed on cancer cell membranes. [66] Though, when melittin coupled

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to avidin becomes inactive but upon release from the conjugate it induces immediate cell lysis. [67] Further, mixture of melittin with the anionic detergent sodium dodecyl sulfate formulated into poly (D), L-lactide-co-glycolide acid) (melittin-loaded nanoparticles) shows better inhibitory action against breast cancer MCF-7 cells [68] (Table 1).
Pegylated immune-liposomes are melittin carriers made by coupling to a humanized antihepatocarcinoma single-chain antibody variable region fragment loaded with a bee venom peptide fraction. [69] A similar immunoliposome is also prepared by using antibody trastuzumab as targeting agent and melittin as main cargo. Both components combat HER2overexpressing human breast cancer cell lines. [70] These nanoparticles in spite of goodness are not suitable for systemic administration because melittin is released in blood vessels during transport. It is disrupted by the lytic peptide. [71] Further, ultra-small diameter melittinnanoparticles (α-melittin-NP <40 nm) are also prepared which work in vivo with few side effects. [72,73] This nanoparticle comprises 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) decorated with the hybrid peptide formed by peptide D-4F and melittin via a GSG linker. This peptide D-4F mimics high-density lipoprotein (HDL). [74] Serine-substituted melittin (Mel-S) showed more cytotoxic effects than asparagine-substituted melittin (Mel-N) against E. coli. [75] Interaction between melittin and the red blood cells is electrostatic that also does collapse of the membrane structure and liberation of the cell contents. [76] Though, peptide remains in a low alpha-helical conformation which forces its stabilization.

Wasp venom
Wasp venom possesses various short peptides and enzymes which are similar to honey bee. It also contains higher percentage of mastoparan and bradykinins. Mastoparan is a membraneactive amphipathic peptide containing Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH2 in its main amino acid chain. [77] It is rich in hydrophobic and basic residues that form its amphipathic helical structures. Mastoparan possess capacity to make pores in membranes that induces a potent mitochondrial permeability transition that affects tumor cell viability. [78] Its mode of action depends on cell type, but it also involves exocytosis. Upon interaction mastoparan induces secretion of histamine from mast cells, platelets and chromaffin cells secrete serotonin and catecholamines. Its action in the anterior pituitary leads to prolactin release. [79] In histamine secretion, mastoparan interference with G protein activity, stimulate the GTPase activity in subunits. It also promotes dissociation of bound GDP from the protein, enhance GTP binding. Mastoparan shortens the lifespan of active G protein and induces G protein-mediated signaling cascade. It leads to intracellular IP 3 release and maintain influx of Ca 2+ . [80] It inserts into the membrane bilayer causing membrane destabilization with consequent lysis. [81] It also perturbs transmembrane signaling. [82][83][84] Upon direct interaction it stimulates phospholipases, does mobilization of Ca2+ from mitochondria and sarcoplasmic reticulum, and causes cell death by necrosis and/or apoptosis. [85,86] Mastoparan induced caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the mice against tumor development. [87] Mastoparan also interacts with the phospholipids found in mitochondrial membrane. It induces permeabilization in cyclosporine A-sensitive and insensitive manners but does not interact with any specific receptors or enzymes. [88] Both PS (phosphatidylserine) and PE (phosphatidylethanolamine) lipids synergistically combine and enhance membrane poration by MP1. Presence of these lipids in the outer leaflet of cancer cells is highly significant for MP1's anticancer action [42] (Table 1).

Change in hydrophobicity
Amino acid substitutions in hydrophobic region of toxins lead to increase in ionic interaction with membranes. Further, hydrophobic interactions also increase target specificity of toxins.
Hydrophobic effect is responsible for the separation of a mixture of oil and water into its two components. It also leads to formation of protein complexes with small molecules and also assists in vesicles formation, protein folding, and insertion of membrane proteins into the nonpolar lipids. [89,90,91,92] In proteins hydrophobic amino acids such as alanine, valine, leucine, isolecuine, phenylalanine, tryptophan and methionine are found in cluster. Normally water soluble proteins possess a hydrophobic core in aqueous medium, and its side chains buried in water. It stabilizes and keep protein molecule in folded state. More exceptionally charged polar side chains are situated on the solvent-exposed surface where they interact with surrounding water molecules. More often, protein folding depends on minimum number of hydrophobic side chains exposed to water. [93,94,95] Further, substitutions of hydrophobic amino acids with non hydrophobic amino acids increase protein folding and formation of hydrogen bonds within the protein which stabilizes protein structure. [96,97] But L-Pro substitutions in polybia-MPI induce significant reduction of antitumor activity because of alpha-helix conformation. Amino acid substitutions in positions 2, 3, 5, 6, 8, 9, and 10 in anoplin analogs increase their surface charge/hydrophobicity that increases hemolytic activity. [98]

Interaction of toxins with protein receptors or protein-protein
For maintaining biological and pharmacological activities membrane-peptide interactions are highly important. More often, membrane potential plays a key role in ionic transport across membrane. Most cell membranes are electrically polarized; they possess inside negative charge nearly 260 millivolts (mV). Their excitability depends on ionic transport across membrane and energy conversion mainly chemical transport through impulse conduction.
During membrane transport some molecules pass through cell membranes because they dissolve in the lipid bilayer. In animal cells a high concentration of K + and a low concentration of Na + relative to the external medium are found. This ionic gradient is generated by Na + -K + ATPase pump which operates specific transport system. Upon hydrolysis of ATP this pump provides the energy required for the active transport of Na + out of the cell and K + into the cell that also generates gradients. ATPase, requires Mg2 + for hydrolysis of ATP.
Modification of surface charges contributes to modulate biological activities of toxins. It also leads to increase the receptor interactions and formation of membrane-peptide complexes. [99] For formation of membrane-peptide complexes, mode of interaction is highly important. It assists in analyzing dynamic properties and the contact residues of the membrane-bound peptide. [99] Mastoparan is an amphiphilic tetradecapeptide extracted from social wasps Polistes flavus that is cytotoxic in nature. It interacts with the lipid bilayer using its hydrophobic side chain. This replacement of hydrophobic side chains with basic amino acids found in MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. [100] Mastoparan activate GTP-binding regulatory proteins (G-proteins) that couple phospholipase C. [79] Mastoparan-X C-terminal 12 residues take α-helical conformation upon binding to the phospholipid bilayer. Similar conformation-activity relationships are also observed in mastoparan analogs as activators of G-proteins. [101] Mastoparan, induces various biological functions including histamine release from rat peritoneal mast cells. In this process acidic residues play an important role in modulating the peptides' lytic and biological activities. [102] More specifically, for the activation of mast cells by mastoparan, at least two positively charged side chains are required on the hydrophilic side of the amphiphilic structure of the peptide. [

Removing toxicity of peptides
Animal toxins despite their toxic effects to mammalian cells they could be used as antibiotics if their toxicity is removed. They can be used as template to design new potential drugs. For example inclusion of an arginine and an isoleucine residue at positions 5 and 8 reduce toxicity of mastoparan, turning it into a potential drug for infectious diseases. [104] They have been proved better candidates due to their lesser toxic effects and higher selectivity upon chemical modification and charge optimization. For lowering the toxic effect, the toxin part of the toxin molecule could be modified either into simple non toxic group, or fused to an antibody or carrier ligand. Such molecules can be used to target malignant brain tumor in patients. Contrary to this, amino acid substitution in toxin peptides increases its biological action. When melittin an insect-derived antimicrobial peptide (AMPs) isolated from Apis mellifera and Apis cerana faces substitution of serine (Ser) to asparagine (Asp). Serinesubstituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Similarly, Mel-N and Mel-S showed different inhibitory effects on the production of IL-6 and TNF-α inflammatory factor in BV-2 cells. [75] The specific biological activity of animal toxins can be practically substitution of amino acids. Though all proteins are inactivated by heat treatment but they become pharmacologically unfit. For example low toxicity of the soybean Agglutinin when given orally, as compared to its rather high toxicity when injected, is noteworthy, because the ToxinPred is a unique in silico method of its kind, which will be useful in predicting toxicity www.wjpps.com Vol 7, Issue 11, 2018. 611

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of peptides/proteins. In addition, it will be useful in designing least toxic peptides and discovering toxic regions in proteins ( Figure 5).

Toxin and lipid interactions
The maintenance of cell membrane integrity is a dynamic process regulated by the living cell in order to provide discrete extracellular and intracellular compartments and within the cell, separation into subcellular organelles. A major component of this integrity is the preservation of the ionic gradients which are necessary for cell function. Toxin molecule insertion into the lipid bilayer generates pores; disturb its ionic equilibrium mainly fine balance of the ion gradients across membrane. It also makes changes in the intracellular water content that results in cell death. A large number of toxin peptides interact directly with lipid components (lipid domains) of the cell membrane and form pores. [105,106] These pore-forming toxins are typically amphipathic polypeptides containing both hydrophilic/polar domain (s) and hydrophobic/non polar domain(s) structures. Though, these vary in size from small peptides to oligomers and up to large macromolecules. These pore forming toxins display increased permeabilization and do disruption of the cell plasma membrane though they show different biological origin, structure or size ( Figure). The pore formation by toxins leads to a colloidosmotic process of the cell followed by swelling. This is a primary event in toxin-cell plasma membrane interaction, starts with pore formation. An example is pardaxin is a small pore forming peptide toxin secreted by Pardachirus marmoratus fish, which targets the plasma membrane via hydrophobic/lipophilic interactions with plasma membrane phospholipids and penetrates into the cell membrane. [107] Furthermore, pore formation triggers few secondary events such as Ca2+ overload, PLA2 activation, eicosanoid production, secretion, endonuclease activation, and cytokine release and protein phosphorylation. All these events constitute secondary cascades of toxin action which eventually lead to cell death. [108] Similarly, protonectin, a peptide isolated from the venom of the wasp Agelaia pallipes pallipes, promotes mast cell degranulation and chemotaxis in polymorphonucleated leukocytes. It possesses amphipathic features which lead to increase in its biological activity. [109] It shows preferential interaction with the micelle, and presents a higher helical structure in aqueous medium. Melittin is a amphiphilic helical molecules that binds with calmodulin. Similarly, mastoporan also binds to the two Ca2+ binding proteins troponin C and calmodulin. [110] Enzyme phospholipase A2, specifically a soluble form (sPLA2) [111] Besides this, membrane-associated iPLA2 also affect number of activities such as lipid metabolism and membrane remodeling, maintenance of mitochondrial integrity, signal transduction, cell differentiation, proliferation and viability. [112] Some of the toxic effects of PLA2 render its binding to specific receptors to which other venom components and the mammalian sPLA2 binds. [113] These sPLA2 enzymes are Ca2+-dependent and contain a His-Asp motif in the catalytic domain. Individual sPLA2 exhibit unique tissue and cellular distribution and provide precursors for pro and antiinflammatory lipid mediator synthesis. It regulates membrane lipid composition. Snake and bee venoms contain different forms of sPLA2 toxins. One of the helices of group III sPLA2 enzymes is hydrophobic that enables its binding to the lipid bilayer membrane. [114] PLA2 found in bee venom consists of a single polypeptide chain of 130 amino acid residues folded by four disulfide bridges and a glycosyl residue at Asp-13 with a molecular weight of 15.8 kDa. The PLA2 from bee venom causes a variety of inflammatory-associated pathologies, including rheumatoid arthritis, septic shock, psoriasis, and asthma. [115] PLA2 stimulates PLA2R1 receptors which mediate the neurotoxic effect. A mild dose of PLA2 (6.2 mg/kg) causes edema, myonecrosis, prolonged the partial thromboplastin time and reducing the plasma fibrinogen concentration. [116] Similarly, melittin isolated from honeybee Apis mellifera is a small linear peptide composed of 26 amino acid residues. Its amino-terminal region predominantly is hydrophobic while its carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property is responsible for its cationic surfactant properties and cytolytic activity. [117] Contrary to this, monomeric melittin does not contain sPLA2 activity by itself. Actually, it facilitates the activation of the endogenous cell housekeeping PLA2 activity of the target via rapid binding to the membrane by hydrophobic interaction with zwitterionic phospholipids. This causes disruption of the phosphate group orientation and changes in physical and steric properties of the phospholipid, which result in dislodging of membrane barrier function. [117] PLA2 forms melittin-phospholipid domains after dissolving into the hydrocarbon region of the bilayer. The highly charged C region and the amino phosphate dipole have a strong electrostatic interaction that results in membrane perturbation, rendering the lipid target www.wjpps.com Vol 7, Issue 11, 2018. 613

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susceptible to PLA2 cleavage. [118] It also leads to alteration of the membrane surface results in antibacterial and hemolytic activity. Melittin stimulates PLA2 in various cells and tissues, mainly human erythrocytes, anterior pituitary tissue, pancreas, rat adenohypophysis, vascular endothelial cells, human leucocytes, platelets [119] and neurons. [120] Melittin dansylated analogue (DNC-melittin) binds to natural membranes and show cytolytic activity. [76] Due to these properties melittin is used as a neurochemical tool to recognize the role of sPLA2 in modulating synaptic transmission. [120] Bee venom group III sPLA2 shows protective immune responses against a wide range of diseases including asthma, Parkinson's disease, and druginduced organ inflammation. [121] This is widely used as a component of anti-venom and for desensitization therapy against bee stings. [122] Due to membrane penetration activity and cytotoxic effects melittin is used in cancer therapy. It is used either as an active ingredient or as an absorption enhancer. [123] Interaction with the plasma membrane Voltage-gated ion channels open or close depending on the setting of voltage gradient across the plasma membrane. Normally for vital functions ions are conducted through the channels down their electrochemical gradient. It depends on ion concentration and plasma membrane potential. These ion channels at large are selectively targeted by certain toxins scorpion [124] and snake venoms. [125] Voltage-Gated Potassium Ion Channels (Kv)The voltage-gated K+ channels are trans-membrane channels specific for K+ ions and are sensitive to voltage changes in the membrane potential. [126] These channels are known mainly for their role in repolarizing (voltage-dependent Kv) the cell membrane following action potentials, which regulate cellular processes such as Ca2+ signaling, cell volume, secretion, proliferation and migration. [127] Kv channels usually have a homo-tetrameric structure. The transmembrane domain, the _-subunit, consists of six helices (S1-S6) forming two structurally and Ion channels are important portals for a variety of toxins interacting with the plasma membrane. [128] Under normal conditions, the physiological role of these channels is to been well established that most Na+, K+, Ca2+ and some Cl channels, are voltage-gated. [129] but others including certain K+ and Clchannels, transient receptor potential (TRP) channels, ryanodine receptors and IP3 receptors, are relatively voltage-insensitive and are gated by second messengers and other intracellular and/or extracellular mediators. [130] Enhancing cytolytic functions Both ionic and peptide interactions assist in enhancement of cytotoxicity in cells. Melittin is an example that it from different conformational change in aqeous medium than mastoparan.
In an aqueous solution, melittin went from a nonhelical form to an alpha helix when

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Besides, cell cytoplasm, cytoskeleton, mitochondria play a major role in regulating cell death, which occurs upon permeabilization of their membranes. Once mitochondrial membrane permeabilization (MMP) occurs, cells die either by apoptosis or necrosis. MMP is regulated by levels of calcium, and cellular redox potential mainly levels of reactive oxygen species.
Besides this mobilization and targeting to mitochondria of Bcl-2 family members are also important factors. [131] Melittin can induce apoptosis of human gastric cancer (GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC. [132] BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death. BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. [133] Melittin is one of the best-studied antimicrobial peptides, and many studies have focused on the membrane underlying its membrane-disruptive activity. melittin is involved in the mitochondria-and caspasedependent apoptotic pathway in C. albicans. Our findings suggest that melittin possesses a dual antimicrobial mechanism, including membrane-disruptive and apoptotic actions. [134] From wasp and honey bee venom potent cytotoxic peptides can be derived that could used after surface modification and to give them target specificity to kill tumor cells. Both mastoparan and mitoparan are cell-penetrating peptides (CPPs) have potential pharmaceutical application in delivering macromolecules into cells. [135] CCPs also affect transport of doxorubicin encapsulating Tf-liposomes across BBB. [136] Transportan and some derivatives have the capacity to carry macromolecules. [137][138][139] Although mastoparan showed efficient translocation, the other CPPs, namely TAT peptide and penetratin, were more efficient. On the other hand, there is a chimeric galanin-mastoparan peptide called transportan, which contains the first 13 amino acids from the highly conserved amino-terminal part of galanin and the 14 amino acids sequence of mastoparan in the carboxyl terminus. [140,141] For targeting can cancer cells recombinant molecules are being made which are used as

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antibody or carrier ligand. Such molecules can be used to target malignant brain tumor in patients. By following similar approach However, non-toxic analog of apamin are made that bears two ornithine residues instead of arginines residues (ApOO). [142] Bee venom can be used by acupuncture method to carry it to relevant sites in function of a specific disease or to acupoints. Melittin tagged with an endosomolytic agent linked to siRNA delivery system effectively stop hepatitis B virus infection. [143] Another molecule mitoparan, transports this potent cytotoxic peptide to the tumor and allows its accumulation in a controlled manner. [144] Another promising and feasible idea tested in vivo to combat cancer has been presented in the form of a patent, which discloses an ultra-small lipid nanoparticle carrying melittin with potential use in clinical practice. [73] Besides cancer treatment wasp venom kinins, polyamines are used in treatment of pain, inflammatory disease, and neurodegenerative diseases such as epilepsy and aversion. [86] Anoplin shows very high cytotoxicity efficacy, arrests MEL cells (murine erythroleukemia) in the G₀/G₁ phase but it could not induce apoptosis in MEL cells. [145] It inhibits proliferation of MEL cells in a dose-dependent and time-dependent manner via disrupting the integrity of cell membrane. Similarly, MP-1, is short cationic α-helical peptide isolated from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. It shows higher content of anionic lipids increases the level of binding of the peptide to bilayers. Polybia-MPI exhibits excellent anticancer activity and remarkably suppresses the growth of sarcoma xenograft tumors. [146] Polybia-MPI /P takes a standard αhelix conformation in the membrane which is important for its cytotoxity on tumor cells. [147] The L-Pro substitution induces a significant reduction of antitumor activity. Polybia-MPI can be used for treatment of prostate cancer and bladder cancer, because of its lower cytotoxicity to healthy body cells. [148]

Scorpion venom
The scorpion venom is a highly complex mixture of salts, nucleotides, biogenic amines, enzymes, mucoproteins, as well as peptides and proteins (e.g. neurotoxins

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functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defense against predators. Scorpion venoms are rich source of potential novel antitumor and anti-cancer therapeutic agents. [149] Scorpion venom fractions and toxin peptides can decrease cancer growth, induce apoptosis and inhibit cancer progression and metastasis in vitro and in vivo. [150] These toxins modulate the ion channels either by blocking the pore of the channel or by altering the voltage gating. Molecules which block the pores have been useful in deciphering the structure of the ion channels. Many scorpion toxins have already been used for probing the voltage gated sodium channels. [149] Based on their molecular size and pharmacological activity, toxin peptides are classified in to two main groups. The first group contains short toxins consisting of 30-40 amino acid residues with 3-4 disulfide bridges, which mainly affect the voltage dependent K+ channels, and the large conductance calcium activated K+ channels. [151] The second group includes long chain toxin peptides of 60-70 amino acids cross linked by 4 disulfide bridges, which mainly have an effect on voltage dependent Na+ channels of excitable cells. [152,153] Insect toxins are  insect toxins which are excitatory in nature and show depressant activity. [154] Several toxins, which specifically affect Na+ and K+ channels, have been exclusively studied with respect to their structure, mode of action and pharmacological properties. [155,156] In addition to it some toxins that specifically target Ca++, K+ and Cl-channels have also been isolated [151,157] (Table 2). A 4.5 kD peptide toxin was isolated from the venom of Palamneus gravimanus, the Indian black scorpion, to block human Kv1.1 channels expressed in Xenopus laevis oocytes. This toxin peptide selectively blocks the human cloned voltage gated potassium channel at a very low concentration (10 nM). This blockage was found to be voltage dependent. Same peptide has shown typical hypertensive symptoms and showe a LD 50 value of 2-mg/kg mice (Table 1).
Venom peptides isolated from several scorpion species showed multiple biological activities such as cytotoxic, antiproliferative and apoptogenic effects on cancer cells. [158] Scorpion venom from Androctonus australis hector (Aah) and its toxic fractions (FtoxG-50 and F3) shows strong effect onon NCI-H358 human lung cancer cells. [159] BmKn-2 exerts selective cytotoxic effects on human oral cancer cells by inducting apoptosis via a p53-dependent intrinsic apoptotic pathway. apoptosis in breast and colorectal cancer cell lines [160] (Table 1).
These venom peptides are highly active and specifically target all major ion channels such as Na + , K + , Cl -, Ca ++ and rynodine sensitive Ca ++ channels [161] and their subtypes both in vertebrates and invertebrates [162] ( Table 2

Development of toxin based therapeutic drug delivery system
Animal toxins are natural weapons which display non-specific cytolytic activity, rapid degradation and excretion when injected in blood. Venom peptides present in wasp and honey bee showed immense cytolytic against cancer cells when conjugated as carrier. A procytotoxic systems was made by using bee venom cytotoxic peptides and its conjugation with poly(l-glutamic acid) PGA polymer through specific cleavage sequences. These were found quite sensitive to over-expressed tumor proteases, such as the metalloproteinase-2 (MMP-2) or cathepsin B. After their selective release cytolytic peptides inside tumor cell operates a spatiotemporal control over tumor cells and kill them successfully. [144] These nanoparticles (NPs) conjugated with toxin peptides showed high stability, uniform size, sufficient drug loading, targeting capability, and ability to overcome drug resistance. [163] NP formulation are used to target glioblastoma multiform (GBM). CTX loaded on NPs enhance the uptake of the

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NPs by GBM cells. It also improves the efficacy of PTX in killing both GBM and GBM drug-resistant cells. A nano complex IONP-PTX-CTX-FL displayed great potential for brain cancer therapy [163,164] (Table 3). Besides natural toxin peptides synthetic analogues of each peptide, were engineered which also showed enhanced cationicity and amphipathicity. These also showed potent anti-proliferative activity against a range of human cancer cell lines. [165] Scorpion venom components are also used to make scaffolds for the development of drugs [166] (Ortiz E et al, 2015) ( Figure 5). From available database on natural toxins new therapeutic molecules can be designed by using structure and activity related information ( Figure 6). Further, their target specificity can be improved by finding anticancer peptides by slight modifications in amino acids, lipid-protein and toxin receptor interactions and a common peptide could possible scorpion, honey bee, and wasp and spider bites due to their long evolutionary relationship and ontology (Figure 7). Thus new putative toxin structures can be developed from naturally secreted toxins by using bio-informatics tools and methods mainly proteomic and transcriptomic library (( Figure 8).

Glioblastoma multiforme (GBM) is a most common malignant central nervous system tumor.
This is highly malignant and aggressive and most lethal human cancers which show very high invasive capacity and lesser therapeutic options. [167] It is fast proliferating and destructive tumor has very low mean survival time (<24months) and develops very silently within the brain microenvironment. These malignant primary brain neoplasm showed mean survival of <24months. Drug chemotherapy of GBM shows low therapeutic efficiency and systemic side effects. [168] Only glioma-specific chloride ion channel represents a specific target for therapy.
These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. These ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues (Du Q et al, 2015) [165] Two nonadecapeptide FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN have shown very strong anti-tumor potential against GBM. [168] Cisplatin-based therapy Cisplatin-based therapy is one of the most important chemotherapy treatments for GBM, although its efficacy is limited due to drug resistance and undesirable side effects. DMCa is a good candidate for clinical platinum-based therapy in GBM treatments and other cancer types. [169] DMCa exhibit strong anti-cancer efficacy compared to cisplatin, especially at low doses. By inducing intracellular oxidative stress, Pt-1-DMCa potentiated platinum-induced www.wjpps.com Vol 7, Issue 11, 2018. 621

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DNA damage and led to enhanced p53 phosphorylation, followed by increased activation of both mitochondrial and death receptor pathways. Decreased phosphorylated AKT and ERK levels were associated with the apoptosis induced by the novel synthesized cisplatin analogue. Thus, a chimera between platinum and a maurocalcine-derived CPP was found a highly successful anti-cancer compound that works by targeting the intracellular redox system. [170] (Table 3).

Chlorotoxin (CTX)-targeted nanoparticles therapy
Chlorotoxin is a disulfide-rich stable peptide derived from Israeli scorpion Leiurus quinquestriatus venom. It shows wider therapeutic potential in treatment of cancer.. It possesses compact shape and is convenient for intracranial delivery. It is specific and efficacious in blocking glioma Cl(-) channel activity [171] CTX-conjugated NPs are preferentially bind to tumor cells and used as a vehicle to deliver anti-cancer drugs specifically to cancer cells1 [172] Chlorotoxin (CTX)-targeted nanoparticles (NPs) work as a targeting ligand that specifically binds to glioma. CTX was conjugated onto iron oxide NPs, quantum dots, and rare-earth upconversion NPs are used in magnetic resonance and fluorescence imaging of glioma (Table 3). CTX-conjugated NPs are also used as carrier system to load anticancer drugs or therapeutic genes for targeted chemotherapy or gene therapy of glioma, respectively. [173] It targets various gated channels i.e. voltage gated chloride channels (GCC), calcium-dependent phospholipid-binding protein Annexin-2, and an inducible extracellular enzyme matrix Metalloproteinase-2 (MMP-2). MMP-2 shows antineoplastic potential. Chlorotoxin possess tumor cell targeting domain and shows different effector functions. [174] Further, for enhancing its action on cancer cells its physical mixture (CTX + Onc) was prepared by conjugating it to a onconase. At high dose this CTX-Onc showed better anti-tumor effect than simple toxin. [175] Similar cytotoxic potential is also reported in Centruroides tecomanus chlorotoxin. [176] In addition, multifunctional nanocomposites are made by using polymeric nanoparticles (PNPs) containing two cytotoxic agents -the drug alisertib and silver nanoparticles. These PNPs have been conjugated with a chlorotoxin, an active targeting 36-amino acid-long peptide that specifically binds to MMP-2, a receptor over-expressed by brain cancer cells. [177] This silver/alisertib@PNPs-chlorotoxin properties with endothelial and angiogenic hotspots disrupting activities. [168] Recombinant AGAP (rAGAP)(Analgesic-antitumor peptide) also affect the migration and invasion of HepG2 cells via a voltage-gated sodium channel (VGSC) β1 subunit and shows strong antitumor activity. [178] (Table 3).

Self-assembled toxin based polymeric nano-structures
Efforts have been made to self-assemble scorpion venom in polymeric nano-structures for controlled delivery of toxins towards tumorous sites. [179] For this purpose, polypeptide extract from scorpion venom (PESV) was combined to Rapamycin. It is taken up by H22 hepatoma cells in mice during autophagy. [180] This PESV combined Rapamycin inhibit the development of H22 hepatoma transplantation tumor in mice. It could be possible by inhibiting the activity of mTOR, enhancing expressions of ULK1, MAP1LC3A, and Beclin1. [180] PESV also inhibit the angiogenesis of H22 hepatoma. The mechanisms might be associated with suppressing the expression of PI3K, P-Akt, HIF-1 alpha, and VEGF-A. [181] PESV (polypeptide extract from scorpion venom) successfully block cell cycle and inhibit angiogenesis directly to inhibit cell proliferation of non-small cell lung cancer cell line A549 mainly through reducing the expression of HIF-1alpha, VEGF and increasing the expression of PTEN. [182] Mechanism of polypeptide extract from scorpion venom (PESV) on promoting anti-tumor effects of cyclophosphamide (CTX). In addition, polypeptide extracts from scorpion venom (PESV) combined to 5-fluorouracil (5-Fu) shows inhibition effects on vasculogenic mimicry (VM) of H2 hepatoma carcinoma cells in mice. [183] Similarly, TsAP-1 and TsAP-2 isolated from Tityus serrulatus showed broad spectrum anticancer potencies which can be significantly enhanced by increasing their cationicity. [184] PESV inhibit the expressions of VEGF and TGF-beta1, promote the maturation of DCs, recover its antigen uptake presentation function, and reverse the immune injury to the body by CTX. It also plays important role in inducing the tumor cell apoptosis. [185] Selectively targeting cancer tissue is one promising strategy.

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cycle in G0/G1 phase. [188] miRNA-based therapeutics is followed for specific killing of tumor and cancer cells in GBM. In this therapeutic approach intravenously-administered chlorotoxin (CTX) is coupled (targeted) with stable nucleic acid lipid particle (SNALP) to formulate anti-miR-21 oligonucleotides. These particles are send to approach towards tumor cells. These particles accumulate preferentially within brain tumors and promote efficient miR-21 silencing. It results in increased mRNA and protein levels of its target RhoB and showing no signs of systemic immunogenicity. This miRNA modulation by the targeted nanoparticles combined with anti-angiogenic chemotherapy shows high success rate in GBM treatment. [167] However, for modulation of tumor response to chemotherapy, a combination of treatment with small interference RNA (siRNA), chlorotoxin and conventional (temozolomide, TMZ) drugs is used. siRNA delivery by targeted nanoparticles resulted in modulating tumor response to chemotherapy in GBM. siRNA interrupt cell signaling, drug act as toxic agent, and CTX contributes apoptotic tumor cell death. [189] Similarly a chlorotoxin-conjugated graphene oxide (CTX-GO) drug delivery system shows better efficacy in glioma treatment. The method is developed by using graphene oxide and laoding doxorubicin CTX-GO (CTX-GO/DOX) via noncovalent interactions. [168] The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells. The combination of CTX-NO and chemotherapeutics also led to decreased cell invasion [190] (Table 3). A new method MiRNA-21 silencing mediated by tumor-targeted nanoparticles combined with sunitinib: is developed for gene therapy of glioblastoma. [191] Further, immunotoxin molecules conjugated to some anticancer drug are to be prepared. For targeting can cancer cells recombinant molecules are being made which are used as targeted toxins, also known as immunotoxins or cytotoxins ( Figure. 9).    Anti-angiogenesis therapy (Zhao QQ et al, 2015).
increase the cytotoxicity in cancer cells (Graf N et al, 2012 siRNA delivery by targeted nanoparticles siRNA interrupt cell signaling, drug act as toxic agent, and CTX contributes apoptotic tumor cell death