Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.     

Distribution of proteinase inhibitors in snake venoms

Potent proteinase inhibitors, which inactivate bovine pancreatic trypsin, α-chymotrypsin, bovine plasma kallikrein and plasmin, were found in several snake venoms. The content of the inhibitor was highest in Vipera russelli venom. Proteinase inhibitors of the same type were demonstrated also in the venoms of five snakes of the Elapidae family; Hemachatus haemachatus (Ringhals cobra), Dendroaspis angusticeps (Green mamba), Dendroaspis polylepis (Black mamba), Naja nivea (Cape cobra) and Naja haje (Egyptian cobra). No proteinase inhibitors were demonstrated in several Crotalidae and Hydrophiidae venoms. Toxic polypeptides such as α-bungarotoxin, cytotoxin I and II (Naja naja venom) and cardiotoxin (Naja naja atra venom) did not show any inhibitory action on typical mammalian proteinases including trypsin, α-chymotrypsin, plasmin and kallikrein.     

Biosynthesis,secretion and in vivo isotopic labelling of venom of the Egyptian cobra, Naja haje annulifera

The venom glands of Elapidae differ from those of the Viperidae by lacking an expanded central lumen; the venom is stored in the tubular lumina as well as inside the cells in densely packed secretion granules. Using isotope tracer techniques, it was found that in the Egyptian cobra (Naja haje annulifera) venom is secreted both from pre-existing and from newly-formed granules. The rate of protein biosynthesis peaks at 4–9 days after venom was extracted (milked) from the glands. Highly labelled toxins (1–10 mCi/mmole protein) were isolated in good yield from the venom of snakes chronically intubated and infused i.p. with (³H)-amino acids. Repeated Fluothane (Halothane) anaesthesias and venom collections had no ill effect on venom yield. The radioactive venom and its component toxins retained full biological potency.     

Cross neutralization of dangerous snake venoms from Africa and the Middle East using the VACSERA polyvalent antivenom. Egyptian Organization for Biological Products & Vaccines

This study was performed to assess the ability of polyvalent snake venom anti-serum, produced by the Egyptian Organization for Biological Products & Vaccines (VACSERA), to neutralize several toxic activities of snake venoms, not only of those included in the antivenom mixture, but also some additional venoms of snakes from Egyptian, African, and Middle Eastern habitats. In general, the results revealed that polyvalent snake venom anti-serum from VACSERA is highly effective in neutralizing Egyptian snake venoms, especially Naja haje, Naja nigricolles, Naja pallida, Cerastes cerastes, Cerastes cerastes cerastes, Cerastes vipera, Pseudocerastes persicus fieldi, and Walterinnisia egyptia. The antivenom was also effective against Naja haje, Walterinnisia egyptia, and Bites aritans from Saudi Arabia. High activity was obtained against venoms from Naja haje, Naja nigricolles, and Naja pallida of Sudan, as well as the African Naja melanoleuca, Naja mossambica, Naja naja oxiana, Bites gabonica, and Vipera lebetina. Only moderate effectiveness was obtained with Echis coloratus and Echis carinatus, and the polyvalent antiserum was ineffective against the venom of Naja nivea.     

Immunological studies on Egyptian cobra antivenin

A monovalent specific antivenin was prepared in horses against the Egyptian cobra (Naje haje) venom; using bentonite as an adjuvant. One ml of the serum tested at the end of the six-month program neutralized 64 ld₅₀ and protected against 53 ld₅₀ in mice. Cross immunological studies were carried out with five elapid, six viperid and one crotalid venoms. The serum proved effective in neutralization of and protection against these venoms. Precipitation reaction lines were obtained using the Ouchterlony technique, with most of the venoms. Identity patterns were established between venoms of N. haje, N. nigricollis, N. flava, N. naja, Walterinnesia aegyptia of the Elapidae as well as those of Echis carinatus and Ethiopian N. haje. Partial identity with the two vipers Cerastes cerastes and Cerastes vipera and nonidentity with Echis coloratus venom. Weak or no reactions were given with the Ethiopian Bitis lachesis, Bitis gabonica and the Japanese Trimeresurus flavoviridis.     

A new type of thrombin inhibitor,noncytotoxic phospholipase A2, from the Naja haje cobra venom

Thrombin is a key enzyme in the blood coagulation cascade and is also involved in carcinogenesis; therefore, its inhibitors are of fundamental and clinical importance. Snake venoms are widely used as sources of proteins that affect blood coagulation. We have isolated a new protein, called TI-Nh, from the Naja haje cobra venom. TI-Nh is a mixed-type inhibitor of thrombin (K_i of 72.8 nM for a synthetic peptide substrate) and effectively inhibits thrombin-induced platelet aggregation with an IC₅₀ value of 0.2 nM. At concentrations up to approximately 50 nM, at which the thrombin-clotting time is substantially prolonged, TI-Nh exerts no detectable effects on both the intrinsic and extrinsic pathways of the coagulation cascade. It does not hydrolyze either fibrinogen or thrombin. Although TI-Nh bears structural features typical of group IB phospholipases A₂ (PLA₂s), it possesses relatively weak enzymatic activity and is nontoxic to PC12 cells at concentrations up to 15 μM. Nevertheless, TI-Nh evokes neurite outgrowth in these cells at a concentration of approximately 1 μM, similar to cytotoxic snake PLA₂s with strong enzymatic activity. TI-Nh is the first thrombin inhibitor found in the venom of the Elapidae snake family, and it is the first phospholipase shown to inhibit thrombin.     

The role of the fibrinolytic enzyme system in the haemostatic defects following snake envenomation

The effect of five Egyptian snake venoms on fibrinolysis both in vitro and in vivo were studied. The viper venoms C. cerastes, C. vipera and E. carinatus lysed fibrin in vitro in both unheated and heated fibrin-agar plates, although this effect was markedly reduced on heated plates. Insignificant fibrin lysis was produced by the cobra venom N. nigricollis and N. haje. The three viper venoms produced lysis of fibrinogen on incubation with the purified material, whereas the cobra venoms produced fibrinogenolysis only after 30–90 min incubation periods.Rabbits treated with lethal doses of N. nigricollis venom displayed blood incoagulability due to an antithromboplastic action; no in vivo fibrinogenolysis could be observed. The venom of N. haje produced insignificant changes in blood clotting and fibrinolysis when injected in rabbits. Rabbits injected with C. vipera venom displayed blood hypocoagulability associated with prolonged thrombin clotting times, indicating fibrinolysis. Likewise, injection of C. cerastes venom produced a weak fibrinolytic state associated with blood hypercoagulability. E. carinatus venom caused blood incoagulability and severe hypofibrinogenemia in rabbits as a result of activation of the fibrinolytic system as well as a direct lytic action of the venom on fibrinogen.     

Immunological studies on polyvalent and monovalent snake antivenins.

Polyvalent antivenin prepared against the Egyptian snake venoms, Naja haje, N. nigricollis, Cerastes vipera, C. cerastes, Echis carinatus and Walterinnesia aegyptia; against the African snake venoms, Dendroaspis polylepis, Bitis gabonica and B. arietans as well as the Asian snake venoms, Trimeresurus flavoviridis. was tested by neutralization and immunodiffusion experiments. The antivenin was of high potency against Egyptian venoms especially C. vipera and C. cerastes, followed by Walterinnesia aegyptia, N. haje, Echis carinatus, N. nigricollis, T. flavoviridis, B. arietans, and B. gabonica.Comparison of the polyvalent antivenin and monovalent B. gabonica antivenin revealed that while the polyvalent serum was of better effectiveness against the Egyptian viper venoms, especially Cerastes venoms, more protection was provided by the monovalent serum against B. gabonica venom.     

Cross‐Neutralisation of the Neurotoxic Effects of Egyptian Cobra Venom with Commercial Tiger Snake Antivenom

Cross‐neutralisation has been demonstrated for haemorrhagic venoms including Echis spp. and Cerastes spp. and for Australia elapid procoagulant toxins. A previous study showed that commercial tiger snake antivenom (TSAV) was able to neutralise the systemic effects of the Egyptian cobra, Naja haje, in vivo but it is unclear if this was true cross‐neutralisation. The aim of the current study was to determine whether TSAV can neutralise the in vitro neurotoxic effects of N. haje venom. Both Notechis scutatus (10 μg/ml) and N. haje (10 μg/ml) venoms caused inhibition of indirect (supramaximal V, 0.1 Hz, 0.2 msec.) twitches of the chick biventer cervicis nerve–muscle preparation with t₉₀ values (i.e. the time to produce 90% inhibition of the original twitch height) of 26 ± 1 min. (n = 4) and 36 ± 4 min.; (n = 4). This effect at 10 μg/ml was significantly attenuated by the prior addition of TSAV (5 U/ml). A comparison of the reverse‐phase HPLC profiles of both venoms showed some similarities with peak elution times, and SDS‐PAGE analysis elucidated comparable bands across both venoms. Further analysis using Western immunoblotting indicated TSAV was able to detect N. haje venom, and enzyme immunoassay showed that in‐house biotinylated polyclonal monovalent N. scutatus antibodies were able to detect N. haje venom. These findings demonstrate cross‐neutralisation between different and geographically separated snakes supporting potential immunological similarities in snake toxin groups for a large range of snakes. This provides more evidence that antivenoms could be developed against specific toxin groups to cover a large range of snakes.     

Fractionation of Egyptian cobra venom

Five toxic fractions were isolated from Egyptian Naja haje venom by filtration using Sephadex G-75. Applying immunodiffusion techniques, fractions four and five were found to contain γ(cardiotoxic)- and α(neurotoxic)-like antigenic activity, respectively. The maximum phospholipase A activity was recorded in fraction three. Chromatographic purification of fractions four and five, using Biorex 70 and various buffers, showed that the best resolution was obtained with ammonium acetate, at pH 6·5. The final concentrated fractions were submitted to immunoelectrophoresis as a test of their purity.     

Hyperglycemic effect of a neurotoxic fraction (F3) from Naja haje venom: role of hypothalamo-pituitary adrenal axis (HPA).

The effect of bolus intravenous injection of sub-LD50 (35 micrograms Kg-1) of the neurotoxic fraction (F3) of the Egyptian cobra Naja haje on the plasma level of ACTH and serum levels of cortisol, insulin, glucose, total lipids, triacylglycerols, free fatty acids, total cholesterol, HDL and LDL-cholesterol, and glycogen content of liver and kidneys were studied in rabbit pretreated with cyproteron acetate (CA) or saline solution and propylene glycol (PG) to elucidate the possible role of the hypothalamo-pituitary adrenal (HPA) axis in the venom fraction-induced hyperglycemia. F3 increased cortisol and insulin level in both groups, whereas ACTH was found to decrease subsequent to the treatment. Serum glucose level was elevated by F3 treatment and this effect was substantiated in CA-treated rabbits. This hyperglycemia was concomitant with a decline in glycogen content of the liver and kidneys. A decline in serum total lipids, triacylglycerols, and free fatty acids was observed following F3 treatment, and this effect was intensified by CA-pretreatment. These data suggest that F3 stimulates glucocorticoid release from adrenocortical cells which, in turn, may modulate both insulin and glucose turnover to maintain hyperglycemia during stress period. The possible underlying mechanisms were discussed.     

Evaluation of the Lethal Potency of Scorpion and Snake Venoms and Comparison between Intraperitoneal and Intravenous Injection Routes

Scorpion stings and snake bites are major health hazards that lead to suffering of victims and high mortality. Thousands of injuries associated with such stings and bites of venomous animals occur every year worldwide. In North Africa, more than 100,000 scorpion stings and snake bites are reported annually. An appropriate determination of the 50% lethal doses (LD₅₀) of scorpion and snake venoms appears to be an important step to assess (and compare) venom toxic activity. Such LD₅₀ values are also commonly used to evaluate the neutralizing capacity of specific anti-venom batches. In the present work, we determined experimentally the LD₅₀ values of reference scorpion and snake venoms in Swiss mice, and evaluated the influence of two main venom injection routes (i.e., intraperitoneal (IP) versus intravenous (IV)). The analysis of experimental LD₅₀ values obtained with three collected scorpion venoms indicates that Androctonus mauretanicus (Am) is intrinsically more toxic than Androctonus australis hector (Aah) species, whereas the latter is more toxic than Buthus occitanus (Bo). Similar analysis of three representative snake venoms of the Viperidae family shows that Cerastes cerastes (Cc) is more toxic than either Bitis arietans (Ba) or Macrovipera lebetina (Ml) species. Interestingly, the venom of Elapidae cobra snake Naja haje (Nh) is far more toxic than viper venoms Cc, Ml and Ba, in agreement with the known severity of cobra-related envenomation. Also, our data showed that viper venoms are about three-times less toxic when injected IP as compared to IV, distinct from cobra venom Nh which exhibited a similar toxicity when injected IP or IV. Overall, this study clearly highlights the usefulness of procedure standardization, especially regarding the administration route, for evaluating the relative toxicity of individual animal venoms. It also evidenced a marked difference in lethal activity between venoms of cobra and vipers, which, apart from the nature of toxins, might be attributed to the rich composition of high molecular weight enzymes in the case of viper venoms.     

Immunological studies on Naja nigricollis antivenin

Monovalent Naja nigricollis antivenin was prepared in horses using venom adsorbed to bentonite (first three injections) followed by increasing doses (up to 50 mg) of unmodified venom. The serum (1 ml) neutralized 51 ld₅₀'s of its homologous venom. Good neutralizing capacity (30–50 ld₅₀'s per ml) was also shown against venoms of N. haje, N. nivea, N. naja, Walterinnesia aegyptia, Cerastes cerastes and Cerastes vipera as well as Echis carinatus venom. For all these venoms, identity patterns, complete or partial, were established using Ouchterlony's technique. Moderate neutralization (22 ld₅₀'s per ml) was shown against Echis coloratus venom which gave a nonidentity pattern. Low neutralization (10 ld₅₀'s per ml) was observed with venoms of Bitis arietans, B. gabonica and Trimeresurus flavoviridis; all these venoms showing very weak or no precipitation lines with Naja nigricollis antivenin.     

Histopathological effects of Naja haje snake venom and a venom gland extract of the scorpion Buthus quinquestriatus on the liver,suprarenal gland and pancreas of mice

Lethal doses of Naja haje snake venom produced central and midzonal hydropic degeneration in hepatic lobules. Buthus quinquestriatus scorpion venom gland extract caused peripheral zonal hydropic degeneration, sinusoidal dilatation and disruption. Fatty degenerative changes started earlier after use of the scorpion preparation. Both preparations depleted glycogen, inhibited succinic dehydrogenase and increased alkaline phosphatase activity in degenerated hepatocytes. Signs of regeneration followed sublethal doses of Naja haje venom. Lethal doses of both venom preparations caused rupture of the capsule of the suprarenal gland and detachment of its cortex. Blood extravasated in the medulla. Zona fasciculata cells were hypertrophied and vacuolated and cortical lipids increased. Depletion of ascorbic acid and medullary catecholamines occurred. After a single sublethal dose the zona reticularis region increased in thickness due to proliferation of its cells. These cells invaded the medullary clumps. Repeated sublethal doses increased the thickness of both the zona fasciculata and reticularis. In the pancreas, lethal doses of both venom preparations produced vacuolization of alpha and beta cells. Partial and complete degranulation were observed in hydropically degenerated beta cells. Alpha cells showed decreased alkaline phosphatase activity.     

Effects of several snake venoms on serum and tissue transaminases,alkaline phosphatase and lactate dehydrogenase

The effect of lethal doses of five venoms (Bitis arietans, Bitis gabonica, Dendroaspis polylepis, Naja nigricollis and Naja haje) on the activities of transaminases, alkaline phosphatase and lactate dehydrogenase were studied. Samples from the serum, liver, heart and kidney were collected 4 hr following lethal venom injections in albino rats. The activity of these enzymes were markedly increased in serum and variably decreased in the liver, heart and kidney after envenomation.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

Isolation and characterization of a toxic phospholipase A2 in the spitting cobra (Naja mossambica mossambica) venom.

N. Martin-Moutot and H. Rochat. Isolation and characterization of a toxic phospholipase A₂ in the spitting cobra (Naja mossambica mossambica) venom. Toxicon17, 127–136, 1979.—A phospholipase A₂ from the spitting cobra (Naja mossambica mossambica) was purified by chromatographic procedures involving gel filtration on Sephadex G-50 and ion exchange on Amberlite IRC-50. The purified enzyme was homogeneous according to physico-chemical criteria as well as chemical purity tests. This protein was characterized by amino acid composition, determination of N- and C- terminal amino acid sequences, isoelectric pH, molecular weight. It exhibits phospholipase activity and toxicity to mice. Positional specificity towards phosphatidylcholine showed that it belongs to the A₂ type. Optimal activity was observed at an alkaline pH and calcium was required for phospholipase activity.