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.

A Neurotoxic Snake Venom without Phospholipase A2: Proteomics and Cross-Neutralization of the Venom from Senegalese Cobra, Naja senegalensis (Subgenus: Uraeus)

The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.

Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom

Objective There has been little focus on the enzymatic and lethal activities of Naja ashei venom and their neutralization by antivenom. This study aimed to determine the snake venom phospholipase A2/svPLA2 activity and brine shrimp lethality of N. ashei venom and their neutralization by two antivenoms (I and II). The venom of other snakes in East Africa including the puff adder (Bitis arietans), green mamba (Dendroaspis angusticeps), black mamba (Dendroaspis polylepis), Egyptian cobra (Naja haje), red spitting cobra (Naja pallida), and the Eastern forest cobra (Naja subfulva) were used for comparison. Results: N. subfulva venom had the highest svPLA2 activity while D. angusticeps venom had the least activity. N. subfulva venom was the most toxic in the 24-hour brine shrimp lethality assay (BSLA), while N. ashei venom was the most toxic in the 48 and 72-hour assays. N. haje venom was the least toxic in all assays. One ml of antivenom I neutralized 0.075 µg of svPLA2 in N. ashei venom compared to 0.051 µg by antivenom II. Antivenom I was ineffective in neutralizing N .ashei venom-induced lethality but 1 ml of antivenom II neutralized 0.21 mg of N. ashei venom.

Distinctive Distribution of Secretory Phospholipases A2 in the Venoms of Afro-Asian Cobras (Subgenus: Naja, Afronaja, Boulengerina and Uraeus)

The protein abundances of phospholipases A2 in cobra venom proteomes appear to vary among cobra species. To determine the unique distribution of snake venom phospholipases A2 (svPLA2) in the cobras, the svPLA2 activities for 15 cobra species were examined with an acidimetric and a colorimetric assay, using egg yolk suspension and 4-nitro-3-octanoyloxy benzoic acid (NOBA) as the substrate. The colorimetric assay showed significant correlation between svPLA2 enzymatic activities with the svPLA2 protein abundances in venoms. High svPLA2 activities were observed in the venoms of Asiatic spitting cobras (Naja sputatrix, Naja sumatrana) and moderate activities in Asiatic non-spitters (Naja naja, Naja atra, Naja kaouthia), African spitters (subgenus Afronaja), and forest cobra (subgenus Boulengerina). African non-spitting cobras of subgenus Uraeus (Naja haje, Naja annulifera, Naja nivea, Naja senegalensis) showed exceptionally low svPLA2 enzymatic activities. The negligible PLA2 activity in Uraeus cobra venoms implies that PLA2 may not be ubiquitous in all snake venoms. The svPLA2 in cobra envenoming varies depending on the cobra species. This may potentially influence the efficacy of cobra antivenom in specific use for venom neutralization.

Snake charming and the exploitation of snakes in Morocco

Traditional activities that potentially threaten biodiversity represent a challenge to conservationists as they try to reconcile the cultural dimensions of such activities. Quantifying the impact of traditional activities on biodiversity is always helpful for decision making in conservation. In the case of snake charming in Morocco, the practice was introduced there 500 years ago by the religious order the Aissawas, and is now an attraction in the country's growing tourism industry. As a consequence wild snake populations may be threatened by overexploitation. The focal species for snake charming, the Egyptian cobra Naja haje, is undergoing both range and population declines. We estimated the level of exploitation of snakes based on field surveys and questionnaires administered to Aissawas during 2003–2014, and compared our results with those of a study conducted 25 years previously. Aissawas use four venomous and four non-venomous species for snake charming and we estimate they harvest a minimum of 4,500 individuals annually, mostly venomous snakes. For exhibition purposes they selectively remove the largest specimens from the wild (i.e. those that could have the highest reproductive output). Compared to the previous data, we detected (1) a reduction in the number of species collected, (2) an increased distance to collecting fields, and (3) an increase in the market price for snakes, after correction for accumulated inflation, signifying a higher demand for these animals.