Cytotoxic activity of Androctonus australis hector venom and its toxic fractions on human lung cancer cell line

Continuous efforts have been made in recent years in the search for new agents to improve the treatment against cancer. Several bioactive components from biological products (such as proteins, peptides and enzymes) that can induce apoptosis in cancer cells have the potential to become suitable candidates as anticancer drugs. In this sense, peptides obtained from scorpion venoms have proven to be valuable tools for the development of new drugs [1, 2].

The current study demonstrated that Aah scorpion venom significantly reduced proliferation of NCI-H358 cells in a dose-dependent manner. The non-small cell lung cancer cell line NCI-H358 was the most sensitive to Aah venom in comparison to MCF7, Hep2 and HeLa cell lines. The variations in response to crude venom on growth of the four cell types after 24 h exposure might be a result of the complex composition of the venom and/or the targets expressed on cancer cells [12]. This observation is consistent with previous studies in which venoms of other scorpions such as Rhopalurus junceus, Androctonus crassicauda and Centruroides limpidus limpidus also induced selective and differential cytotoxicity against different human malignant cell lines [12, 36, 37].

According to these results, the cytotoxic potential of Aah toxic fractions (FtoxG-50 and F3) was assessed only on the most sensitive cell line to the crude venom (NCI-H358). Cell treatment with increasing concentrations of the toxic fractions (FtoxG-50 and F3) during 24 h resulted in a dose-dependent drop of cell viability. The F3 fraction showed more cytotoxic effect towards NCI-H358 cells than FtoxG-50. Interestingly, F3 fraction did not significantly affect the normal human lung fibroblast compared to toxic fraction FtoxG-50. The difference in the cytotoxic effect between the two tested fractions on lung cancer cells and normal fibroblastic cells could be due to their different composition and/or different cellular targets.

Indeed, accumulated evidence has indicated that several Kv channel subtypes are widely expressed in the plasma membranes of numerous cells and that these channels are involved both in the regulation of proliferation and apoptosis [38, 39]. It was reported that margatoxin, a selective Kv1.3 blocker, inhibited the cell proliferation of human lung adenocarcinoma A549 cell line by cell cycle arrest in G1 phase [40]. Jang et al. [41] showed cell growth inhibition in A549 cells after exposure to dendrotoxin-k, a Kv1.1 blocker, without affecting cell proliferation of normal MRC5. These authors reported the very low expression of Kv1.1 mRNA and protein in MRC-5 cells compared to A549 cells. Therefore, in the present study, the F3 fraction seems to induce a selective cytotoxicity on NCI-H358 lung cancer cells without any significant effect on normal lung fibroblast suggesting the overexpression of Kv channels on the plasma membranes of NCI-H358 cells.

Additionally, cytotoxicity events were confirmed by the increase of LDH leakage in the Aah venom and toxic fraction-treated cells which is usually correlated with loss of cell membrane integrity [42]. In order to assess the molecular mechanism underlying F3 fraction induced-cytotoxicity, ½ IC₅₀ (13.52 ?g/mL), IC₅₀ (27.05 ?g/mL) and 2 IC₅₀ (54.1 ?g/mL) concentrations were selected for the subsequent experiments.

Besides the obtained cytotoxicity, F3 fraction induced inhibition of cell proliferation after testing over a longer treatment period with the clonogenic assay in a dose-dependent manner. Cells exposed to different concentrations of F3 fraction exhibited morphological changes evidenced by: alterations in cell monolayer with areas devoid of cells, rupture of membranes and release of cytosolic contents. We also observed round and shrunk cells, which are morphological features of apoptosis. Similar morphological alterations were observed after treatment with fraction FI isolated from Tityus discrepans scorpion venom on human breast carcinoma cell line SKBR3 [15].

Apoptosis is a highly regulated process that occurs in almost all living organisms that selectively eliminates transformed cells. Consequently, the induction of apoptosis by cytotoxic natural compounds is considered as the main key and efficient strategy for cancer therapy and new drug development [26, 43]. To elucidate whether F3 fraction inhibits the proliferation of NCI-H358 cells by inducing apoptosis, treated cells were examined after staining with the DNA binding fluorochrome Hoechst 33258 and were analyzed for DNA fragmentation and caspase-3 activity. Our results showed that cells exposed to increasing concentrations of F3 fraction displayed nuclear morphological alterations which are indicative of apoptosis characterized by chromatin condensation, DNA fragmentation, cell shrinkage and compartmentalization of the dead cells into apoptotic bodies associated with the decrease of cell viability.

Breakdown of the nucleus is a hallmark of apoptosis that occurs during the early phase of this mode of cell death [26]. This includes the condensation of chromatin and associated fragmentation of the DNA followed by breakdown of the entire nucleus. The caspase family plays an important role in the initiation and execution pathways of cell apoptosis, in which caspase-3 acts as the central protease regulator and is required for the progression of apoptosis that is activated during the cascade [44, 45]. It was observed that caspase-3 activity was significantly increased in F3 fraction treated cells and inhibited by its specific inhibitor Ac-DEVD-CHO. All these results suggest that F3 fraction has an apoptosis-inducing effect on NCI-H358 cells, which is supported by several studies that reported the ability of some venoms from scorpions and spiders to trigger apoptosis through DNA fragmentation and caspase-3 activation [14, 36, 45–47]. Furthermore, NAC, (a general free radical scavenger) significantly reduced the F3 fraction-induced apoptosis in NCI-H358. Therefore, we can infer that reactive oxygen species (ROS) may be involved in the F3 fraction-mediated apoptotic process.

ROS are the key mediators of cellular oxidative stress and have an important role in tumor cell damage and mitochondrial stability. Imbalance redox status of ROS levels may harm major cellular components such as DNA, proteins, lipids and membranes leading to oxidative damage and cell death [48]. To determine whether the apoptosis of NCI-H358 cells was induced by ROS production, we quantified intracellular ROS by DCF fluorescence after cell exposure to ½ IC₅₀, IC₅₀ and 2 IC₅₀ concentrations of F3 fraction for 1, 4 and 24 h. The obtained results showed an increase in ROS production in exposed cells to F3 fraction in dose and time-dependent manner with significant increase after 24 h of treatment for all tested concentrations (1.27, 3.29, 3.71 fold increase at ½ IC₅₀, IC₅₀ and 2 IC₅₀ concentrations respectively). Moreover, NAC significantly inhibited the intracellular ROS production, which was completely counteracted at 1 h and 4 h. This result suggests that ROS may be a key early signal of F3 fraction-induced apoptosis. Similar to our present results, it has been reported that toxins from various animal venoms could induce intrinsic apoptosis through ROS upregulation, a situation that could be prevented by pretreatment with antioxidants [43, 49–54].

Our data showed also that F3 fraction enhanced the production of nitrite, a primary product of NO metabolism in lung cancer cells. It has been previously demonstrated that nitric oxide can increase the oxidative stress by the production of reactive nitrogen species (RNS), which are a variety of nitrogen containing molecules that are typically derived via NO reactions [55]. Excessive RNS generation contribute to biomembrane damage including mitochondrial membrane and in the formation of permeability transition pore [46, 47, 55]. High levels of NO can also alter protein functions through S-nitrosylation and/or nitration of regulatory proteins and increased Fas density on some tumor cell surface such as CaP cells or SW480 human colon carcinoma cells [55, 56]. Earlier studies also reported the involvement of nitrosative stress in the venoms induced apoptosis [46, 47].

Malondialdehyde is a final metabolite of lipid peroxidation and was formed from a variety of unsaturated fatty acids in biological membranes stimulated by ROS and RNS overproduction [57]. Protein-bound carbonyls represent a marker of overall protein oxidation, as they are formed early during oxidative stress conditions in blood, tissues and cells [48, 57, 58]. In the present study, we found that F3 fraction could increase the MDA and protein carbonyl levels in the NCI-H358 cells in a dose-dependent manner. These results clearly indicate that lipid peroxidation of cell membranes and protein oxidation were induced in response to ROS and RNS generation after F3 fraction treatment. Enhancement of these stable peroxidation products could be an essential factor of apoptosis increasing cellular oxidative stress [44, 58, 59].

Mitochondrial membrane potential (??m) is a key parameter for many mitochondrial functions including ion transport, ATP production and ROS generation [48]. On the other hand, disruption of ??m promotes mitochondrial dysfunction, oxidative damage and various apoptotic processes [60]. In the present study, loss of mitochondrial membrane potential (??m) was evaluated in NCI-H358 by using the cationic lipophilic dye, JC-1, which has been widely used to detect alteration of mitochondrial membrane integrity considered as one of the early events of apoptosis. We observed a dose-dependent decrease in ??m after 4 h of treatment with ½ IC₅₀, IC₅₀ and 2 IC₅₀ concentrations of F3 fraction. The alteration of ??m was indicative of the involvement of the mitochondria in the apoptotic processes induced by F3 fraction. Previous studies also reported the involvement of ??m alteration in venom induced mitochondria damage and cell death [14, 43, 46, 47, 52, 53]. Moreover, the collapse of the ??m was attenuated, but not completely abolished, by NAC pretreatment which suggests that F3 fraction induced ??m collapse with further ROS generation resulted from mitochondrial membrane damage. Similar results were obtained with cardiotoxin 3 from the cobra Naya naya atra and Pelagia noctiluca crude venom on neuroblastoma cells [49, 53].

High levels of oxidative damage can be caused by not only oxidative stress, but also by the dysfunction of the cellular repair system, which may modify the cell defense system, provoking cell death [57, 58]. Cellular antioxidant defense systems including SOD, catalase, and GSH may prevent disturbances in ROS homeostasis, or reduce the effect of oxidative stress in cells [44, 57]. Thus, we further investigated the cellular antioxidant defense systems in F3 fraction treated cells by assessment of such antioxidants. Our results showed that F3 fraction significantly reduces the activity of cellular antioxidant enzymes such as SOD and catalase, and significantly depleted intracellular GSH. The alteration in the antioxidant system could reflect the excessive reactive oxygen and nitrogen species overproduction and oxidative damage due to F3 fraction cells treatment. Previous reports also indicated that Odontobuthus doriae venom and plancitoxin I isolated from the venom of crown-of-thorns starfish Acanthaster planci reduce the cellular antioxidant level in response to high oxidative stress [44, 46].

The present findings suggest that the F3 fraction exhibits a potent ability to promote ROS generation in NCI-H358 cells by eliciting oxidative stress and depleting cellular antioxidants (SOD, catalase, GSH) (Fig. 10). This dual property is a promising approach to make the tumor cells more vulnerable to further oxidative stress induced by exogenous ROS-generating agents such as F3 fraction. Currently, several anticancer therapeutic agents are known to stimulate oxidative stress and thus kill tumor cells in a preferential manner [48]. Besides, the F3 fraction-induced mitochondrial dysfunction – as a consequence of excessive ROS production and ??m disruption (Fig. 10) – would modulate the opening of the mitochondrial permeability transition pore resulting in release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space into cytosol, and then trigger to caspase cascades activation and apoptosis [60].