Purified Lesser weever fish venom (Trachinus vipera) induces eryptosis, apoptosis and cell cycle arrest

Accidents caused by the sting of Trachinus vipera (known as Lesser weever fish) are relatively common in shallow waters of the Mediterranean. Symptoms after the sting vary from severe pain to edema or even tissue necrosis in some cases. Here we show that purified Lesser weever fish venom induces eryptosis, the suicidal erythrocyte death, and apoptosis of human colon carcinoma cells. The venom leads to erythrocyte shrinkage, phosphatidylserine translocation and increased intracellular Ca2+, events typical for eryptosis. According to mitochondrial staining cancer cells dyed after the activation of the intrinsic apoptotic pathway. Trachinus vipera venom further causes cell cycle arrest.

Scientific RepoRts | 6:39288 | DOI: 10.1038/srep39288 phosphatidylserine translocation to the cell surface 16 . To this end, erythrocytes were incubated for 48 h in Ringer solution without or with Trachinus vipera venom (10-500 μ g/ml). In order to estimate the alterations of cell volume, forward scatter was determined in flow cytometry and as illustrated in (Fig. 1A,B), the exposure to the venom was followed by a significant decrease of forward scatter (at 500 μ g/ml). Accordingly, venom Erythrocytes were maintained in Ringer solution followed by treatment or not for 48 h with 10 to 500 μ g/ml of venom. The forward scatter of erythrocytes was estimated by flow cytometry. (A) Illustrates representative dot plots (control was labeled in green and 500 μ g/ml venom in red), while (B) report quantitative data. Data are reported as means ± SEM (n = 9). (C,D) Effect on phosphatidylserine exposure. Erythrocytes (control and treated ones) was labeled with annexin-V for the assessment of apoptosis-associated parameters (phosphatidylserine exposure). (B) Illustrates representative dot plots (control was labeled in green and 500 μ g/ml venom in red), while (C) report quantitative data. Data are reported as means ± SEM (n = 9). (E,F) Effect of venom on erythrocyte Ca 2+ activity. Erythrocytes (control and treated ones) was labeled with Fluo3-AM for the assessment of erythrocyte cytosolic Ca 2+ concentration. (E) Illustrates representative dot plots (control was labeled in green and 500 μ g/ml venom in red), while (F) report quantitative data. Data are reported as means ± SEM (n = 9). ***(p < 0.001) indicate significant difference as compared with non-treated erythrocyte (ANOVA). administration was followed by erythrocytes shrinkage. Phosphatidylserine exposing erythrocytes were identified utilizing annexin-V-binding and as shown in (Fig. 1C,D), at 48 h, the percentage of annexin-V-binding erythrocytes increased particularly at 500 μ g/ml. Thus, venom administration led to erythrocyte cell membrane scrambling with translocation of phosphatidylserine to the cell surface. Since both, cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface are stimulated by increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ), further experiments estimated the effect of Trachinus vipera venom on [Ca 2+ ] i . To this end, erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence was determined by flow cytometry. The exposure of the erythrocytes to venom was followed by an increase of Fluo3 fluorescence at 500 μ g/ml (Fig. 1E,F). Consequently, the venom increased the concentration of cytosolic Ca 2+ . These findings disclose that Lesser weever fish venom triggers eryptosis.

The Trachinus vipera venom kills HCT116 cells in vitro.
To investigate the effect of Trachinus vipera purified venom on cells in vitro, we first tested different concentrations on HCT116 cells using the WST-1 viability assay. Cells were seeded in the 96 well plates and then treated with the Lesser weever fish venom (50, 100, 500 and 1000 μ g/ml) for 24, 48 and 72 h. HCT116 cells displayed a growth inhibition in response to the treatment at 72 h and with an IC 50 of 460 + /− 40 μ g/ml of Trachinus vipera venom ( Fig. 2A). In a separate series, a cells count was performed. Cells were seeded on coverslips glasses and treated. After 72 h cells were fixed and nuclei were stained with Hoechst. The nuclei count using image J showed a similar result to WST-1 test, i.e. 500 μ g/ml treated wells/cells contained (after wash and fixation) about half of the cells number counted in control condition (Fig. 2B). Consistent with these results, 500 μ g/ml (and more with 1000 μ g/ml) reduced the clonogenic potential of HCT116 cells, as determined in clonogenic assays, a test reflecting long term effect/toxicity 17 (Fig. 2C,D). These results confirm that the venom of Trachinus vipera starting at 72 h/500 μ g/ml exert toxic and anti-proliferative effects on cancer cells. Mechanisms underlying cell death induced by Trachinus vipera venom. To understand how Lesser weever fish purified venom induces HCT116 cells mortality, we performed a cells co-staining with the vital dye propidium iodide (PI) and the mitochondrial transmembrane potential (Δ ψ m ) sensor DiOC 6 (3). Indeed, the PI cell incorporation shows loss of cell membrane integrity and in consequence cell death while loss of mitochondrial transmembrane potential is a sign of early stage apoptosis. The combination of these two parameters is an indication of cell death 18 . The frequency of dying (DiOC 6 (3) low PI low ) and dead (DiOC 6 (3) low PI high ) cells analyzed by flow cytometry was markedly increased among venom treated cells to reach about 32% at 500 μ g/ml of venom. The pre-apoptotic fraction (DiOC 6 (3) low ) constitutes 75% of the total death. (Fig. 3A,B). We confirmed the FACS data with microscopy. Indeed, living labeled cells with the MitoTracker ® Orange CMTMRos (to show the loss of mitochondrial transmembrane potential on microscope) were co-stained with Hoechst to visualize the nucleus after fixation and the loss of granularity was evaluated 19 . Treated cells lost about 10 to 20% of granularity, which is corresponding to the proportion of cells that entered early apoptosis (Fig. 3C,D). Moreover, the analysis of the cell cycle profile, showed an appearance of a sub-diploid population (Sub G 1 ) corresponding to apoptotic bodies 20 in the treated compared to control cells (Fig. 3E,F). Altogether, the marked loss of mitochondrial transmembrane potential and the accumulation of apoptotic bodies in treated cells suggest that the Trachinus vipera venom induces death after activation of the mitochondrial pathway of apoptosis.
Trachinus vipera venom cause cell cycle arrest. We further investigated the cell cycle perturbation after treatment with the fish venom. Cells were treated with the purified venom (500 μ g/ml) and analyzed compared to non-treated cells using flow cytometry. A clear cycle arrest on G 1 phase of treated cells was noted. Indeed, the profile showed an accumulation of 20% more cells in G 1 at 72 h of treatment with venom ( Fig. 4A,B). Moreover, in response to treatment, cells exhibited a marked increase (more than twice) of cyclin E protein abundance (the cyclin E accumulated in G 1 phase and early S phase) (Fig. 4C,D). On the other hand, the incorporation of the thymidine analog 5-ethynyl-20-deoxyuridine (EdU) into DNA was significantly reduced by the treatment, inducing decrease in cycling and duplicating (Fig. 4E,F). At the same time, the mitotic index was estimated with or without treatment. The simple count of mitotic cells with microscopy showed a decrease in the percentage of mitosis (about 25% comparing to control) (Fig. 5A,B). Also, the treated cells presented a decline in cyclin B1 (the cyclin associated with the G 2 /M phase) (Fig. 5C) as well as the mitotic-specific phosphoepitope MPM2 (a specific marker of mitotic entry) (Fig. 5D). The phosphorylation of histone H3, another indicator of ongoing mitosis, is also reduced in treated compared to control cells (Fig. 5E,F). Collectively, the data suggest cell cycle arrest and accumulation of treated HCT116 cells in an interphasic stage.

Discussion
Driven by the incidence and pattern of injuries caused by Trachinus vipera venom, we decided to investigate the pharmacological potential of the Lesser weever fish venom in two models that are erythrocytes and Colon Carcinoma HCT116 cells. Fish specimens were collected and the venom was extracted from the dorsal fins by mechanical techniques and dialysis (Fig. S1). The venom induced apoptosis of human colon carcinoma HCT116 cells. According to the WST-1 test, cell count and Clonogenic assays, Lesser weever fish venom compromizes the survival of HCT116 cancer cells. The WST-1 assay detects the production of formazan activity and thus shed light on the metabolism of the cells 21 . Moreover, cell count and Clonogenic assay demonstrate the potential of the venom to arrest cell proliferation (Fig. 2). The venom clearly provokes apoptosis. There are two main pathways that lead to apoptosis: the death receptor pathway initiated by tumor necrosis factor receptors which is the extrinsic pathway 22 and the mitochondrial or the intrinsic pathway, which involves mitochondria and Bcl-2 family members 23 . The intrinsic pathway is initiated by loss of the mitochondrial transmembrane potential which leads to opening of the mitochondrial permeability transition pores and release of effectors including cytochrome c and apoptosis inducing factor from the mitochondria into the cytosol. Cytochrome c triggers the proteolytic activity of caspase-3 and caspase-9 in the cytosol, then the activation of caspases degrade (poly [ADP-ribose] polymerase PARP) and caspase-activated-DNase, which initiates DNA degradation 24 . Our findings, in HCT116, demonstrated that the Lesser weever fish venom kills cells by the activation of the intrinsic pathway of apoptosis (loss of mitochondrial transmembrane potential and membrane integrity and the degradation of nucleus) (Fig. 3). The effect of the purified venom was also tested on erythrocytes. Red blood cells (erythrocytes) lack nuclei and mitochondria, and as consequence should be insensitive to the suicide death provoked by the mitochondrial pathway of apoptosis 25 . However, erythrocytes can undergo suicide via another pathway. This particular death is baptized eryptosis, characterized by cell shrinkage, blebbing and cell membrane scrambling with phosphatidylserine translocation to the cell surface. Eryptosis can be induced by ceramide 26 , oxidative stress 16 , energy depletion 16 , as well as stimulation of some kinases including casein kinase 1α , Janus-activated kinase JAK3, protein kinase C, and/ or p38 kinase 16 . Another important trigger of eryptosis is activation of caspases. Indeed, despite the inability of red blood cells to synthesize proteins, they contain functional apoptotic caspases, in particular caspase 3 [27][28][29][30] . Moreover, erythrocytes contain Fas, FasL, Fas-associated death domain FADD and the active caspase 8 27,28 . Eryptosis is induced by the activation of Fas-signaling complex (binding of transmembrane FasL to its receptor Fas or the association between FADD and Fas) in upstream of caspase 8 activation [31][32][33] . However, the main signaling pathway documented as inducer of eryptosis is increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ) 16 . Calcium can directly cause the phosphatidylserine membrane translocation (cell scrambling) 34 . Moreover, calcium can activate K + channels with an efflux of KCl from the cell causing cell shrinkage 35 . In addition, increased cytosolic concentration of calcium is followed by activation of the cysteine endopeptidase calpain, which degrades membrane proteins and causes cell membrane blebbing 36 . Thus, size and granularity of erythrocytes were evaluated, the membrane phosphatidylserine exposure and the concentration of the cytosolic Ca 2+ were assessed and we found that the treatment induces eryptosis, the suicidal erythrocyte death (Fig. 1). In our study, we demonstrated also that treated human carcinoma cells HCT116 undergo cell cycle arrest. Indeed, the control of the progression of the cell cycle of cancer cells is an effective strategy for cancer therapy because deregulated cell-cycle control is a fundamental aspect of cancer for many common malignancies 37 . The findings outlined previously indicate that treated cells remain in G 1 phase and exhibit a high level of Cyclin E, which is the Cyclin accumulated in late G 1 phase of the cycle 38 . Also, the incorporation of thymidine analogue EdU dramatically decreased, which is a sign of the arrest of DNA duplication and thus disruption of cell proliferation 20 . Furthermore, after treatment, the amount of Cyclin B1 decreased, which is the Cyclin accumulated in late phase S and mitosis 39 . As well, the phosphorylation of the two epitopes MPM2 and pH3, that are markers of mitosis 40 , declined leading to a large reduction of the mitotic cells (Figs 4 and 5).
Presumably, piscine venoms generally possess few toxins among their compounds which are active 3 . Weever fish venom is a complex mixture of many substances including several peptides, proteins of high molecular weight such as kinin or kinin like substances, serotonin, adrenaline, noradrenaline, histamine, and several enzymes with a wide spectrum of biological activities 2,41 . The composition of Trachinus Draco venom is very  This is the first report evaluating the semi-purified venom of Trachinus vipera in vitro on erythrocytes and HCT116 cells. The venom induces eryptosis characterized by cell shrinkage and phosphatidylserine translocation to the cell surface and Ca 2+ entry. Moreover, Lesser weever fish venom provokes cells cycle arrest and apoptosis on HCT116 cells. Further studies must be conducted to identify the different components of this venom and their chemical structures.

Materials and Methods
Fish specimens. Specimens of Lesser weever (Trachinus vipera) were provided by the same fisherman and wholesaler from Kelibia port in the Cap Bon coast which is located North-East of Tunisia (36°51′ N-11°05′ E). Fishes were collected, identified, and shock-frozen and kept in − 20 °C for venom extraction 14 . We do confirm that all experiments were performed in accordance with relevant guidelines and regulations.
Venom extraction and dialysis. The venom was extracted from the dorsal fins of fish. To that end, fins were crushed 10 minutes in a mixer, and then proceeded to sonication at a frequency of 47 kHz during 30 min at 20 °C. Sonication helped to burst the lipid bilayer and to release the cells contents. Afterwards, the crushed mixture was centrifuged at 5000 rpm/10 min/4 °C. The supernatant was collected for dialysis against MilliQ water using dialysis cellulose membrane of 8 kDa cutoff. The dialysate was filtered through 0,22 μ m, then lyophilized and stored at − 20 °C until further use. For the simultaneous quantification of plasma membrane integrity and mitochondrial transmembrane potential (Δ ψ m), cells were collected and stained with 1 μ g/mL propidium iodide (PI), which only incorporates into dead cells, and 40 nM 3,3′ -dihexyloxacarbocyanine iodide (DiOC 6 (3), a Δ ψ m-sensitive dye (Molecular Probes-Invitrogen), for 30 min at 37 °C. For the assessment of cell cycle distribution, cells were collected, washed once with 0.1% (w/v) D-glucose (Sigma-Aldrich) in PBS and then fixed by gentle vortexing in ice-cold 75% (v/v) ethanol (Carlo Erba Reagents) for 30 sec. After overnight incubation at − 20 °C, samples were centrifuged and PBS washed to remove ethanol and stained with 50 μ g/mL PI in 0.1% (w/v) D-glucose in PBS supplemented with 1 μ g/mL (w/v) RNase A (Sigma-Aldrich) for 30 min at 37 °C. Afterwards, samples were incubated for at least 2 h at 4 °C before cytofluorometric analysis. For the simultaneous measurement of DNA content and histone H3 phosphorylation or cyclin E 1 levels, cells were fixed with 75% (v/v) ethanol in PBS, permeabilized with 0.25% (v/v) Tween 20 in PBS and co-stained with 10 μ M 4′ ,6-diamidino-2-phenylindole DAPI (Molecular Probes-Invitrogen) and a rabbit antiserum specific for phosphorylated histone H3 (rabbit polyclonal IgG1 #06-570, Millipore-Chemicon International), or a mouse antiserum specific for Cyclin E (mouse monoclonal IgG1 #551159, BD Biosciences). For the EdU assay, cells were incubated with 10 μ M EdU for 30 min at 37 °C, fixed, permeabilized and stained with the Fluorescent dye azide (Click-iT TM reaction cocktail, from Invitrogen) and DAPI according to the manufacturer's instructions. Cytofluorometric acquisitions were performed by means of a FACSCalibur (BD Biosciences) or a FACScan (BD Biosciences) cytofluorometer equipped with a 70 μ m nozzle or with a Gallios cytofluorometer (Beckman Coulter).

Erythrocytes, cell lines and culture conditions.
WST-1 cell viability assay. The effect of the Trachinus vipera purified venom was determined by the use of WST-1 assay. HCT116 cells were seeded in 96-well plates (5000 cells/well). 24 hours later, cells were treated with different concentrations of the venom (50, 100, 500 and 1000 μ g/ml) diluted in phenol red-free media (100 μ l). After 24 h, 48 h and 72 h WST-1 assay reagent (Roche Applied Science, Mannheim, Germany) was subsequently added (10 μ l) to each well and cells were incubated for 4 hours at 37 °C before the absorbance lecture. Each well was measured at the wavelength of 450 nm and reference wavelength of 690 nm, using a scanning multiwell spectrophotometer (Synergy 2). Statistics were calculated using Student's t-test assuming unequal variances and the mean ± SEM is presented. Each experiment was performed in triplicate and repeated three times. Immunofluorescence. Immunofluorescence microscopy was performed according to conventional procedures. Briefly, cells were fixed with methanol 100% for 10 minutes at − 20 °C and then washed with PBS before nucleus staining with a dilution of Hoechst 33342 (1/5000 in PBS) or 4′ ,6′ -diamidino-2-phenylindole (DAPI, 1/5000 in PBS) 10 minutes at ambient temperature. For staining mitochondria, cells were labeled during 30 minutes with the MitoTracker Orange CMTMRos (Invitrogen) (1/1000) then fixed and stained with Hoechst to visualize the nucleus. Microphotographs with the objective 20 and 40 were taken for analyses and count. Images were analyzed with the open source software Image J (freely available from the National Institute of Health, Bethesda, MD, USA at the address http://rsb.info.nih.gov/ij/).

Clonogenic survival assays.
To evaluate clonogenic survival, HCT116 cells were seeded at two different concentrations (100 and 200 cells/well) in 24-well plates. Twenty-four hours later, cells were treated with different concentrations of the Trachinus vipera purified venom (50, 100, 500 and 1000 μ g/ml) for one week. Colonies were then fixed/stained with an aqueous solution containing 0.25% (w/v) crystal violet, 70% (v/v) methanol and 3% (v/v) formaldehyde (Carlo Erba Reagents) and counted. Only colonies made of > 30 cells were included in the quantification. For each treatment, the survival fraction (SF) was estimated according to the formula: SF = number of colonies formed/number of cells seeded.