Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties

Background Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). Methods The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. Results All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C. gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C. gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells. Conclusion The cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.

The use of genomic and proteomic approaches had permitted a rapid increase in the number of sequences from cnidarians deposited in protein and gene databases [30][31][32]. Some of these toxins have been used for the development of anticancer molecules. One interesting example is the hemolytic toxin (HT) from Stichodactyla helianthus sea anemone which was conjugated with an antibody towards an antigen expressed on immature T lymphocytes (IOR-T6) producing an o-hemolytic hybrid IOR-T6-HT that showed toxicity against CEM cells expressing the IOR-T6 antigen and non-toxic effects for K562 cells without the antigen [33].
Additionally, several marine natural products are able to inhibit the toxic effects of snake venoms, such as extracts from Plocamium braziliense [34], Canistrocarpus cervicornis [35] and seaweed Prasiola crispa [36]. The marine extracts that also inhibit PLA 2 activity include manoalide [37], vidalols, and a group of terpenoids that contain masked 1,4-dicarbonyl moieties. Furthermore, the biotechnological potential of PLA 2 inhibitors may provide therapeutic molecular models that exert antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes [38,39].
This study aimed to evaluate toxicological activities and their efficacy against tumor and snake-venom toxic activities from five Caribbean Sea cnidarian species of the hydrozoa class: Millepora alcicornis, Plexaura homomalla and Cnidarians of the anthozoa class: Condylactis gigantea (total and body wall), Stichodactyla helianthus, and Bartholomea annulata.

Materials and reagents
The synthetic fluorescent substrates Acyl 6:0 NBD phospholipids, NBD-phosphatidylcholine (PC) and NBD-phosphatidic acid (PA) were purchased from Avanti Polar Lipids Inc. (USA). The reagents used in the electrophoresis, salts and other reagents were obtained from Sigma Chemical Company (USA).

Cnidarian extracts
The cnidarians specimens were collected in the coast of Havana City during a one-year period. The extracts of corals were obtained as previously described by [40], whereas anemone extracts were obtained according to [41]. Protein quantitation was based on the Bradford method (BioRad) using bovine serum albumin (BSA) as a standard. Electrophoresis SDS-PAGE 12.5% (m/v) was carried out as previously described [42]. 500 μg samples C. gigantea (body-wall), C. gigantea (total), M. alcicornes, S. helianthus, P. homomalla and B. annulata were pretreated in reducing conditions (SDS plus β-mercaptoethanol) at 100°C for 5 min. Gels were stained with 0.1% Coomassie brilliant blue R-350 in ethanol: acetic acid (5:1, v/v) for 15 min and discolored in 10% acetic acid. The molecular mass was estimated by interpolation from a linear logarithmic plot of relative molecular mass versus distance of migration using standard molar mass markers (SDS7 Sigma-Aldrich).

Phospholipase activity
The Phospholipase A 2 (PLA 2 ) activity was measured using the indirect hemolytic assay on agarose gels containing red blood cells and egg yolk phospholipids [43]. The hemolytic activity was evaluated spectrophotometrically using suspensions of fresh human RBC (red blood cells) as previously described [44,45]. PLA 2 activity was evaluated also through the hydrolysis of synthetic fluorescent phospholipid, using the fluorescent substrate Acyl 6:0 NBD phospholipid, NBDphosphatidylcholine (NBD-PC). The assay was perfor med using a spectrofluorimeter (Shimadzu, RF-5301PC, software RFPC) with excitation and emission wavelengths of 460 and 534 nm, respectively. The enzymatic activity of each cnidarian extract was evaluated for 250 s after the addition of substrate (3.3 μg/mL, final concentration) in a reaction medium containing 50 mM Tris-HCl, and 8 mM CaCl 2 , pH 7.5, at room temperature.

Proteolytic activity assay
Proteolytic activity upon fibrinogen was measured as described by [46] with some modifications. Fibrinogen (70 μg) diluted in PBS was incubated with different amounts of cnidarian extracts diluted in 20 μL buffer (pH 7.5) at 37°C for 2 h. The reaction was stopped with 20 μL of a solution containing 10% (v/v) glycerol, 10% (v/v) β-mercaptoethanol, 2% (v/v) SDS, and 0.05% (w/v) bromophenol blue. Fibrinogen hydrolysis was demonstrated by SDS-PAGE using 12% polyacrylamide gels. Proteolytic activity upon casein was measured as described by [47]. Cnidarian extracts (100 and 500 μg) were incubated for 30 min at 37°C in a solution of 0.1 M Tris-HCl pH 9.0 containing 1% casein. After the incubation period, 1.5 mL of 30% TCA was added to each sample to stop the enzymatic reaction and centrifuged at 340 x g for 25 min. Then, the samples were read on a spectrophotometer at a wavelength of 280 nm. One unit of protease activity was defined as the amount of enzyme that produces an increase in absorbance of 0.001 units/minute at 280 nm.

Hemorrhagic activity assay
Hemorrhagic activity was quantitatively estimated by the method of [48] with some modifications. Groups of six Swiss mice (18-22 g) were shaved on the back and then intradermally (i.d.) injected with different doses of cnidarians extracts or snake venoms, in 50 μL of phosphate buffered saline (PBS). After 2 h, animals were anesthetized and euthanized. The shaved back skin was removed and the hemorrhagic halo diameter was measured. The minimum hemorrhagic dose (MHD) was obtained from the mean of these diameters (mm). The MHD is defined as the dose of snake venom or extract that produces a hemorrhagic lesion of 10 mm diameter after 2 h.

Coagulant activity assay
The clotting time was determined by mixing 20 μL of the samples (in 0.15 M NaCl, pH 7.4) with 200 μL of citrated bovine plasma at 37°C. The B. jararacussu snake venom (20 μg) was assayed in order to determine the minimum coagulant dose (corresponding to the time between 1 and 1.2 s -100% activity). For the neutralization trials, the snake venom was previously incubated with different cnidarian extracts for 30 min at 37°C, at different proportions (1:5, 1:10 and 1:30, w/w).

Neuromuscular blocking
Mice were euthanized by exsanguination after previous cervical dislocation. Phrenic-diaphragm (PD) preparation was removed and mounted vertically in a conventional isolated organ-bath chamber containing 15 mL of physiological solution of the following composition (mmol/L): NaCl, 135; KCl, 5; MgCl 2 , 1; CaCl 2 , 2; NaHCO 3 , 15; Na 2 HPO 4 , 1; glucose, 11. This solution was bubbled with carbogen (95% O 2 and 5% CO 2 ). The preparation was attached to an isometric force transducer (Grass, FT03) for recording the twitch tension. The transducer signal output was amplified and recorded on a computer via a transducer signal conditioner (Gould, 13-6615-50) with an Acquire Lab Data Acquisition System (Gould). The resting tension was 5 g; indirect contractions were stimulated by supramaximal pulses (0.2 Hz, 0.5 ms) delivered from an electronic stimulator (Grass-S88 K) and applied to the phrenic nerve by means of a suction electrode. The preparation was allowed to stabilize for 45 min before the addition of a single concentration of toxin [49].

Muscle-damaging activity Morphological analysis
At the end of the myographic study, the diaphragm muscle was removed from the bath and frozen in liquid nitrogen. Transverse sections (8 mm thick) were cut out at − 20°C in a cryostat and stained with hematoxylin and eosin (HE) prior to examination by light microscopy [50]. Muscle damage was quantified in HE stained preparations, using an Analysis Imaging System (Leica, Qwin). The number of fibers with lesions was expressed as a percentage of the total number of cells (muscle damage index), in three non-overlapping non-adjacent areas of each muscle, observed at the same magnification.

Creatine kinase release
The creatine kinase (CK) assay was carried out using the CK-UV kinetic kit from Sigma Chem. Co. Different cnidarian extracts were injected (i.m., 50 μL) into Swiss male mice weighing 18-22 g (n = 6). The control animals received 0.15 M PBS. After 3 h, the blood from the tail was collected in heparin-coated tubes and centrifuged for plasma separation. The amount of CK was then determined using 4 μL of plasma, which was incubated for 3 min at 37°C with 1.0 mL of the reagent. Enzyme activity was expressed in international units per liter (IU/L), with one unit of activity corresponding to phosphorylation of 1 μmol of creatine/min at 25°C.

Edema inducing activity
Groups of six Swiss male mice (18-22 g) were injected in the sub plantar region with different doses of cnidarian extracts in 50 μL of PBS. After 0.5, 1 and 3 h, the paw edema was measured using a low-pressure spring caliper (Mytutoyo-japan) [51,52]. The zero time values were then subtracted and the differences reported as median % ± S.D.

Cytotoxic activity
Tumor cell cytotoxic activity of cnidarian extracts on human acute T-cell leukemia (Jurkat) and B16F10 cell lines were assayed using the MTT method according to [53]. Cells were dispersed in 96-well plates at a density of 1 × 10 5 cells per well. After 24 h of culture, the media were removed and fresh media, with or without different concentrations of samples, were added into the wells and incubated for 24 h. The extracts were evaluated at 1000, 100 and 10 μg/mL concentrations using Vincristine as positive control (100 μg/mL). Results were expressed as a percentage (%).

Statistical analysis
Results were expressed as mean ± S.D. Data was analyzed by ANOVA complemented by the Tukey-Kramer test, using the statistical program GraphPad 5.0. Values of p < 0.05 were considered significant.

Results and discussion
The SDS-PAGE analysis in denaturing conditions of the cnidarian extracts showed the difference between extracts of C. gigantea (body-wall), C. gigantea (total), S. helianthus, B. annulata, M. alcicornes and P. homomalla. Considering that the extracts were obtained from the entire organism, it should be noted that the anatomy of M. alcicornes and P. homomalla is different from that of anemones. For this reason, the method of protein extraction must be differentiated for these organisms. Thus, it is possible that in the 500 μg extract applied to the electrophoresis, a low protein yield made it impossible to visualize bands on the polyacrylamide gel (Fig. 1a).
The extracts of S. helianthus and C. gigantea (bodywall) hydrolyzed the NBD-PC substrate that is specific for the PLA 2 enzymes, which was not observed for the other extracts tested (Fig. 2b). Martins and coworkers [54] isolated a PLA 2 (CgPLA2) composed of 14 kDa dimers and 29 kDa monomer from the C. gigantea extract.
Another study reported showed that Sticholysin I and II, two 19 kDa pore-forming cytokines, present in the S. helianthus extract, low activity against specific substrates for phospholipase activity when tested alone [55].
However, fractions enriched with these two molecules together demonstrated a significant increase in phospholipase activity [56]. Although CgPLA 2 and Sticholysin I and II are known to exert phospholipase activity, it should be emphasized that the experiments were carried out with total extract. It is possible that these molecules are responsible for the hydrolysis promoted against the NBD-PC substrate, but we do not rule out the existence of other molecules that are components of the extract, which alone or in clusters may be acting in the hydrolysis of the NBD-PC substrate.
The extracts of C. gigantea (body wall) and B. annulata were able to partially hydrolyze the α and β chains of fibrinogen; however, S. helianthus, M. alcicornis and P. homomalla were incapable of hydrolyze fibrinogen efficiently (Fig. 1b). The fibrinogenolytic assay was carried out using 50 μg of the cnidarian extracts, whose proteolytic activity upon casein was evaluated; furthermore, the extract of B. annulata (100 μg) hydrolyzed casein at 80 U/min. The extracts of C. gigantea (body wall) and B. annulata at 500 μg hydrolyzed casein at 118 and 170 U/ min, respectively (Fig. 2c).
As to the hemorrhagic effect, extracts of M. alcicornis and P. homomalla induced bleeding at a concentration of 150 μg (Fig.4a) and inhibited bleeding induced by Bothrops snake venom. All cnidarians extracts tested inhibited the hemorrhagic activity induced by B. moojeni venom at a ratio of 1:30 w/w, showing approximately 40% inhibition in the presence of extracts of C. gigantea (body wall), P. homomalla and M. alcicornis (Fig.4c). Interestingly, the B. neuwiedi venom extracts did not inhibit hemorrhage (Fig. 4b).
Coagulant activity was not induced by cnidarian extracts. However, all cnidarian extracts except M. alcicornis inhibited the coagulant activity induced by B. jararacussu venom at the ratios of 1:5 and 1:10 w/w. In this assay, the B. annulata extract at a 1:30 w/w ratio showed the greatest ability to delay the clotting time of citrated plasma after the addition of B. jararacussu venom for more than 40 min (Table 1). The other extracts tested at the 1:30 w/w ratio presented lower inhibitory effect (Fig. 5). Additionally, C. gigantea body-wall extract at a concentration of 200 μg/mL were  able to inhibit thrombin-induced coagulation. This activity can be produced by the presence of protease inhibitors, as reported by [64], who showed the presence of protease inhibitory activity in the extract from the marine sponge Xetospongia muta (Poriphera) and in the sea anemones B. granulifera and B. annulata. According to these authors, this activity was dose-dependent and the molecule responsible for the inhibition had a low molecular weight. Di Bari and coworkers [65] also described the presence of protease inhibitors in aqueous extracts of marine sponges; all extracts were able to inhibit the activity and expression of matrix metalloproteinases (MMP-2 and MMP-9) in mice astrocyte culture. At 3 h after the injection of 50 μg of cnidarian extracts into the gastrocnemius mouse muscle, a slight myotoxic effect from the extracts of M. alcicornis and P. homomalla was observed, approximately 22% above those observed for the controls injected with PBS alone. The C.
gigantea (body-wall), C. gigantea (total) and S. helianthus presented increased activity, while extract of B. annulata did not show myotoxic effect.
Some studies have demonstrated the toxic effects of marine animals such as coral Millepora alcicornis, which causes systemic reactions in the kidney, lung and liver [8], and Millepora complanata, which presents non-peptide toxins highly lethal to mice with LD 50 of 4.62 μg/g body weight [66]. The other cnidarian extracts tested, C. gigantea (body-wall), C. gigantea (total) and S. helianthus, showed a more pronounced myotoxic effect, smaller only compared with to those induced by B. jararacussu venom and the myotoxin BthTX-I, which are highly myotoxic (Fig. 6a). The edema induction was observed only in the presence of extracts from P. homomalla and S. helianthus (Fig. 6b).   In isolated neuromuscular preparation, extract from S. helianthus (100 μg/mL) induced a time-dependent blockage of indirect twitches (Fig. 7). After 300 min, the twitch amplitude was reduced by about 84%. In contrast, at the same concentration, extracts from C. gigantea (body wall) and C. gigantea (total) did not affect the indirectly evoked twitches. Morphological and morphometric analyses revealed an absence of significant damage in diaphragm muscles exposed to S. helianthus and C. gigantea total extracts (Fig. 8). On the other hand, a slight, but significant level of damage was observed in muscles exposed to C. gigantea body wall extract (Fig. 8). Fig. 7 Effects of S. helianthus extract (100 μg/mL) upon indirect evoked twitches on mouse phrenic-diaphragm preparation. The ordinate represents the % amplitude of twitches relative to the initial amplitude. The abscissa indicates the time (min) after the addition of the extract to the organ bath. Vertical bars represent the SEM; *indicates the point from which there are significant differences relative to control (p < 0.05) Fig. 8 Light micrographs of mouse diaphragm muscles submitted to hematoxylin and eosin staining. Control muscle (a) and muscles exposed to extracts of C. gigantea (body-wall) (b), C. gigantea (total) (c) and S. helianthus (d). Note the general normal appearance of fibers with polygonal aspect (f) and endomysium (en). Muscle damage index of (C) and (D) (4.3 ± 1.3, n = 5 and 5.5 ± 1.1, n = 4, respectively) were not significantly different from that of (A) (2.0 ± 0.4, n = 5). However, muscle damage of B (5.9 ± 0.8, n = 5) was significantly higher than that of A. (ANOVA, p ≤ 0.05). The remaining cnidarian extracts did not show such activity The cytotoxic activity of cnidarian extracts upon the human tumor cell lines JURKAT (leukemia T) and B16F10 (melanoma) (Fig. 9a and b) showed that all extracts presented anti-tumor activity against JURKAT cells, except the extract B. annulata. Additionally, extracts of C. gigantea (body-wall) and S. helianthus showed significant anti-tumor activity at concentrations of 1000 μg/mL, of which the latter still showed cytotoxic activity of 50% even at the lowest concentration evaluated (10 μg/mL). As to their activity on B16F10 cells, only the extract of C. gigantea (body wall) and M. alcicornis showed anti-tumor activity at concentrations of 1000 and 100 μg/mL. Some authors proposed that this cytotoxic activity on tumor cell lines is associated with the induction of apoptosis considering the fact that some enzymes isolated from animals exhibit hydrolytic activity by altering the cell membrane [67]. Another study reported the cytotoxic effect of extracts from the marine sponge Polymastia janeirensis on a human glioma line (U138MG); in the experiment, both aqueous and organic extracts induced cell death by apoptosis and necrosis [68]. Similar results with extracts from the sponge Hyattella cribriformis in ethyl acetate, which exhibited potent growth inhibition of tumor cells as sarcoma, ovarian cancer, colon and breast cell lines [69].

Conclusions
The neutralization of the clotting induced by B. jararacussu snake venom and the inhibition of the hemorrhagic activity induced by B. moojeni venom were demonstrated by the majority of the cnidarian extracts tested, whereas the ability to inhibit thrombin-induced coagulation was shown by the C. gigantea (body wall). Together with the anti-tumor effect against JURKAT cells demonstrated by all cnidarian extracts tested and the specificity shown against B16F10 cells, these findings constitute important evidence that cnidarians extracts are a rich source of bioactive molecules that should be studied in order to produce data for the development of new alternatives for snakebite envenomation and cancer therapies.

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the authors (CSO, CASC, RDS, DLR, SM), upon reasonable request.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.