Summary

Natural products

Animal health

Vector control

Case report

Cytotoxicity of Rhopalurus junceus Cuban scorpion venom and its fractions on human tumor cell lines

Alexis Díaz García 1*
Luis Morier Díaz 2
Hermis Rodríguez Sánchez 2
Yamira Caballero Lorenzo

1 Department of Research, Biopharmaceutical and Chemical Production Laboratories. (LABIOFAM).
2 Department of Microbiology, Cell Culture Laboratory, "Pedro Kouri" Institute of Tropical Medicine.
* E-mail: alexisdg@infomed.sld.cu

Scorpion venom has been used in traditional medicine in some countries such as China and India to treat various illnesses. The wider uses include treating seizures, pain and cancer [1,2]. In natural conditions, the venom of the scorpion, is an opalescent fluid, milky white, with a pH 7.12, containing inorganic salts, mucopolysaccharides, enzymes and a variety of proteins including peptides with molecular masses lower than 8 kDa [3 ]. Recent scientific publications recognize the potential of scorpion venom in the treatment of cancer in which it has been observed throughout in vitro studies the inhibition of cell growth and the existence of apoptosis as cell death event [4.5]. In Cuba, the venom of the Rhopalurus junceus scorpion, endemic to the island, has been used for therapeutic purposes since the early nineteenth century, when was expended the so-called "oil of scorpion" which was said to reverse the retention of urine [ 6].However, is was not until the early 80’s in the twentieth century  in Guantanamo, that glimpsed the potential of this scorpion venom, as an anti-tumor agent from empirical studies [7]. To date, the composition of the venom of this scorpion and its effect on tumor cells is not known, so in this work we decided to determine the cytotoxicity of the Rhopalurus junceus scorpion venom and its protein fractions on cultures of tumor and normal human cell lines. 

Materials and methods

Scorpions: The scorpions of the Rhopalurus junceus species used in the experiments are in the escorpionario belonging to the Biological-Pharmaceutical Laboratories (LABIOFAM). Adult scorpions were used which were kept at room temperature and fed with insects, mainly crickets (crickets) and Periplaneta americana (cockroach) provided once a week. 

Venom: The venom was obtained by electrical stimulation of the scorpions’ stings, with periods of at least 21 days without extracting venom. The obtained venom was conveniently diluted in distilled water, centrifuged at 15 000 rpm for 20 minutes for the removal of constituents such as mucus and cellular residues. Finally, the supernatantwas stored in 1.5 mL vials and stored at -20 ° C until use. Previous studies have shown that storage at this temperature does not significantly affect the characteristics of the extract. 

The protein concentration was calculated in all cases by the modified Lowry method [8]. 

Low-pressure liquid chromatography (CLBP): The venom was dissolved in 0.1 M ammonium acetate (NH4Ac) by vortex and centrifuged at 15 000 rpm for 20 minutes. The supernatant was injected onto a Superdex 75 HR 10/30 gel filtration column, with dimensions of 10 x 300 mm implemented on an AKTA FPLC system (Amersham Pharmacia Biotech). The column was equilibrated with 0.1 M of NH4Ac and the material eluted in the same solvent at a flow rate of 0.5 mL / min. The elution of the material was monitored at a wavelength of 280 nm for 72 min. Several chromatographic runsand fractions were performed with similar retention times, collected, merged, dialyzed, and the concentration was calculated as described previously. The separation and collection of venom fractions were performed at room temperature. 

Determination of relative molecular masses:  to determine the relative molecular mass was used a commercial kit (Amersham Pharmacia Biotech) containing: ribonuclease A (13.7 kDa), chymotrypsinogen (25 kDa), ovalbumin (43 kDa), albumin (67 kDa) and blue dextran 2000 and were run under the poison’s same conditions. From retention times and known molecular masses a calibration curve was constructed to determine the relative molecular masses of different protein fractions obtained during the chromatography. 

Polyacrylamide gel electrophoresis: Venom samples and fractions were analyzed in protein electrophoresis under no reducing conditions, in a 16% separating gel and 4% stacking gel and were run in an electrophoresis chamber (Biorad, Mini- PROTEANR ll) [9]. In each well was added an amount equivalent to 50 μgof venom and 10 μgof each fraction. The molecular weight was estimated by intermediate molecular weight markers, in parallel run with the samples. The run was performed at 120 V, 400 mA for 2 hours. The gel was colored with Coomassie Blue G-250 (Sigma) and a distinctive solution was used to visualize the protein bands. 

Cell lines: In the study were used three tumor cell lines: Hela (ATCC CCL-2, human cervical carcinoma), Hep-2 (ATCC CCL-23; human larynx carcinoma), NCI-H292 (ATCC CRL-1848; lung carcinoma) and diploid line MRC-5 (ATCC CCL-171, human lung fibroblasts).The cell lines Hela, Hep-2 and MRC-5 were grown in culture flasks in minimum essential medium (MEM, Sigma), supplemented with 2 mM of glutamine and nonessential amino acids and 10% of  fetal bovine serum (SFB). The NCI-H292 line was grown in RPMI-1640 (Sigma) supplemented with 2 mM of glutamine and 10% of SFB. Each line was incubated in a humid atmosphere at 37 ° C at 5% CO2 until monolayer formation. Each cell line was detached by treatment with 0.25% of a trypsin solution and prepared at a concentration of 2 x 105 cells / mL. 

Cytotoxicity assay: The effect of the venom was detected by the method of Mosmann [10]. The test was performed on polystyrene plates of flat bottom and 96 wells for cell culture (Corning Inc. costar R). In each well was added 50 mL of each cell line and incubated in a 5% CO2 atmosphere at 37 ° C for 24 hours. After this time, 50 mL of culture medium were added for each cell line, previously dissolved with venom to get a final concentration of 0.25, 0.5, 0.75 and 1 mg / mL in each well. The effect of the fractions was evaluated in Hela, NCI-H292 and MRC-5 cells. The final concentration of the fractions was determined from the percent that each one represents in the venom. All cell lines had a final concentration of 104 cells / well and the SFB was used in the medium at a 10%. 

The plates werereincubatedin 5% CO2 atmosphere at 37 ° C for three days. After this time were added 10 mL of a sterile solution of methyl 3 - (4,5-dimethylthiazol-2-yl)-25difenil tetrazolium (MTT) (Sigma) at 5 mg / mL in a sterile phosphate buffersolution in each well and incubated under the same conditions for 3 hours. Finally, 100 L / well were added of dimethyl sulfoxide (DMSO, Sigma) and incubated for 10 minutes. The absorbance was determined in an ELISA microplate reader Dynex Technologies MRX Revelation at a wavelength of 560 nm with 630 nm reference. Each concentration was performed in triplicate and the test was repeated three times. 

The concentration-response curves were obtained by picturing the absorbance versus the different concentrations of venom. Cytotoxicity was expressed as CC50, the concentration of the venom that causes a 50% decrease in the number of viable cells (MTT absorbance) compared with untreated controls. CC50 values of venom and the fractions were determined from the concentration-effect curves (% cell proliferation) through linear regression analysis, using the statistical package GraphPad Prism 4.03 version (GraphPad Software, Inc.). 

Apoptosis:  For the analysis of DNA fragmentation, Hela, Hep-2, and NCI-H292 cell lines, were cultured in 12 wells plates and incubated at 37 ° C, 5% CO2 for 24 hours in a humid atmosphere. After this time, 0.5 mg / mL were applied in each cell line and incubated under the same conditions for 48 hours and were used as controls, wells culturedwith cell lines without venom. The DNA was extracted at 24 and 48 hours, using the WisardR Genomic DNA Purification Kit (Promega) 11purification system. The extracted DNA was run in a submarine electrophoresis at 70 V, 30 mA for 1-2 hours using a 1.2% agarose. 

Statistical analysis: All data were analyzed for normal distribution. The analysis of variance(ANOVA) was performed followed by a Multiple Range Test of Duncan using the statistical package GraphPad Prism 4.03 version (GraphPad Software, Inc.). In all cases the difference was considered significant for p <0.05. 

Results 
Molecular exclusion chromatography: Figure 1 shows the chromatogram of the venom and the fractions obtained. The venom was eluted for 72 minutes at a constant flow rate of 0.5 mL / min monitored at 280 nm. The chromatographic profile showed a range of elutionfrom 19 minutes to 72 minutes. The FI fraction represents the union of proteins from 14 kDa to 70 kDa. The chromatographic profiles had an average of eight peaks. The FII, FIII and FIV fractions showed relative molecular masses of 8 kDa, 4 kDa and <3 kDa, respectively. 

Polyacrylamide gel electrophoresis: Figure 2 shows the electrophoretic pattern of the venom and the fractions obtained by gel filtration chromatography. The electrophoresis showed the presence of two bands identified at 45 kDa, 66 kDa and 29 kDa. The majority band was located below 14 kDa, showing that most of the venom content is made of low weight molecular proteins. The FI fraction showed an electrophoretic pattern similar to the full venom. The fractions F-II, F-III and F-IV showed a single band below 14 kDa, which proves that it corresponds to low weight molecular proteins. 

Cytotoxicity:  The evaluation of the cytotoxic activity of the venom was done in three tumor cell lines and in a human lung diploid line. 

The results showed that the venom affected significantly (p <0.05) the growth of tumor cells compared to the normal cells (Table 1).The fractions obtained were grouped into 4 groups and were evaluated in Hela, NCI-H292 and MRC-5 cell lines. FI turned out to be more toxic to MRC-5 while F-II showed similar levels of sensitivity for the three cell lines. The F-III showed a significant inhibition of tumor cell growth (p <0.05) compared to the normal cells, similar to that observed for the full venom. The F-IV fraction just showed CC50 significantly lower (p <0.05) than normal cells for NCI-H292 cells (Table 1). In all cases (venom, FIII, FIV) the NCI-H292 cell line was the most sensitive.

Table 1. CC50 values of the venom and the fractions for the tested cell lines. The range of retention times, the proportions of each fraction in the venom and the relative molecular weights are presented. Tr: retention time; Ap / At: area of peaks / total area; MR: relative molecular masses obtained from a calibration curve of Tr versus protein molecular mass of known molecular mass * p <0.05 difference statistically significant compared to MRC-5 cell line.

Apoptosis: the induction of apoptosis, due to the effect of the venom, was observed by the pattern of DNA fragmentation (Figure 3).In all three cell lines was observed the event of apoptosis as a death cell mechanism, increasing as the incubation time with the poison increased. This indicates that the higher the incubation time is more cells die as a result of this event. 

The morphological change occurred in the Hela tumor cell line, due to the toxicity of the venom; this is shown in Figure 4. The culture formed a homogeneous monolayer in the control without venom (Figure 4A). The application of venom at low concentrations resulted in loss of typical cell morphology and breakage of the monolayer. The increase in the concentrations of venom caused cellular changes associated with cell elongation and irregularity of the cell borders (Figure 4B). The highest concentrations caused total destruction of the monolayer cell, fusion of cell membranes and formation of large vacuoles by the joint ofthe cytoplasmic contents of various cells and the formation of cincitios. Cells with increasing volume were also observed as well as necrotic cells with pyknosis and cellular residuesin the culture medium (Fig. 4C, 4D).

Figure 4. Effect of Rhopalurus junceus scorpion venom on Hela cell line at different concentrations. A) Cell line without the application of venom. Homogeneous monolayer characteristic morphology. B) 0.5 mg / mL of venom. C) 0.75mg/mL of venom. D) 1 mg / mL of venom.

Discussion 

Throughout this paper was presented the partial characterization of Rhopalurus junceus Cuban scorpion venom. The results of the protein electrophoresis showed the presence of protein bands between 29 kDa and 67 kDa and a band below 14 kDa which corresponds with other works on scorpion venom in which most of its content is of low molecular weight proteins [12.13] 

The total venom and F-III and F-IV fractions showed a significant and differential cytotoxicity to tumor cells. The F-IV fraction only showed significant toxicity to NCI-H292. The present active principles in F-III fraction which cause significant reduction of cell viability in Hela may be at a lower concentration or not present in FIV fraction. On the other hand, the high sensitivity of NCI-H292 cell line in all cases (venom, FIII, FIV) could be due to more than one component capable of acting in the venom of a scorpion on these cells. The FIII and FIV fractions showed relative molecular masses lower than 4 kDa. Scorpion venoms contain proteins with molecular masses below 8 kDa and are considered toxins [13]. The physiological action of these toxins is exerted mainly on ion channels present in the cell membranes of mammals and insects and some are specific to each species [14.15]. In cancer, in some types of tumors, the proliferation, migration and invasiveness are associated with overexpression in cell membrane ion channels [16] and cell receptors [17]. Blocking the physiological activity of these ion channels and receptors inhibits the proliferation and migration of these cells [18]. The high affinity of the venom of some scorpions through these ion channels and the increase in the density of these channels in the membranes of tumor cells facilitates a greater selectivity and therefore an increase in toxicity towards these cells [19]. This feature could be related to the differential toxicity of the venom and F-III and F-IV fractions on the cell lines studied. 

The event of apoptosis was detected in tumor cell lines. The occurrence of DNA degradation, after 24 hours of incubation, in HeLa cell lines indicated that this one is more likely to develop apoptosis. The highest concentrations of the venom caused an increase in cell volume and high pyknosis which may indicate the presence of necrosis. These results are consistent with those obtained by Omran MAA [20] who using the venom of the L. quinquestriatus scorpion on eukaryotic cell lines 293T and C2C12 in different concentrations, observed  that only in a subpopulation of the total number of cells  occurred  the event of  apoptosis whereas in the remaining population other events took place including necrosis. Other authors agree that there is a double road that leads to cell death. An event characterized by apoptosis and the other by necrosis, which is normally masked by the first [21.22]. 

Conclusion 
In this work the venom of the Rhopalurus junceus scorpion showed a significant toxicity on tumor cells of epithelial origin. Additionally, its molecular mass fractions lower than 4 kDa, showed similar cytotoxicity on this cell type. Although this work was not directed to the identification of cellular targets, the fact that there is a close relationship between ion channels and tumor proliferation, metastasis, migration and invasiveness and that peptides present in scorpion venom may suggest that the active principles of the Rhopalurusjunceus venom act the same way. Further studies should be carried out for the identification of active principles and its relationship with ion channels. 

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Figure 1. Chromatographic profile of Rhopalurus junceus scorpion venom separated by CLBP. The fractionation was performed using a Superdex 75 HR 10/30 molecular exclusion column
Figure 1. Chromatographic profile of Rhopalurus junceus scorpion venom separated by CLBP. The fractionation was performed using a Superdex 75 HR 10/30 molecular exclusion column
Figure 2. Protein electrophoresis of the total Rhopalurus junceus  scorpion venom content and fractions. The run was performed at a 16% of separating gel. M) Molecular mass marker (14 kDa-97 kDa). V) Total venom. FI, FII, FIII, FIV) fractions obtained in CLBP.
Figure 2. Protein electrophoresis of the total Rhopalurus junceus scorpion venom content and fractions. The run was performed at a 16% of separating gel. M) Molecular mass marker (14 kDa-97 kDa). V) Total venom. FI, FII, FIII, FIV) fractions obtained in CLBP.
Figure 3. Agarose gel electrophoresis (1.2%) of DNA extracted from tumor cell lines. 1) molecular weight marker Lambda DNA / Hind III. 2-4) DNA Hela cell line; 2: DNA control without venom, 3: DNA extracted at 24hours, 48hours extracted DNA. 5-7) DNA cell line NCI-H292; 2: DNA control without venom, 3: DNA extracted at 24hours, 48hours extracted DNA. 8-10) DNA cell line HEp-2, 2: DNA control without venom, 3: DNA extracted at 24hours, DNA extracted at 48hours.
Figure 3. Agarose gel electrophoresis (1.2%) of DNA extracted from tumor cell lines. 1) molecular weight marker Lambda DNA / Hind III. 2-4) DNA Hela cell line; 2: DNA control without venom, 3: DNA extracted at 24hours, 48hours extracted DNA. 5-7) DNA cell line NCI-H292; 2: DNA control without venom, 3: DNA extracted at 24hours, 48hours extracted DNA. 8-10) DNA cell line HEp-2, 2: DNA control without venom, 3: DNA extracted at 24hours, DNA extracted at 48hours.