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\title{Cytotoxic Effect of Rhopalurus junceus Scorpion Venoms on the HeLa Cell Line}
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             \author[1]{Arianna  Yglesias-Rivera}

             \author[2]{Arlett Rubio  Suárez}

             \author[3]{Jose Raul  Torres-Viltres}

             \author[4]{Rayner  Ochoa-Cardentey}

             \author[5]{Hermis  Rodríguez-Sánchez}

             \author[6]{Georgenis  García-Oliva}

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\date{\small \em Received: 13 December 2019 Accepted: 31 December 2019 Published: 15 January 2020}

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\begin{abstract}
        


Introduction: Venom composition in both sexes of Rhopalurus junceus scorpion has the same major components but it is different in many compounds. However, it is unknown whether the sex of this scorpion influences in its biological activity.Objective: To compare the cytotoxic effect of this scorpion from both sexes on the HeLa tumor cells.Materials and Methods: Protein content and gel image were analyzed by SDS-PAGE and Image J 1.46 software, respectively. HeLa and Vero cells were treated with each sex and a mixture of both sexes of this scorpion venom. Percentage of cell viability and the morphological changes were determined by MTT assay and phase contrast microscopy, respectively.

\end{abstract}


\keywords{Rhopalurus junceus scorpion venom, female, male, mixture of two sexes, heLa.}

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\section[{Introduction}]{Introduction}\par
The scorpion venom is a highly complex and heterogeneous mixture of compounds, mainly proteins and peptides \hyperref[b0]{(Ahmadi et al., 2020)}. Around 2000 scorpion species have been described and only 30 species of the Buthidae family are considered dangerous to humans (Desales-Salazar et al., 2020). In a study carried out with two scorpions from Buthidae family: Androctonus finitimus and Hottentota tumulus, it was shown that the quantity and quality of extracted venom were associated with temperature, diet and the extraction method \hyperref[b12]{(Tobassum et al., 2018)}. Besides, venom composition can be influenced by different factors like sex, geographical location, age, time intervals of extraction and others \hyperref[b13]{(Pucca et al., 2014)}. For example, males and females of Tityus nororientalis scorpions produce venoms with different composition and activity (De \hyperref[b3]{Sousa et al., 2010)}. venom decreases the viability of tumor cells of epithelial origin and has no cytotoxic effect on normal cells (Díaz- \hyperref[b4]{García et al., 2013)}. In a proteomic comparative analysis of male and female R. junceus scorpion venom, from their 200 components just 63 were common and the most abundant component appeared in both sexes \hyperref[b11]{(Rodríguez-Ravelo et al., 2015)}. Previous studies by our group, using a mixture of venom of female and male scorpions in the same proportion and similar laboratory conditions have demonstrated their cytotoxic and apoptotic effect against cancer cells (Díaz-García et al., 2013; Díaz-García et al., 2015; Díaz-García et al., 2017; Yglesias-Rivera et al., 2019). Considering the described scenario, the objective of the present study was determine if there are differences among R. junceus scorpion venoms from female and males individually and mixture. 
\section[{II.}]{II.} 
\section[{Methods}]{Methods} 
\section[{a) Scorpion Venom source}]{a) Scorpion Venom source}\par
Female and male adults of Rhopalurus junceus scorpions, collected in Isla de la Juventud (Cuba), were kept in captivity for at least one month before venom extraction by electrical stimulation. Scorpions were maintained under Bioterium conditions in individual ancer is among the leading causes of death worldwide and has a major impact in both developed and underdeveloped countries (Siegel et al., 2020). Specifically, cervical cancer is one of the main public health problem affecting middle-aged women, particularly in developing countries \hyperref[b1]{(Arbyn et al., 2020)}. Conventional antitumor therapies used in clinical practice are surgery, radiotherapy and chemotherapy (Somayeh et al., 2017). These treatments are effective only to some extent, as they are not applicable in all cases and the undesirable side effects often make them impractical \hyperref[b17]{(Topcul and Cetin, 2014)} Rhopalurus junceus (R. junceus) belongs to Buthidae family and is an endemic scorpion from Cuba. Preclinical studies have shown that R. junceus scorpion Keywords: Rhopalurus junceus scorpion venom, female, male, mixture of two sexes, heLa. plastic containers at 23 ±1°C temperature, 60 ± 10 \% relative humidity and 12:12 h light-dark cycle, in the laboratories belonging to the Entrepreneurial Group of Biopharmaceuticals and Chemicals Productions (LABIOFAM). Bioterium conditions about management of scorpion colonies and collection of venom have been approved by the Ministry of Science, Technology and Environment of Cuba (CITMA 20/2016). Three groups of scorpions containing 50 female (G1), 50 male (G2) or a mixture of 25 females with 25 males (G3) were used. Venom was dissolved in distilled water and centrifuged at 15000xg for 15min. The supernatant was filtered by using a 0.2?m syringe filter and stored at -20 o C until used. The protein concentration was calculated by Lowry Modified Method \hyperref[b10]{(Herrera et al., 1999)}. 
\section[{b) SDS-PAGE and determination of molecular weight (MW)}]{b) SDS-PAGE and determination of molecular weight (MW)}\par
Electrophoretic analysis of each pooled venom was carried out according to the previous method (Díaz-García et al., 2015) with 4\% stacking gel and 16\% separating gel under non-reduced and reduced (2mercaptoethanol, 95°C, 10 min) conditions using an electrophoresis chamber (Biorad). All samples were dissolved in a sample buffer (50mM Tris-HCl, pH 6.8, 0.1M DTT, 10\% glycerol, 2\% SDS, and 0.1\% bromophenol blue). In each well, 50 ?g of venom was applied and a protein MW marker was used. The run conditions were 120 V to free current for two hours. The gels were stained with Coomassie Brilliant Blue G-250 and were subsequently rinsed with Methanol: Acetic acid: Water (45:10:45). The gels were photographed and analyzed using ImageJ 1.46 software. 
\section[{c) Cell line and culture}]{c) Cell line and culture}\par
HeLa (cervix adenocarcinoma ATCC CCL-2?) cell line was maintained in minimum essential medium (MEM). Vero (normal African green monkey kidney ATCC CRL-1586?) cell line was maintained in Dulbecco's modified Eagle's medium. The mediums of both cell lines were supplemented with 2 mM of glutamine and non-essential amino acids, 10\% of fetal bovine serum (SFB) and penicillin-streptomycin 100 UI/mL -100 µg/mL. The cells were grown in a humidified atmosphere, 5\% CO 2 at 37ºC. 
\section[{d) In vitro cell viability assay (MTT assay)}]{d) In vitro cell viability assay (MTT assay)}\par
The effect of scorpion venom on cell viability was determined by the MTT Assay  {\ref (Mosmann, 1983)}. HeLa cells (1x10 4 /well) and Vero cells (1x10 4 /well) were plated in 50?l of medium/well in 96-well culture plates (Costar Corning, Rochester, NY) and incubated overnight in a humidified atmosphere of 5\% (v/v) CO 2 at 37 o C. After incubation, 50?l of venom was dissolved in medium at final concentration of 0.0625, 0.125, 0.25, 0.5 and 1mg/mL and was added in five well for every concentration. Cells without scorpion venom were used as untreated control. After 72h of incubation, 10?l of 5mg/mL of sterile MTT was added per well and incubated for another 3h. The supernatant was carefully removed, 150?l DMSO was added per well and incubated for 15min at 37 o C. The absorbance was determined in a microplate reader (ELISA MRX Revelation Dynex Technologies 560nm with 630nm as reference). Absorbance from untreated cells was considered as 100\% of growth and used for viability calculation. The effect of scorpion venom on the viability for human cell lines panel was expressed as the percentage of viability, using the formula: \%viability= A 560-630nm of treated cells/ A 560-630nm of control cells x 100\%. The IC 50 values (venom concentration that causes 50\% reduction of the cell) from cancer cells were determined. The experiments were performed three times by triplicate. 
\section[{Phase-contrast microscopy}]{Phase-contrast microscopy}\par
After treatments, cells were washed with PBS and morphological changes in culture were then observed under microscope IX-71 (Olympus Corporation, Tokyo, Japan). Images were captured using the camera DP-72 (Olympus Corporation, Tokyo, Japan) and 10X objectives. 
\section[{f) Statistical analysis}]{f) Statistical analysis}\par
Kruskal-Wallis non-parametric test and Dunn's multiple comparison tests was used to compare different assays. Two-way Anova and Bonferroni posttest were performed to analyze differences in MW, protein band intensity and cell viability. The IC50 value was determined by interpolation of tendency line from linear regression curve. GraphPad Prism version 5.01 for Windows, (GraphPad Software, San Diego California, USA) for p<0.05 was used for all analysis. 
\section[{III.}]{III.} 
\section[{Results}]{Results}\par
There was no significant difference among total protein concentration of female (G1), male (G2) and the mixture of both sexes (G3) of R. junceus scorpion venom in our experimental conditions (Figure \hyperref[fig_1]{1}). The values of Mean ± SD for G1, G2 and G3 were 8.3 ± 1.1 mg/mL, 8.5 ± 2.9 mg/mL and 8.2 ± 1.6 mg/mL, respectively. However, the electrophoretic analysis of protein content from G1, G2 and G3 under non-reduced and reduced conditions demonstrated similarities and differences among experimental groups (Figure  {\ref 2}).  The graphic represents the mean ± SD of total protein concentration values of three independent experiments with female (G1), male (G2) and female + male (G3) R. junceus scorpion venom. Data were analyzed using Kruskal-Wallis followed by Dunn test.\par
Figure  {\ref 2}: SDS-PAGE analysis of male, female and the mixture of both sexes R. junceus scorpion venom. A) Nonreduced SDS-PAGE. B) Reduced SDS-PAGE. G1: female, G2: male, G3: female + male R. junceus scorpion venom. Separating gel 16\%, stacking gel 4\%. Molecular weight protein marker from 10 kDa-175 kDa was used Six bands were observed in non-reduced (Figure  {\ref 2A}) and reduced (Figure  {\ref 2B}) conditions of SDS-PAGE, in the venom of female and male scorpions. While in the case of the venom obtained from the mixture of scorpions of both sexes, six bands were observed under non-reduced conditions and seven under reduced conditions. The comparison of molecular weight (MW) and protein band intensity among the groups of R. junceus scorpion are presented in Table  {\ref 1}. Non-reduced electrophoresis conditions showed five similarities in the MW of (55, 45, 39, 28 and 19 kDa) from G1 and G2 scorpion groups. However, in these conditions was observed a difference between them. A band of 11kDa was displayed only in G2 and 12kDa band only in G1 and G3. The molecular weights observed in non-reduced conditions of the mixture of both sexes were: 52, 45, 39, 28, 19 and 12 kDa.\par
Electrophoresis under reduced conditions displayed two resemblances (at 44 and 11 kDa) and four differences between the protein MW of G1 and G2 groups. An 11kDa coincident band was observed in all groups. There were no statistically significant differences between the molecular weight of the bands in both electrophoretic conditions for G1 and G2. The appearance of 47kDa band in G3 group was the only statistically significant difference in MW respect to G1 and G2 groups.\par
Regarding to intensity values, statistically significant differences were found between both G1 and G2 groups for the bands at 12 and 11 kDa (p <0.001); for G3 group in the bands at 52 (p <0.05) and 12 kDa (p <0.001) concerning to G1 and G2 groups. All these results were based in electrophoretic analysis under non-reduced conditions. In reduced conditions, the Year 2020  
\section[{Global}]{Global} 
\section[{Table 1: Comparison of MW and intensity of protein band in female (G1), male (G2) and female + male (G3) of R. junceus scorpion venom}]{Table 1: Comparison of MW and intensity of protein band in female (G1), male (G2) and female + male (G3) of R. junceus scorpion venom}\par
Legend: Data were analyzed using two-way ANOVA followed by Bonferroni test: a?: p<0.05 (G1), a???: p<0.001 (G1); b?: p<0.05 (G2), b???: p<0.001 (G2) and c?: p<0.05 (G3), c???: p<0.001 (G3).\par
Viability was significantly reduced on the HeLa tumor cell line treated respect to untreated control after 72h for 0.25 mg/mL (p<0.05), 0.5 mg/mL (p<0.001) and 1 mg/mL (p<0.001) for G2 group. The same results were observed for G1 and G3 at 0.5 mg/mL (p<0.01) and 1 mg/mL (p<0.001) (Figure \hyperref[fig_3]{3A}). The effect on the Vero cell line resulted in no significant difference in cell viability between treated and untreated cells for all groups and concentrations studied (Figure \hyperref[fig_3]{3B}). However, no statistically significant differences were observed for the percentages of cells viability among all groups and concentrations of R. junceus scorpion venom evaluated in HeLa and Vero cell lines. The IC 50 values found for HeLa cells were not significantly different among G1 (1.13mg/mL), G2 (1.034 mg/mL) and G3 (1.175 mg/mL) groups. In Vero cell line, no cytotoxic effect was observed in all concentration tested and the theoretical IC 50 values were higher than 1 mg/mL: 3.1, 3.5 and 2.6 mg/mL for G1, G2 and G3, respectively (Table  {\ref 2}). 
\section[{Table 2:}]{Table 2:}\par
The IC 50 values on the HeLa and the Vero cell lines exposed to G1, G2 and G3 of R. junceus scorpion venom. Values represent the mean ± SD derived from three independent experiments. Data showed as >1 means No effect.\par
The morphological changes induced at 1 mg/mL of G1, G2 and G3 of R. junceus scorpion venom are shown in the Figure \hyperref[fig_4]{4}. All of them induced a loss of membrane integrity on HeLa cells; meanwhile Vero cells were not affected by scorpion venom from all studied group. Studies about venom recovery in Androctonus finitimus and Hottentota tumulus scorpions kept in the laboratory, demonstrated better yield and quantity by electrical method than manual method. Also, it was revealed the influence of diet and temperature on venom production \hyperref[b12]{(Tobassum et al., 2018)}. However, there are others parameters affecting venom consistency such as sex, geographical location, age and time intervals for extraction \hyperref[b13]{(Pucca et al., 2014)}. Regarding this last parameters, it has been reported that extended periods of Cuban R. junceus venom collection was positively correlated with the regeneration of venom composition and the increase of cytotoxic effect against A549 lung cancer cells (Díaz-García et al., 2019). On the other hand, 200 individual molecular masses were identified in male and female R. junceus scorpion venom from which 63 are identical in both sexes \hyperref[b11]{(Rodríguez-Ravelo et al., 2015)}.\par
The present study, demonstrated that total protein concentration was similar among female, male and the mixture of both sexes of R. junceus scorpion venom. Moreover, the numbers of bands in all sex studied groups were identical from both electrophoretic conditions, with no statistically significant differences in the molecular weight in female and male scorpions. Nevertheless, unique bands were observed in each one of the sexes. with poisonous animals how Cerastes cerastes snake, where specific bands were found in male (42 and 39 kDa) and female (46 and 44kDa) venoms  {\ref (Sarhan et al, 2017)}. However, in the study with Cerastes cerastes snake only was shown the MW in the electrophoretic analysis. While we also determined, the intensity of each one of the bands obtained under both electrophoretic conditions. Statistically significant differences were observed between both sexes (G1 and G2) for the intensity of the bands to 12 kDa (under non-reduced conditions) and 11 kDa (under non-reduced and reduced conditions).Previous proteomic studies with R.junceus scorpion venom disagree to current study where the scorpion venom was kept in captivity. That study evaluated by HPLC and mass spectrometry, the venom of females and males scorpions kept in its natural medium. As results, the relative abundance of identical components was different among the genders (Rodríguez-Ravelo et al., 2015). We have already reported the majority band below 14 kDa in the electrophoretic profile \hyperref[b5]{(Díaz-García et al., 2015)}. Several authors that work with scorpion venom have reported the presence of the mixture peptides in a diffuse area conformed by small molecules lower than 14 kDa, due to the little resolution power of SDS-PAGE technique, these peptides cannot be observed separated \hyperref[b9]{(Hernández-Betancourt et al., 2009)}. This is the first study which compares the electrophoretic profiles of this venom from different sexes and the combination of both sexes (same proportion) from scorpion maintained under conditions of captivity. Previous studies with no same proportion have been done (Díaz-García et al., 2013; Díaz-García et al., 2017; Díaz-García et al., 2019). One of the important contributions of this study was the demonstration that morphological changes and loss of viability on the HeLa tumoral cell line; and the lack of cytotoxicity on the Vero cell line induced by R.junceus scorpion venom was independently of gender. This biological effect could be possible because the most abundant components are present in both sexes (Rodríguez-Ravelo et al., 2015). Also, the bands of the most intensity in all sex studied groups corresponded to proteins with low MW that they are the main responsible for their therapeutic potentialities. However, next studies are needed to compare this biological effect of venom from scorpions with different sexes with other tumor cells.\par
V. 
\section[{Conclusions}]{Conclusions}\par
In our experimental conditions, there were similarities in the protein concentration and some differences in the electrophoretic profile of female, male and the combination of both sexes of Rhopalurus junceus scorpion venom. However, the cytotoxic effect of Rhopalurus junceus scorpion venom is maintained regardless of the sex of the scorpions on the HeLa tumor cell line.\begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-2.png}
\caption{\label{fig_0}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-3.png}
\caption{\label{fig_1}Figure 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-4.png}
\caption{\label{fig_2}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3}\includegraphics[]{image-5.png}
\caption{\label{fig_3}Figure 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-6.png}
\caption{\label{fig_4}Figure 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-7.png}
\caption{\label{fig_5}}\end{figure}
 		 		\backmatter   			 
\subsection[{Acknowledgments}]{Acknowledgments}\par
The authors are grateful to Technical and scientific Support Department of Tropical Medicine Institute "Pedro Kourí". The authors thank the assistance of the personnel from the scorpion Bioterium especially to personal of scorpion bioterium facilities LABIOFAM-Isla de la Juventud. 			  			 
\subsection[{Conflict of Interest}]{Conflict of Interest}\par
The authors declare no conflict of interest. 
\subsection[{Funding}]{Funding}\par
Authors confirm that this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.			 			  				\begin{bibitemlist}{1}
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\end{document}
