Rhopalurus Junceus Venom Modulates the Expression of E6/E7 Viral Oncogenes of HPV-16 in SIHA Tumor Cell Line

Introduction: E6 and E7 are viral oncoproteins of Human Papilloma Viruses (HPV) that can modulate several innate pathways related to the immune system blocking the production of cytokines such as IL-1β. Until now, it is unknown if the ability of the Rhopalurus junceus scorpion venom to decrease the viability of tumor cells is related to the modulation of the expression levels of HPV16 viral oncogenes E6 and E7 (16E6E7). Objective: To evaluate in vitro the antitumoral and modulatory properties of the Rhopalurus junceus scorpion venom in cervix cancer cells positive for HPV16. Materials and Methods: Cell viability was analyzed by MTT assay and morphological changes were observed by phase contrast microscopy in SiHa cell line treated with scorpion venom for 72h. The expression of 16E6E7 and p53 genes were examined trough real time RT-PCR and end-point RT-PCR, respectively, after 24 and 48h of treatment of SiHa cells with R. junceus scorpion venom. Finally, the supernatant was harvested and quantified for IL-1β by ELISA after 24 and 48h of incubation. Results: R. junceus scorpion venom decreased the viability of SiHa cells and induced morphological changes. Analysis of expression of 16E6E7 and p53 by RT-qPCR and RT-PCR, respectively, showed a reduction of E6 and E7and increase in p53 genes expression after 24 h of the incubation in the treated cells respect to the untreated control. While the production of IL-1β by the SiHa cells treated with 0.5 mg/mL and 1 mg/mL of R. junceus scorpion venom, increased significantly at 48 h (p <0.05). Conclusion: R. junceus scorpion venom has antiviral potential by modulating the in vitro expression of the E6 and E7 oncogenes that lead to a cytotoxic effect on the cervical cancer positive for HPV16.


Introduction
Human Papilloma Virus (HPV) comprises a diverse group of DNA viruses belonging to the Papillomaviridae family and represents one of the sexually transmitted diseases (STD) with the highest incidence [1]. Viral oncoproteins E6 and E7 can deregulate several innate pathways related to the immune system that block the production of cytokines such as IL-1β [2,3]. In spite that prophylactic vaccines have been developed to prevent HPV infection, these are not effective on active HPV infections, showing no therapeutic effect [4]. In women, cervical-uterine cancer is the second most frequent in underdeveloped countries, while worldwide it is the fourth in incidence and mortality [5]. This fact is highly linked to persistent infections with HPV genotype 16 (HPV16) [6]. Tumor cells often show high expression of E6 and E7 and in many cases with integration of viral DNA into the host cell genome [7]. The E6 viral oncoprotein of HPV16 promotes the degradation of the tumor suppressor gene p53, which prevents the induction of cell cycle arrest and apoptosis of cervical-uterine tumor cells [8]. The conventional antineoplastic treatments used are not applicable in all cases and produce undesirable side effects [9]. Natural products have been identified as promising sources of drugs for cancer treatment and prevention [10]. For example, curcumin, witaferin A, and epigallocatecin-3-gallate are cytotoxic for cervical cancer cells positive for HPV and negatively modulate the expression of one or both viral oncoproteins (E6 and E7), resulting in the loss of the transforming phenotype and the inhibition of cell growth [11,12].
The Rhopalurus junceus scorpion (R. junceus) is an endemic species from Cuba that belongs to the Buthidae family. In vitro studies have shown that it exerts a selective cytotoxic effect on cancer cell lines of epithelial origin without affecting normal cells [13]. The antineoplastic effect of this scorpion venom includes the induction of apoptosis, which was observed in the cervical cell line HeLa positive for HPV18 [13] and in the metastatic breast cancer cell line MDA-MB-231 [14]. However, it is unknown whether the ability of the venom R. junceus to decrease the viability of cervicaluterine tumor cells is related to the modulation of the expression levels of HPV16 viral oncogenes E6 and E7 and IL-1β.

Venom Source
Female and male adults Rhopalurus junceus scorpions, collected in Isla de la Juventud special municipality from Cuba were kept in captivity for at least 1 month before venom extraction by electrical stimulation. Scorpions were maintained under bioterium conditions in individual plastic containers at 23 ±1°C temperature, 60 ± 10 % relative humidity and 12:12hr light-dark cycle in laboratories belonging to Laboratories of Biopharmaceuticals and Chemicals Productions (LABIOFAM). The obtaining, bioterium condition, management of scorpion colonies, and collection of venom have been approved by the Ministry of Science, Technology and Environment of Cuba (CITMA 20/2016). Venom was dissolved in distilled water and centrifuged at 1650 g for 15min. The supernatant was filtered by using a 0.2μm syringe filter and stored at -20 °C until used. The protein concentration was calculated by the Lowry modified method [15].

Cell Line and Culture
SiHa (cervix squamous cell carcinoma grade II ATCC HTB-35™) cell line was maintained in minimum essential medium (MEM), supplemented with 2mM of glutamine and non-essential amino acids, 10% of fetal bovine serum (SFB) and penicillin-streptomycin 100UI/mL -100µg/mL. The cells were grown in a humidified atmosphere, 5% CO 2 at 37 ºC.

Isolation and Quantification of Total RNA
The SiHa cell line (1x10 5 cells/mL) was grown in flat-bottom 24-well cell culture plates and incubated for 24h and 48h with 0.25mg/mL, 0.5mg/mL and 1mg/mL of R. junceus scorpion venom diluted in culture medium. At the end of each incubation period, the culture supernatant from each well was collected (three replicates of each treatment were mixed) and 140μL were taken from each sample and to perform automatic RNA extraction using the commercial Qiamp viral RNA kit (Qiagen) using the Qiacube equipment (Qiagen) following the manufacturer's instructions. The concentration of total ribonucleic acid (RNA) in the samples was determined using a Biophotometer (Eppendorf, Germany).

RT-qPCR Analysis of E6 and E7 Genes from VPH-16
The commercial QuantiFast Multiplex RT-PCR kit (QIAGEN, USA) was used for the analysis. The reaction mix (for 10 reactions)

RT-PCR Analysis of p53 Gene Expression
Reverse transcription and Polymerase Chain Reaction (RT-PCR) were performed in a single step using the commercial OneStep RT-PCR kit (Quiagen Inc. Germany). A concentration of 100 ng of total RNA was used for each treatment. PCR amplification was carried out in a Ther-mal cycler (AUXILAB, Spain). β-actin gene amplification was performed as an internal control gene. The primer sequence for RT-PCR was 5´-CCTTCCTGGGCATGGAGTCCTG-3´ and 5´-GGAGCAATGATCTTGATCTTC-3´ for β-actin, 5´-GGGTTAGTTTA-CAATCAGCCACATT-3´ and 5´-GGCCTTGAAGTTAGAGAAAATTCA-3´ for p53. The initial step of reverse transcription was carried out at 30min at 50 °C, while the initiation step of the PCR at 95 °C for 15 min. PCR conditions were 40 cycles at 94 °C for 60sec, at 60 °C for 60sec at 72 °C for 1min. Amplified PCR products were subjected to electrophoresis at 70V in 1.5% (w/v) agarose gel for 90min. The gels were examined and the intensity of each band was measured by using ImageJ 1.46 software.

In Vitro Cell Viability Assay (MTT Assay)
The effect of scorpion venom on cell viability was determined

Phase Contrast Microscopy
The cells were washed with PBS after 72h of treatment.
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.

Determination of IL-1β Levels in the Culture Supernatant
The

Statistical Analysis
Results are presented as the mean ± standard error media (SEM). Significant differences were considered for p<0.05. times, respectively, with respect to the untreated control ( Figure   3B). There was a tendency to increase IL-1β production by the SiHa cells treated with R. junceus scorpion venom was shown (Figure 4). Statistically significant differences were only observed between the untreated cells and the treated with 0.5 and 1mg/mL of R. junceus scorpion venom after 48 h of incubation (p <0.05).    Note: The mean ± standard deviation of the IL-1β concentration present in supernatant of SiHa treated with 0.25, 0.5 and 1 mg/mL of R. junceus scorpion venom for 24h (A) and 48h (B). Control represents cells treated with culture medium. Significant differences *p<0.05.

Discussion
In women, cervical-uterine cancer is the second most frequent in underdeveloped countries, while worldwide it is the fourth in incidence and mortality [5]. This incidence is highly linked to persistent infections with HPV16 [6]. Preclinical studies have demonstrated the antitumoral effect of scorpion venoms [16] and their antiviral potentialities [17]. R. junceus scorpion venom decreases the viability of epithelial tumor cells such as cervical cancer cell line positive for HPV16 [13]. However, it is unknown  [19], which showed 100% cell viability after exposure to a dose range C. limpidus scorpion venom (0.5-4mg/mL) higher than the evaluated range in this study.
The reduction of the viability in SiHa cells induced from R. junceus scorpion venom is associated with the appearance of morphological changes in this cell line. The results show that the highest concentration of this venom causes the loss of cell morphology and a total rupture of the monolayer with respect to untreated cells. These morphological changes agree with previous results reported for this scorpion venom in tumor cells HeLa [13] and MDAMB231 [14]. Furthermore, a similar morphological change is induced by Tityus serrulatus scorpion venom in SiHa and HeLa cells [18]. Currently, this is the first study that demonstrates the influence of R. junceus scorpion venom in the reduction of E6 and E7 viral genes of HPV16 on the SiHa cells. Previously, it was showed that E6 and E7 oncoproteins are expressed jointly in the course of HPV infection with oncologic high risk (HPV-HR) and in the subsequent development of cancer [20]. Our results agree with another research based in natural products, such as the curcumin which is able to inhibit the expression of E6 and E7 viral oncogenes Previously, our group of work had been reported that this venom induces apoptosis in [13] and in MDA-MB-231 [14] tumor cells.
Treatment with this scorpion venom causes an overexpression of the pro-apoptotic genes p53, bax, noxa, puma, caspase 3 and p21; as well as in a decrease in the expression of the anti-apoptotic genes bcl-2 and bcl-xL in HeLa [13] and MDAMB231 [14]. While aloeemodin promotes apoptosis dependent of p53 in HeLa and SiHa cells [22]. A better understanding of p53-mediated apoptosis is essential, based in the use of scorpion venom, as it is promising for potential clinical applications. Also, it is necessary to elucidate the possible promotion of other p53 independent apoptotic cell death mechanisms induced by R. junceus scorpion venom in cervicaluterine cancer cell lines positive for HPV16.
An interesting result in this study was the increase of IL-1β in SiHa cells treated with R. junceus scorpion venom, which was statistically significant at concentrations of 0.5 and 1mg/mL of this venom after 48h of incubation. Previous study showed that 16E6E7 block IL-1β production in HPV16 infected keratinocytes and in cervical cancer derived cells lines [8]. The E6 oncoprotein forms a complex with ubiquitin ligase E6-AP and the tumor suppressor p53, leading to degradation of pro-IL-1β and impaired the IL-1β production [3]. Therefore, the inhibition of IRF6 by E6 is a strategy escape used by HPV16 to block the production of IL-1β. The fight between oncoviral persistence and host immunity is focused in the regulation of IL-1β [8]. For that reason, the increase of IL-1β suggests its antiviral potentialities against HPV16 as a consequence of the exposure of SiHa cells to the R. junceus scorpion venom [22].