Molecular Biology Reports https://doi.org/10.1007/s11033-020-05503-6 REVIEW Cervical cancer and potential pharmacological treatment with snake venoms Alejandro Montoya‑Gómez1 · Leonel Montealegre‑Sánchez1 Eliécer Jiménez‑Charris1 · Herney Andrés García‑Perdomo2 · Received: 27 November 2019 / Accepted: 6 May 2020 © Springer Nature B.V. 2020 Abstract Cervical cancer is the fourth most common cancer worldwide in women. Apoptosis reactivation has become the main strat‑ egy for decreasing cancer proliferation. There is a need to extend the search for new drugs to implement more effective and less toxic strategies for cervical cancer treatment. Research has been carried out to find new drugs that have minimal side effects and that focus on the tumor microenvironment, particularly in the induction of cellular apoptosis and cell migration and the inhibition of angiogenesis. Potent toxins from snake venoms have shown potential as sources for the synthesis of new drugs with such characteristics. The present work aimed to describe cervical cancer characteristics, associated risk factors, current treatments and to highlight the effects of toxins isolated from the venom of snakes of the Viperidae family on cervical cancer cell lines. Keywords Anticancer agent · Toxins · Cytotoxicity · Apoptosis · Tumor cell lines · Translational medicine Introduction Cervical cancer (CC) is considered a global public health problem. In 2018, 569,847 new cases of CC were reported worldwide, and approximately 311,365 deaths were directly attributable to this type of tumorigenic disease [1]. In Colombia, according to the Cali Cancer Registry, 8963 cases of CC were registered from 1962 to 2007. Between 2007 and 2011, there were a total of 4462 new CC reports and 1861 deaths [2]. The mortality rate standardized by age between 2009 and 2011 was 7 per 100,000 women [3]. Therefore, the high morbidity associated with this cancer type gener‑ ates a significant socioeconomic impact on the country. The previous observations pose a challenge for the health system that highlights a need to design and implement management * Alejandro Montoya‑Gómez [email protected] * Eliécer Jiménez‑Charris [email protected] 1 Grupo de Nutrición, Facultad de Salud, Universidad del Valle, Calle 4B # 36–00, Edificio 116, Oficina 5002, Cali, Colombia 2 Departamento de Cirugía/Urología, Escuela de Medicina, Universidad del Valle, Cali, Colombia policies to reduce the incidence of and mortality from this cancer. Pelvic radiotherapy (PR), chemotherapy (CWCC), and brachytherapy are the most common interventions to treat CC patients [4]. In general, 50% to 60% of patients achieve a cure with this approach, but local recurrence and tumor pro‑ gression to metastasis after treatment are frequent outcomes, especially in patients with late diagnosis and advanced stage tumors. In that sense, there are few effective treatments for patients with recurrent CC. Deaths often occur after patient withdrawal from this kind of treatment to avoid a significant decrease in their quality of life due to severe pain, bleeding, and other debilitating symptoms. Currently, efforts are being made to improve the healing potential of PR with more pre‑ cise tumor targeting and intensification [5]. However, it is unlikely that this approach alone will solve the problem [6]. Chemotherapy is not specific and can affect both tumor and healthy cells [7]. There are medications and alternative treatments used to counteract the adverse effects induced by these drugs. However, this strategy is not sufficient or may even induce other side effects that add discomfort to patients [8]. In that sense, until now, no drugs have been developed that attack cervical carcinoma cells selectively without affecting healthy tissue viability. Some reviews have highlighted studies on molecules isolated from snake 13 Vol.:(0123456789) Molecular Biology Reports venoms and their effects on different types of cancer cells [9–11]. Nonetheless, there are no reviews that describe the effects of these molecules on CC cells. Thus, the purpose of this manuscript was to describe bioprospecting studies of snake venom toxins with potential use in CC treatment. Cervical cancer epidemiology Cancer is one of the leading causes of nonviolent death, both in developing countries and in developed countries. Lung, prostate, colorectal, stomach, and liver cancer are the most common cancer types in men, while breast, colorectal, lung, cervix and thyroid cancer are the most common among women [12]. Globally, there were 530,000 cervical cancer cases in 2012, with 85% of these occurring in less developed regions [13]. Approximately 11.5 million people will die of cancer in 2030 [14]. Cervical cancer originates in the cervix epithelium (squa‑ mocolumnar junction that may include external squamous Fig. 1 Chronological progression of cervical cancer. Genetic and epi‑ genetic factors modify basal cells in the cervical epithelium. Dyspla‑ sia can progress from mild to severe, in which case it is recognized as a precursor lesion. The late stage, known as carcinoma in situ (CIS) or cervical intraepithelial neoplasia grade 3 (CIN III), is characterized 13 cells, internal glandular cells, or both) with cervical intraepi‑ thelial neoplasia grade 2 or 3 (CIN II or CIN III) as a precur‑ sor lesion (Fig. 1). CIN and carcinoma in situ (CIS) can turn into invasive cancer [15]. Precursor lesions are the initial manifestation. They usually exhibit slow and progressive evolution over time, which usually occurs in stages. Lesions evolve to cancer in situ when they only involve the epithe‑ lial surface and then to invasive cancer when the compro‑ mised tissue crosses the basement membrane and invades the stroma of the cervix [16]. The classification of precancers and CC is performed according to the appearance of the tissues under the micro‑ scope. The two most common CC types are squamous cell carcinoma and adenocarcinoma [17]. Most CCs are squa‑ mous cell carcinomas (9 out of 10 cases). They originate from cells in the ectocervix, most frequently in the transfor‑ mation zone (where the ectocervix joins the endocervix). The rest of the CCs are adenocarcinomas, which originate from glandular cells (the endocervix). Commonly, earlystage CC does not manifest signs or apparent symptoms. by induction of angiogenesis and recruitment of immune cells. In this stage, there is a transformation to invasive cancer with alterations in the surrounding cells and an increase in angiogenesis and immune cell recruitment processes Molecular Biology Reports However, in more advanced stages, vaginal bleeding, unu‑ sual vaginal discharge, pelvic pain, dyspareunia, and post‑ coital hemorrhage may occur [18]. When detected early, treatment is relatively simple and effective [19]. Therefore, the higher social risk for the acquisition of potentially fatal disease derives from the lack of periodic gynecological examinations and Papanicolaou exams [15, 20]. All types of solid tumors, such as CC, require the acti‑ vation of the angiogenesis process, which is necessary to receive oxygen and nutrients supplying their metabolic requirements. The progression of cervical intraepithelial neoplastic lesions to invasive carcinoma is associated with angiogenesis. The previous study showed an increase in the density and size of the vessels in the stroma-epithelial cervi‑ cal contact zone (Fig. 1). Angiogenesis promotes the overex‑ pression of vascular endothelial growth factors (VEGF) and the expression of extracellular proteases, including matrix metalloproteinase 9 (MMP-9) [21, 22]. Smith-McCune and Weidner [23] showed that neovascularization occurs in a series of steps that facilitate the diffusion of proangiogenic compounds towards dysplastic tissue. For this reason, it is necessary to find molecules that affect tumor proliferation not only aimed at reactivating the apoptotic process but also aimed at altering the proangiogenic potential of cancer cells. Human Papillomavirus infection and other risk factors The main risk factor for CC is infection by human papil‑ lomavirus (HPV), outperforming other known risk factors [24–27]. Transient HPV infection is common in young women, and its persistence leads to an increased risk of pre‑ cancerous and cancerous lesions [28–30]. Currently, more than 200 types of HPV viruses have been recognized on the basis of their DNA sequences [31]. Certain types of HPV can cause warts on or around the female and male genital organs, as well as in the anal area. Based on an association with precursor lesions, HPVs can be classified as low-risk and high-risk. Low-risk HPV includes types, but is not lim‑ ited to 6, 11, 42, 43, and 44. High-risk HPV includes types such as 16, 18, 31, 33, 35, which are strongly linked to cervi‑ cal intraepithelial neoplasia and CC [31]. Epidemiological evidence links more than 50 types of HPV that infect the genital tract. Types 16 and 18 are the most closely related to the presentation of high-grade dys‑ plasia and CC. HPV type 18 is the most commonly associ‑ ated with cervical adenocarcinoma, while HPV 16, followed by HPV 18, is the most frequent types detected in squamous cell carcinoma [32, 33]. The presence of integrated forms of HPV 16 increases with the severity of cervical neoplasia, although in some women with the invasive disease, episomal forms of this type have been detected. In contrast, integrated forms of HPV 18 are almost always associated with highgrade cervical intraepithelial neoplasia (HGCIN) and inva‑ sive disease [34]. Aguayo et al. described two conditions for the conversion to malignant tumors: the interruption of intracellular and extracellular pathways generated by the presence of HPV and the expression of oncoproteins for the maintenance of the malignant phenotype [35]. The viral DNA of HPV-16 contains eight open reading frames (ORFs) and a regulatory region called the long con‑ trol region (LCR). It contains an origin of replication and transcriptional regulatory elements [36]. The first region after LCR contains 6 ORFs corresponding to the E6, E7, E1, E2, E4, and E5 genes, which encode proteins necessary for virus replication and cell transformation. The last two regions code for viral capsid proteins: L1, the main struc‑ tural protein, and L2, the binding protein for DNA encapsu‑ lation [37]. In terms of integration, HPV (mainly HPV-16) probably has first contact with the cell, through the polysac‑ charide heparan sulfate and then enters the cell mediated by receptors such as alpha-6 integrin [38]. Once the virus integrates, there is a disruption of the synthesis of the E2 protein, which is a repressor of the p97 promoter; this causes overexpression of the E6 and E7 proteins, which induces the loss of P53 and pRb proteins. In cervical carcinogen‑ esis, HPV integration in basal cells generates deregulation of the expression of these two viral oncogenes. This kind of deregulation is considered a critical event for the progression towards the disease [35]. Another factor strongly implicated in the development of cervical cancer is the sensitivity of the tissue to estrogen [39–42]. The transformation zone is a highly estrogen-sen‑ sitive area of the genital tract, where most HPV injuries and their sequelae occur. This contrasts with the fact that there are other areas of the genital tract with low estrogen sensitiv‑ ity that, when infected with HPV, show equal or higher loads of HPV without the development of a neoplasm [43, 44]. These observations suggest that this hormone plays a vital role in CC development, and there is a dynamic amplifica‑ tion of HPV and estrogen effects. In the transformation zone, 16 α-hydroxyestrone metabolites prolong estrogen effects. This compound is genotoxic, and it comes from the conver‑ sion of estradiol. This conversion increases in the presence of HPV-16 [42]. Estrogen stimulates the expression of onco‑ genes in HPV-infected cervical cell lines [45]. The previ‑ ous affirmation strengthens the hypothesis of the synergistic effect of both factors in the development of cervical cancer. This information is supported by studies showing a role for the estrogen receptor Erα in the development of CC in murine models and by other studies showing the relationship between increased expression of the E6 and E7 oncogenes and the presence of estrogen [41]. The role of the receptor has been analyzed in other studies, and it has been found that its expression is relatively constant in tumor-associated 13 Molecular Biology Reports stromal fibroblastic cells, with which it is possible that the progression of CC is stimulated [40]. Regarding the type of HPV most likely to cause CC in the presence of estrogen, it was found in a recent study that HPV-16 is more strongly associated with the development of CC than other types of HPV; in addition, it has an additive effect with high endog‑ enous estradiol levels that leads to an increase the risk of developing CC [39]. In addition, other risk factors, such as smoking [46], pro‑ longed use of oral contraceptives [47, 48], a high number of full-term pregnancies [49], early age at the first intercourse, a high number of sexual partners [50], nutritional status, immu‑ nosuppression [51, 52], and exposure to diethylstilbestrol in the uterus [53], have been shown to be related to CC. Cervical cancer interventions and treatments The pathophysiology of CC involves very long periods, and when detected early, there is ample opportunity for action, making its prevention or cure possible. Preinvasive lesions, which in most cases present mild dysplasia, should be moni‑ tored rather than treated because spontaneous regressions are very common (Fig. 1). Treatment schemes are defined according to age and can include control tests such as colpos‑ copy [19, 20, 54, 55]. A specialist colposcopist or gynecolo‑ gist should assess HPV-related disturbances in pap smears to make a definitive diagnosis and define the intervention [19, 20, 54, 55]. Currently, there are two types of procedures: ablation and excision. Cryotherapy is the most widely used method of ablation and involves the application of low tempera‑ tures to reach a freezing point that will lead to cell death. It is a 90% effective method, but the main limitation is the lack of a biological sample for histopathological studies [19, 20, 54, 55]. For the excision procedure, the electrosur‑ gical technique uses a fine electric wire loop to completely remove the affected neoplastic area of the cervix. It is 95% effective for high-grade dysplasia (large and endocervical lesions). Additionally, it allows for obtaining histologi‑ cal samples for staging cancer [19, 20, 54, 55]. Clinicians treat patients according to the tumor classification, general health conditions of the patient, stage of the disease, and the decision of the patient [20]. Radiotherapy, chemother‑ apy, palliative care, pain therapy, and rehabilitation are among the nonsurgical therapies currently used to treat CC, all necessary for the comprehensive care of women affected by CC [56]. 13 Side effects of nonsurgical treatments used in CC Radiation therapy is a treatment that uses high doses of radiation to kill cancer cells and shrink tumors. External radiation can be used alone to treat areas of spread cancer or as a primary treatment for CC in patients who cannot tolerate chemoradiation. Common side effects of external radiation therapy include fatigue, stomach problems, diar‑ rhea or soft stools, nausea and vomiting, and skin changes [15, 17]. Pelvis radiation can also irritate the bladder, causing discomfort and frequent urges to urinate (radiation cystitis). Radiation can affect the vulva and vagina, caus‑ ing them to be tender and painful and sometimes causing bloody discharge. It can also affect the ovaries, causing changes in menstrual periods and even early menopause. In addition, radiation can cause low blood counts, which can lead to anemia and leukopenia, increasing the risks of serious infections. Blood counts are usually decreased when patients are given chemotherapy with radiation. Additionally, fatigue and nausea tend to worsen [15, 17]. Another type of radiation therapy is called brachytherapy or internal radiation therapy. This intervention is offered as adjuvant therapy for the treatment of CC. In brachytherapy, radiation only travels a short distance, so the main effects of radiation are on the cervix and the walls of the vagina. The most common side effect is irritation of the vagina, which becomes flushed and sensitive to pain. Brachytherapy can also cause many of the same side effects caused by external radiation [17]. Vaginal dryness and pain during intercourse can be long-term side effects of radiation (both brachyther‑ apy and external radiation). In addition, pelvic radiation can also weaken the bones and cause increased susceptibility to fractures. Hip fractures are the most common and may occur 2 to 4 years after radiation [17]. Drugs currently used in chemotherapy destroy cancer cells but also cause damage to normal cells, which can result in the appearance of undesirable effects. These side effects depend on the period of exposure, type, and dosage of medications administered. Some common side effects of chemotherapy may include nausea and vomiting, loss of appetite, hair loss, mouth ulcers, and tiredness. In addition, blood cell counts could decrease; therefore, the probability of infections increases. There is a shortage of platelets; then, blood loss or bruising can occur after minor cuts or injuries. Additionally, dyspnea can appear due to the decrease in red blood cell levels [15, 17]. When given chemo- and radiation therapies, side effects such as nausea, fatigue, and diarrheas are often more severe than with either treatment alone [15, 17]. Prema‑ ture menopause and infertility can also occur permanently. Molecular Biology Reports In addition, medications such as paclitaxel and cisplatin can produce nerve damage outside the brain and spinal cord [57]. This condition, called peripheral neuropathy, sometimes causes symptoms mainly in the feet and hands, such as numbness, pain, burning, tingling, sensitivity to cold or heat, and weakness. In most cases, these symptoms are relieved or can disappear once the treatment finishes, but in some women, they may persist for a long time [15]. Until now, there have been no strategies involving chemoor radiotherapies that do not involve any side effects in patients receiving these types of treatments. Therefore, the search for alternative antitumor molecules with greater specificity against CC is required. In that sense, natural sources of pharmacologically active molecules such as snake venoms are promising areas for drug discovery. Finding new treatments will help to fight against this type of pathophysiological problem that affects the female population. Snake venoms Snakes are reptiles without limbs and, thus, have developed diverse strategies to obtain their food and defend themselves against possible predators. Some of them developed huge muscles to constrict their prey. Others developed venom glands and hollow fangs to inoculate biomolecules that can paralyze and digest their prey. Snake venoms contain hundreds of pharmacologically active molecules, including organic and mineral components (histamine and other aller‑ gens, alkaloids, polyamines), small peptides, and proteins [58, 59]. Although venoms of the Elapidae and Viperidae family may contain more than 100 protein components, these proteins belong to a few large families of proteins with enzymatic activity (­ Zn2+-dependent metalloproteases, serine proteases, type II phospholipases ­A2, l-amino acid oxidase) and proteins without enzymatic activity, such as natriuretic peptides, disintegrins, inhibitors of Kunitz type proteases, cystatin, type C lectins, vascular growth factors and cysteine-rich secretory proteins (CRISPs) [60]. The biological effects of snake venoms are complex since every component has different actions and can act in concert, generating significant pathophysiological effects. Venoms can be classified as neurotoxic or hemorrhagic/ myotoxic, according to the main effects on animals. In the first group are the venoms of the Elapidae family, which possess a wide variety of enzymes of phospholipase type ­A2 ­(PLA2). These isoenzymes show different biological effects, mainly characterized by presynaptic and postsynaptic neu‑ rotoxic activity, cardiotoxicity, and hypotensive, convulsive, and edema-inducing activities [58]. Snake venoms of the Viperidae family contain proteins that cause myotoxicity and interfere with the hemostatic and tissue repair systems by inhibiting platelet aggregation and causing hemorrhages. Consequently, envenomation by these snakes usually pro‑ duces persistent bleeding [61]. To develop studies that explore the effects of snake venom components on tumor cells, at least three experi‑ mental phases are required (Fig. 2) [62]. The first phase involves venom extraction and fractionation. The latter is performed by diverse techniques, such as reverse-phase high-performance liquid chromatography (HPLC). Then, proteomic analysis can be applied to assign the molecules to known protein families by a combination of techniques such as SDS-PAGE, mass spectrometry, and N-terminal sequencing. Once the isolated and characterized molecules are available, it is possible to develop the second experi‑ mental phase. It consists of determining whether the mol‑ ecule of interest has cytotoxic effects on tumor and nontu‑ mor cells. Some studies include an in silico phase, in which a three-dimensional model of the molecule of interest is obtained mainly by homology modeling. Subsequently, this process evaluates the interaction of the molecule of inter‑ est with three-dimensional models of receptors and other protein types and molecules of biological interest available in structural databases. The third experimental phase con‑ sists of determining the possible mechanisms of cytotoxicity induced by the molecule of interest on cancer cells. The use of immunological techniques, flow cytometry, and confocal microscopy helps to determine mechanisms of cell death and morphological alterations in tumor cells. In addition, it is important to determine the ability of proteins to alter the metastatic ability of these cells by inhibiting proliferation, adhesion, migration, invasion, or new vessel formation. Cell migration and Matrigel invasion assays, as well as wound healing and proangiogenesis assays, are among the most common in vitro tests used in this phase [62]. Snake venom and its effect on CC cells Despite the toxicity of snake venoms, there are also many reports of their antitumor effects against cervical cancer cell lines (Table 1). For example, in a study carried out in Bra‑ zil, the effect of the whole venom of two species of Bothrops on cervical cell lines, SiHa and HeLa, was evaluated. The results revealed that venoms from both snakes induced apoptosis in both cell lines. The authors observed cell cycle arrest and depolarization of the mitochondrial membrane in the G0/G1 phase in experiments with HeLa cells, a cell line representative of the most common type of CC [7]. In a study conducted in Korea, the total venom of Vipera lebetina turanica inhibited the proliferation of CaSki cells, a line of squamous epithelial carcinoma, and C33A cells (adenocar‑ cinoma cell line) in a dose-dependent manner. The venom induced apoptosis by increasing the expression of DR3 and 13 Molecular Biology Reports Fig. 2 The experimental design of Jiménez-Charris et al. [62, 79, 80] was applied to purify and identify Pllans-II, an acidic phospholipase ­A2, and to determine its anticancer effects on cervical adenocarci‑ noma cells. In the first phase of the study, snake venom was obtained by manual extraction (a), the protein cocktail was separated by RPHPLC (b), and the abundance of each family of proteins was deter‑ mined (c). In the second phase, they evaluated the cytotoxic activity of Pllans-II on nontumor cells such as C2C12 myoblasts (d), and a three-dimensional model was constructed based on the amino acid sequence (e). In the third phase, the ability of Pllans-II to induce apoptosis in HeLa cells was determined by an annexin-V/propidium iodide assay (f), and the formation of apoptotic bodies was corrobo‑ rated by scanning electronic microscopy (g). In addition, to investi‑ gate the ability of Pllans-II to inhibit new vessel formation, its effect on HUVECs was evaluated by a Matrigel tube formation assay (h) DR5, receptors for tumor necrosis factor, as well as the expression of the proapoptotic proteins Bax and caspase-3 (cleaved form), -8 and -9. A decrease in the expression of the antiapoptotic proteins Bcl-2, CIAP-1, and XIAP, evidenced by the exposure of cell lines with different doses of venom, accompanied previous findings [63]. The transcription fac‑ tor NF-κB and the p50 nuclear subunit, normally expressed in greater amounts in tumor tissues and CC cell lines than in healthy cervical tissues, were also affected by treatment with V. lebetina turanica venom in both cell lines. It inhib‑ ited the phosphorylated form of IκB in the cytoplasm and the nuclear expression of subunits p50 and p65; therefore, NF-κB bound to DNA in both cell lines. This finding could explain the overexpression of proapoptotic proteins and the subexpression of anti-apoptotic proteins. Regarding how V. lebetina turanica toxins block NF-κB, studies have shown that snake venom toxins can bind to sulfhydryl groups of the p50 subunit or IKKs, inhibiting the activity of NF-κB in other cancerogenic lines [64, 65]. Finally, the antitumor activity of the venom was evaluated in vivo in a xenograft mouse model subcutaneously trans‑ planted with CaSki cells. The authors found a 30–40% decrease in tumor mass and overexpression of DR3, DR5, and caspase-3, -8, and -9 in venom-treated mice compared to control mice. Additionally, inhibition of NF-κB, which was in concordance with the results obtained in cell lines. These results regarding the in vitro and in vivo down‑ regulation of NF-κB and overexpression of DR3 and DR5 receptors induced by V. lebetina turanica venom could 13 Molecular Biology Reports Table 1 Effect of snake venoms on cervical cancer cells Snake Cervical cancer cell line Principal effects on cervical cancer cells Bothrops jararaca and Bothrops erythromelas SiHa and HeLa In SiHa: Cytotoxicity [7] In HeLa: arrest in the G0/G1 phase of the cell cycle, and depolarization of the mitochondrial membrane Dose-dependent toxic effect on both cell lines [63] Increase in the expression of the receptors for tumor necrosis factor DR3 and DR5 Increase in the expression of the pro-apoptotic proteins Bax, caspases 3 [cleaved form], 8 and 9 Decrease in the expression of the antiapoptotic proteins Bcl-2, CIAP-1, XIAP Inhibition of the phosphorylated form of IκB in the cytoplasm, as well as the nuclear expression of the subunits p50 and p65, and therefore, blocking the binding of NF-κB to DNA Decrease between 30 and 40% of the tumor mass [63] Overexpression of receptors DR3, DR5, and in Caspase proteins 3, 8, 9 in tumor tissue Inhibition of NF- κB in tumor tissue Vipera lebetina turanica CaSki and C33A Vipera lebetina turanica Xenographic mice with CaSki cells subcutaneously trans‑ planted indicate potential targets for the development of new treat‑ ments for CC, especially in resistant tumors. Antitumor effects of peptides on cervical cancer cells Because of their small size, specificity, selectivity, and sta‑ bility, peptides have structural and pharmacological char‑ acteristics that make them very interesting in biomedical research. Peptides isolated from snake venoms are rich in disulfide bonds and have a higher affinity for other molecules than do synthetic peptides. However, the use of peptides iso‑ lated from snake venoms is an emerging area of study [10]. Only four studies have explored the effect of peptides or molecules derived from snake venom on CC cells (Table 2). In 1994, Sheu et al. isolated a peptide containing the RGD domain from the venom of Trimeresurus flavoviridis (later named Protobothrops flavoviridis) [66]. This peptide, named References triflavin, inhibited the binding of HeLa cells to fibronectin, fibrinogen, vitronectin, and other extracellular matrix com‑ ponents in a dose-dependent manner, with greater affinity than a synthetic peptide with the RGD motif. Additionally, it also inhibited the binding to laminin, collagen type I and type IV, but to a lesser extent. Integrins such as α5β1 and αVβ3, used by cells to adhere to fibronectin and vitronectin of the extracellular matrix, respectively, were identified as potential targets of triflavin. However, the cytotoxicity tests showed that triflavin did not affect the viability of the cells, which suggests that the inhibitory effect on adhesion does not imply cytotoxicity. Zare and Sarzaeem reported the effect on HeLa cells of the peptide ICD-85, generated from the venoms of the Ira‑ nian snake Agkistrodon halys and the scorpion Hemiscorpius lepturus [67]. This peptide showed a dose-dependent cyto‑ toxic effect on the HeLa cell line and a nonsignificant effect on the viability of nontumor lung cells when the dose was less than 50 µg/mL. This cytotoxic effect was related to the Table 2 Effects of peptides and peptide derivatives from snake venoms on cervical cancer cells Peptide Snake Cervical cancer cell line Principal effects on cervical cancer cells References Triflavin Protobothrops flavoviridis HeLa [66] ICD-85 Agkistrodon halys HeLa Nanoparticles Agkistrodon halys of ICD-85 HeLa Inhibition of cellular binding to extracellular matrix components, without cytotoxicity Cytotoxicity related to induction of apoptosis Increase in levels of caspase-8 protein expression Greater cytotoxicity than for ICD-85, related to induction of apoptosis Increase in levels of caspase-8 protein expression Less damage to the cytoplasmic membrane than for ICD-85 peptide [67] [68] 13 Molecular Biology Reports Effects of isolated proteins on cervical cancer cells mitochondrial membrane permeabilizes and cytochrome C is released. Additionally, the induction of apoptosis in HeLa cells was accompanied by an increase in reactive oxygen species (ROS) and the translocation of the Bad and Bax proapoptotic proteins from the cytosol to the mitochondria, promoting the release of apoptogenic mitochondrial pro‑ teins. Coimmunoprecipitation assays showed that there was an interaction between the Bcl-xL and Bad proteins and a reduction in the binding between Bcl-xL and Bak in HeLa cells treated with ACTX-8. The increase in the interaction between Bcl-xL and Bad blocked the antiapoptotic signal promoted by Bcl-xL, while the reduction in the interaction between Bcl-xL and Bak allowed Bak to participate in the permeabilization of the mitochondrial membrane, promot‑ ing apoptosis. Texeira et al. reported the characterization of an LAAO (LAAOcdt) isolated from the venom of Crotalus durissus terrificus [72]. The authors evaluated the cytotoxic effect of the protein on nine tumor cell lines, including HeLa and SiHa. LAAOcdt resulted in more cytotoxicity to HeLa cells, and they found overexpression of p-H2AX, a protein related to early DNA fragmentation. In SiHa cells, the cytotoxicity was less evident, as flow cytometry showed no effects on the cell cycle and western blot analyses showed no evidence of the overexpression of apoptogenic proteins. These results suggest that apoptosis may not be the mechanism of cell death that explains the cytotoxic effect exerted by LAAOcdt on both CC lines. l‑Amino acid oxidases (LAAOs) Phospholipase ­A2 ­(PLA2) These flavoenzymes catalyze the oxidative deamination of l-amino acids, generating alpha-ketoacids, ammonium, and hydrogen peroxide. The enzymes are widely distributed in several organisms, and the LAAOs of snake venoms are among the most widely studied. The abundance of these proteins in the venom of some snakes, as well as the ease of purification, make them an interesting object of study in enzymology, structural biology, and pharmacology [69]. To date, three studies have reported the effects of this family of proteins isolated from snake venoms on CC cells. The first publication is a 1996 study from Yonsei University (Korea), in which purified LAAOs from the venom of Agkistrodon halys adhered to the cytoplasmic membrane and generated cytotoxic effects on HeLa cells. However, this cytotoxic effect was the lowest reported among the effects induced in leukemic lines [70]. In 2004, Zhang and Wei reported the effect of an LAAO isolated from the venom of Agkistrodon acutus, ACTX-8, on HeLa cells [71]. This LAAO induced apoptosis through the intrinsic pathway, evidenced by the alteration in the mitochondrial membrane potential, as well as the activa‑ tion of caspase-3 and -9. This process occurs when the These components are between the most abundant in snake venoms and belong to a large family of enzymes that hydro‑ lyze glycerophospholipids in the sn-2 position of the glyc‑ erol backbone, releasing lysophospholipids, arachidonic acid, and fatty acids. These proteins are distributed widely in nature in different secretions and biological fluids, such as those generated by inflammatory processes, pancreatic secretions, and tears. They can also be found in venoms of arthropods, mollusks, and snakes, fulfilling essential roles for the biological performance of these animals in their natu‑ ral environment [73]. In the case of the P ­ LA2s present in snake venoms, effects on cellular processes such as prolifera‑ tion, migration, angiogenesis, and cell death, among others, have been widely explored in the search for pharmacological applications and the design of new drugs [74]. PLA2s found in snake venoms of the Viperidae family belong to group II, which can be catalytically active if they have an Asp residue in position 49 (catalytic region) or devoid of this activity if the enzyme has a Lys residue in this position, which affects its ability to bind the ­Ca2+ cofac‑ tor [75]. Cytotoxicity against cancer cells has been reported for both types of ­PLA2s, suggesting that cytotoxicity is not induction of apoptosis with no alterations in the integrity of the cytoplasmic membrane or morphological changes in cells characteristic of the apoptotic process (rounded cells and granulation) when treating the cancer cells with the ­IC50 of the peptide. Additionally, in HeLa cells (and not noncan‑ cer cells), there was a dose-dependent increase in the expres‑ sion of the caspase-8 protein when cells were treated with ICD-85; this caspase is an essential component of apoptosis mediated by cell death receptors. These results allowed the authors to conclude that ICD-85 selectively induces apopto‑ sis in HeLa cells and that one of the apoptotic mechanisms induced by the peptide is the activation of caspase-8. One year later, the same research group reported the effects of a preparation of the peptide ICD-85 with nano‑ particles on HeLa cells. In this study, the cytotoxic effects of the nanoparticles were significantly greater than those of ICD-85 alone, and the alteration to the cytoplasmic mem‑ brane integrity was lower, which indicated less necrotic effects. Nanoparticles induced the expression of the cas‑ pase-8 protein more potently than ICD-85, indicating that treatment with the nanoparticles prepared from ICD-85 was more effective than treatment with ICD-85 alone [68]. The antitumor effects of peptides and peptide derivatives from snake venoms on CC cells are summarized in Table 2. 13 Molecular Biology Reports necessarily related to the enzymatic activity of phospholi‑ pases and that they are probably interacting with cell surface receptors affecting intracellular pathways [76]. However, it is still a matter of research to establish which type of recep‑ tors and signaling pathways P ­ LA2s of snake venoms interact with cancer cells. Most of the effects of P ­ LA2s on CC cells have been assessed with Crotoxin, a heterodimeric protein purified from the venom of the South American snake Crotalus durissus terrificus [77]. Crotoxin generated a significant dose-dependent cytotoxic effect on the ME-180R CC lines with higher expression levels of EGFR compared to A431, a line of squamous cell carcinoma. Muller et al. [78] also evaluated the cytotoxicity of Crotoxin on nine tumor cell lines, including two lines of CC (HeLa and SiHa). Despite being cytotoxic for several carcinogenic cells, including HeLa cells, at a dose of 30 µg/mL, SiHa cells were resist‑ ant to Crotoxin effects and did not present alterations in the cell cycle, apoptosis, or the expression of proteins related to proliferation or DNA damage. Interestingly, Crotoxin was innocuous in nontumoral cells [78]. Recently, an acidic Asp49-PLA 2 isolated from the Colombian Porthidium lansbergii lansbergii snake venom showed cytotoxic activities on CC cell lines. This protein, Pllans-II, inhibited the viability of HeLa and MCF-7 cells (breast adenocarcinoma) in a dose-dependent manner, while it was shown to be innocuous on mouse myoblasts, umbili‑ cal cord vascular endothelial cells (HUVECs), and breast epithelial cells (MCF-10A). Treatment with one I­ C50 of Pllans-II on HeLa cells generated cell cycle arrest in the G0/G1 phase, apoptosis induction and a decrease in the migratory capacity of these cells. HeLa cells treated with Pllans-II presented a downregulation of BIRC5, Bax, and BCL2 genes and upregulated BCL2L1 and caspase-8. These results suggest the activation of the extrinsic apoptosis path‑ way and the possible interaction of P ­ LA2 with membrane receptors involved in apoptosis induction. Pllans-II was demonstrated to interact with α5 and β1 integrin subunits on the HeLa cell membrane and showed an angiostatic effect on HUVECs, with no changes in VEGF concentrations [62]. Pllans-II also affected the viability of CaSki cells in a dosedependent manner, with an ­IC50 very similar to that in HeLa cells (100 µg/mL vs. 98 µg/mL, respectively, unpublished data). Pllans-II also induced cell cycle arrest at the G2/M phase, induced apoptosis, and inhibited adhesion and migra‑ tion. These effects on CaSki cells could also be related to interactions with membrane receptors since treatment with the protein did not alter the mitochondrial membrane potential. The authors observed the interaction between the C-terminal region of Pllans-II (noncatalytic) and the α5β1 integrin in silico by using protein–protein modeling analy‑ ses. The antitumor effects of snake venom proteins on CC cells are summarized in Table 3. Conclusion Cervical cancer is a multifactorial disease that significantly affects women worldwide. The studies included in this review show that some snake venoms and their components can induce cytotoxic effects on CC cells. Most of these studies have been developed around the isolation of previously identi‑ fied molecules and the investigation of the mechanisms under‑ lying the cytotoxic effects, which include the alteration of the cell cycle, induction of the cell death by extrinsic or intrinsic apoptosis pathways, and inhibition of the metastatic ability of cancer cells (cell adhesion, migration, and angiogenesis). These findings provide new perspectives on drug discovery and development for cancer treatment and can be used as a basis for the design of more potent molecules. These studies can also help to reveal cellular mechanisms that can be inves‑ tigated in greater depth for a better understanding of carcino‑ genesis and the development of metastasis. Given that there are few studies on snake venom proteins in cervical cancer, no preclinical or clinical studies are available in the literature to determine their in vivo safety and efficacy. Therefore, more research efforts are needed to delve into the mode of action of the components of snake venoms in cervical cancer cells, thus elucidating mechanisms that so far have not been described. In addition, some limitations for future research on the potential use of snake venom in CC treatment include the correct delivery of the active components to the desired anatomical sites and the limited amounts of purified com‑ ponents available for proof of concept and in vivo tests. These problems could be assessed by evaluating the use of nanomaterials for better bioavailability of the active com‑ pounds and the use of recombinant technology or chemical synthesis for their design and large-scale production. The use of these new technologies opens doors for increasing future preclinical and clinical studies evaluating the use of snake venom components in the treatment of CC and other types of cancer. 13 Molecular Biology Reports Table 3 Effects of proteins from snake venoms on cervical cancer cells Protein Snake Cervical cancer cell line Principal effects on cervical cancer cells References LAAOs Agkistrodon halys HeLa [70] Crotoxin ­[PLA2] ACTX-8 [LAAO] Crotalus durissus terrificus Agkistrodon acutus ME-180R HeLa LAAOcdt [LAAO] Crotalus durissus terrificus HeLa and SiHa Crotoxin ­[PLA2] Pllans-II [Asp-49-PLA2] Crotalus durissus terrificus Porthidium lansbergii lansbergii HeLa HeLa Pllans-II [Asp-49-PLA2] Porthidium lansbergii lansbergii CaSki Adheres to the cytoplasmic membrane and generates a cytotoxic effect on HeLa cells Cytotoxicity Alteration in the mitochondrial membrane potential, as well as the activation of caspases 3 and 9, which induces intrinsic pathway apoptosis Activation of reactive oxygen species Translocation of Bad and Bax proapoptotic proteins from the cytosol to the mitochon‑ dria, which would promote the release of apoptogenic mitochondrial proteins Interaction between Bcl-xL and Bad proapop‑ totic proteins, and reduction in the interac‑ tion between Bcl-xL and Bak antiapoptotic proteins Cytotoxicity on both cell lines, but a stronger effect on HeLa cells In HeLa: overexpression of p-H2AX, a pro‑ tein related to early DNA fragmentation In SiHa: overexpression of P21 protein Cytotoxicity Cytotoxicity Cell cycle arrest in the G0/G1 phase of the cell cycle Apoptosis induction Decrease on the migratory capacity Downregulation of BIRC5, Bax and BCL2 genes Up-regulation of BCL2L1 and CASP8 genes Interaction with α5 and β1 integrins Cytotoxicity Arrest in the G2/M phase of the cell cycle Apoptosis induction Decrease on the adhesion and migratory capacity In Silico interaction with α5β1 integrin Funding There is no funding source. 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