**1. Introduction**

Ophidian accidents constitute a serious public health problem in Brazil, with an average of 29,000 cases and 125 deaths reported every year (Brazilian Ministry of Health, 2019) [1]. Approximately 80% of ophidian accidents are caused by snakes of the Viperidae family, more specifically of the *Bothrops* genus [2]. Among them, about 25% lead to death or sequels capable of generating temporary or permanent incapacity for work and customary activities. Venom from the *Bothrops jararaca* (*B. jararaca*) snake is a complex mixture composed of proteins, peptides, amino acids, nucleotides, lipids, and carbohydrates that

Montoni, F.; da Silva Santos, W.; Calacina, H.M.; Lima, I.F.; Cajado-Carvalho, D.; Ferro, E.S.; Nishiyama-Jr, M.Y.; Iwai, L.K. Bothrops Jararaca Snake Venom Modulates Key Cancer-Related Proteins in Breast Tumor Cell Lines. *Toxins* **2021**, *13*, 519. https://doi.org/ 10.3390/toxins13080519

**Citation:** Kisaki, C.Y.; Arcos, S.S.S.;

Received: 5 July 2021 Accepted: 19 July 2021 Published: 25 July 2021

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present a range of different actions when they are isolated or together [3–7], leading to hemotoxic, cardiotoxic, cytotoxic, or neurotoxic effects [8,9]. Several previous reports have defined the proteomics composition of *Bothrops* venoms [5,10–16]. These studies have shown that *Bothrops* venoms are composed of various classes of toxin, including metalloproteinases, serine proteinases, phospholipases A2, and C-type lectins, the most abundant components participating in the local and systemic envenomation effects.

The venom of *B. jararaca* engenders three main activities: proteolytic, coagulant, and hemolytic. The proteolytic activity causes degradation of extracellular matrix proteins, plasma, and cell surface [17], which represents an important factor for the clinical characterization of a bothropic accident [2,18]. In addition, venom can cause local tissue lesion, myonecrosis, edema, cardiovascular alterations, hypovolemic shock, coagulation alteration and renal alterations, resulting from the combined action of the enzymatic and toxic activity of the venom [19]. About 90 to 95% of the dry weight of the *B. jararaca* venom is composed of a complex mixture of proteins, mainly metalloproteinases, serine proteinases, phospholipases (PLA2), and L-amino acid oxidases. The metalloproteinases comprise most of the venom composition [20,21]. They are proteolytic enzymes associated with fibrinolysis and coagulation, and they are involved in cell migration and tissue repair, besides being related to pathological effects such as cancer [22,23]. In terms of therapeutic interventions, protease inhibitors have been shown to inhibit homeostasis and thrombosis by acting on the coagulation cascade [24]. The third major component of the venom, PLA2, is an enzyme capable of hydrolyzing the ester bonds at the sn2 position of glycerolphospholipids, releasing arachidonic acid, important for the biosynthesis of many mediators involved in inflammation, such as prostaglandins, thromboxanes, and leukotrienes [25]. Finally, the L-amino acid oxidases (LAAOs), which make up about 1 to 9% of the venom composition [26], are flavoenzymes belonging to the class of oxidoreductases, which produce alpha-keto acid, hydrogen peroxide, and ammonia [27,28]. However, when there is a high production of hydrogen peroxide, it has been found that L-amino acid oxidases can induce apoptosis in mammalian endothelial cells [29].

Snake venom constituents have been isolated and studied for their therapeutic potential in the treatment of various diseases. One example is Eptifibatide, marketed as Integrilin, derived from the *Echis carinatus* snake venom and produced by Millennium Pharmaceuticals and Schering-Plow. It is used as an antiplatelet drug [30]. Another example is the angiotensin I converting enzyme inhibitor Captopril produced by Bristol-Myers Squibb whose active component was derived from *B. jararaca* venom. It is used for the treatment against hypertension and renal insufficiency [31,32]. In addition to the potential use of the derivatives of snake venom toxins in the treatment of non-malignant diseases [33], several studies have described anti-tumorigenic characteristics of snake venom, stating that snake venom may be capable of inhibiting cell proliferation and promoting cell death by different means: inducing apoptosis in cancer cells by increasing the influx of Ca2+, inducing the release of cytochrome C, decreasing or increasing the expression of proteins that control the cell cycle, and causing damage to cell membranes [26,34–36]. With the goal of searching novel therapy against cancer, studies have characterized the proteins, peptides or enzymes derived from snake venom to identify components that are capable of interfering with the transport of substances or signal transduction across the membrane or disrupting the cell membrane [35,37].

With the rapid advances of nano-scale liquid chromatography (nLC) and mass spectrometry (MS) technologies in the last two decades, nLC-MS/MS-based proteomics analysis has been widely applied as a powerful tool for biomarker discovery to improve cancer therapy [38,39]. A number of studies have characterized the biochemical and physiological action of venom or isolated venom derivatives on cell lines or tissues [40–43]. In addition, several works have shown proteomic changes of cancer cell lines upon drug treatment that suggest molecular mechanisms of drug action, including diverse effects on proteasome regulation, metabolic processes, and oxidative stress [44,45].

However, to the best of our knowledge, there has been no report that describes the effects of *B. jararaca* snake venom treatment on breast cancer-related cell proteome. In the present study, nLC-MS/MS was used to characterize the effects of sub-toxic doses of *B. jararaca* snake venom on two different breast cancer cell lines MCF7 and MDA-MB-231. MCF7 and MDA-MB-231 are non-metastatic and metastatic tumor cell lines, respectively. They are characterized by a high degree of glycolytic efficiency that promotes the interaction between the tumor cell and the extracellular matrix [46]. Although both cell lines are from breast origin, they are molecularly distinct. MCF7 are estrogen and progesterone receptors positive and HER2 negative, while MDA-MB-231 are triple negative (estrogen receptor, progesterone receptor, and HER2 negatives) and prone to cytotoxic agents because of their impaired DNA repairing capability which is in part due to mutation in the p53 gene [47,48]. Proteomic changes observed herein upon treatment with *B. jararaca* snake venom in these cell lines highlight proteins and cell pathways that could be targeted in cancer therapy.

### **2. Results**

#### *2.1. The Cytotoxicity of B. jararaca Snake Venom in MCF7 and MDA-MB-231 Cells*

The *B. jararaca* venom cytotoxicity assay on MCF7 and MDA-MB-231 cell lines was monitored using the WST-1 reagent. This analysis showed that although cell viability was similar between both MCF7 and MDA-MB-231 cell lines, they had different venom resistance profiles where MDA-MB-231 cells showed to be more resistant to the venom when compared to the MCF7 cells. Although both cell lines started to die at doses higher than 2.5 μg/mL, at the 5.0 μg/mL of venom, only about half of the MDA-MB-231 cells have died while most of the MCF7 have died at this venom concentration (Figure 1). Lethal concentration 50 (LC50) was determined as 4.50 μg/mL for MCF7 and 4.76 μg/mL for MDA-MB-231 cell line. Interestingly, the treatment of cells with the low dose at 0.5 μg/mL of venom also killed more MCF7 cells compared to the MDA-MB-231 cells when compared to the venom doses of 0.63 μg/mL and 1.25 μg/mL (Figure 1).

**Figure 1.** Cytotoxicity assay of (**a**) MCF7 and (**b**) MDA-MB-231 cell lines treated with *B. jararaca* snake venom ranging from 0 to 20 μg/mL for 24 h. Experiment was performed using the WST-1 reagent kit.

#### *2.2. Optical Microscopy Analysis of MCF7 and MDA-MB-231 Cells under B. jararaca Venom Treatment*

Optical microscopy analysis at 10× magnification of MCF7 and MDA-MB-231 cell lines treated with concentrations higher than 2.5 μg/mL of *B. jararaca* snake venom showed cellular morphological changes such as cell shrinkage and cell birefringence change (Figure S1). At the 20 μg/mL of venom treatment all MCF7 cells detached from the plate, whereas the MDA-MB-231 cell line continued to show morphological death-like changes, but the cells did not detach from the plate (Figure S1).

Based on the cytotoxicity assays and visualization of cell morphology changes through the microscope images, two working concentrations, representing a low dose of 0.63 μg/mL and a high sub-toxic dose of 2.5 μg/mL of venom, were selected for further proteomics analysis.
