*2.9. Exclusive Proteins*

We further analyzed proteins exclusively identified in one or two of the three conditions in both MCF7 cell line (Table S4a, Figure S8) and MDA-MB-231 cell line (Table S4b, Figure S8). Proteins identified only on PBS treated cells (control group), suggesting that the expression of the proteins was inhibited with the addition of venom, allowed us to identify 28 proteins in the MCF7 cell line from which we highlight proteins: eukaryotic peptide chain release factor subunit 1 (ETF1), serine/arginine repetitive matrix protein 2 (SRRM2), PHD finger-like domain containing protein 5A (PHF5A), and lamina-associated polypeptide 2 (TMPO). The analysis of the MDA-MB-231 cell line allowed us to identify 41 proteins exclusively expressed at the control group from which we highlight Plasminogen activator inhibitor 1 (SERPINE1), MHC class I antigen (HLA-C), Transcription factor BTF3 (BTF3L4), Cytochrome c oxidase subunit 6C (COX6C), Myb-binding protein 1A (MYBBP1A), and MYCBP protein (MYCBP) (Figure S8a, Table S4).

Of the proteins identified exclusively when cells were treated with 0.63 μg/mL of venom there were 28 proteins from MCF7 cell line, from which we highlight Cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1), Latexin (LXN), Vesicle-associated membrane protein-associated protein B/C (VAPB), Cysteine and glycine-rich protein 1 (CSRP1), and DDB1- and CUL4-associated factor 7 (DCAF7). From MDA-MB-231 cell line, 20 proteins were identified from which we highlight Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), low molecular weight phosphotyrosine protein phosphatase (ACP1), Transcription factor A, mitochondrial (TFAM), and Rae1 protein homolog (RAE1) (Table 1 and Table S4, Figure S8b).

Proteins identified exclusively at the high dose treatment of 2.5 μg/mL of venom included 17 proteins detected in the MCF7 cell line, from which we highlight 26S proteasome non-ATPase regulatory subunit 6 (PSMD6) and 60S ribosomal protein L36 (RPL36). Similarly, we identified 44 proteins in MDA-MB-231 cell line from which we highlight protein Ubiquitin carboxyl-terminal hydrolase (HEL-117) (Figure S8c, Table S4).

We also identified proteins that were expressed exclusively when cells were treated with low and high doses of *B. jararaca* venom. From the MCF7 cell line, we identified 40 proteins from which we highlight: Serine/threonine-protein kinase ATR (ATR) and Jupiter microtubule-associated homolog 1 (JPT1). From the MDA-MB-231 cell line, we identified 22 proteins from which we highlight Angiomotin (AMOT) and Small nuclear ribonucleoprotein G (SNRPG) (Table 1, Tables S3 and S4, Figure S8d).

Among the proteins that were identified in the control group and at the 2.5 μg/mL venom treatment, we highlight: Small nuclear ribonucleoprotein Sm D1 (SNRPD1) in MCF7, and Epididymis secretory protein Li 71 (Hel-S-71) and Isoform Far upstream elementbinding protein 3 (FUBP3) in MDA-MB-231 cell line (Figure S8e, Table S4). Of proteins that were identified in the control group and low 0.63 μg/mL dose of venom, we highlight Serine/arginine-rich splicing factor 10 (SRSF10) in MCF7 cell line, and Histone H2B type 2-E (HIST2H2BE), 60S ribosomal protein L34 and L37a (RPL34, RPL37A) in MDA-MB-231 cell line (Figure S8f, Tables S3 and S4).

#### **3. Discussion**

The present study successfully identified cancer-related proteins that undergo significant changes upon *B. jararaca* venom treatment of MCF7 and MDA-MB-231 cells, including SNX3, HEL-S-156an, UQCRC1, RPL36, and ATR identified in MCF7 cell line and H3C15/HIST2H3, HEL-S-132P, MTCH2, TFAM, KCTD12, RPL34, and RPL37A identified in MDA-MB-231 cell line, and histone H3F3B and LAP3 in both cell lines.

With the discovery of rattlesnake venom's antitumor activity in 1931 by Essex and Priestley [115] and later the angiotensin-converting enzyme inhibitor drug Captopril in 1981, developed originally from *B. jararaca* snake venom [116–118], several groups have been focusing on to the therapeutic potentials of bioactive compounds in snake venoms by isolating and characterizing components of the venom and analyzing their pharmacological potential that may lead to the development of more efficacious drugs [119,120].

Snake venoms have a complex mixture of proteins that can account for 95% of the total dried weight. Metalloproteases, serine proteases, LAAOs, and PLA2s are some of the most abundant proteins present in bothropic venoms, and the toxic action of these proteins are responsible for myotoxic effects, disruption in the coagulation cascade through thrombin-like proteins (serine proteases), imbalance in the blood homeostasis by the action of some metalloproteinases, apoptosis induction, and changes in the protein expression in oxidative stress and energy metabolism by the increase of Ca2+ influx. Several studies have revealed that these enzymes are critical regulators of cancer pathologies in several types of cancers including breast cancer [121–127]. Moreover, it has been previously shown that *B. jararaca* venom may have an important antitumor effect on Ehrilich ascites tumor cells in vivo and in vitro [36].

Aiming at the drug development, several groups have used proteomics approach to observe up- or down-regulated effects of proteins resulting from disease activity or side effects of the treatments [44,45,128–131]. Moreover, the quest for the identification of biological markers or biomarkers using venom that may help in the early detection of pathologies such as cancer, and the quest for products able to evaluate the metastatic potential and propose new forms of treatment to tumors, remains an important goal of research groups and pharmaceutical companies.

Proteomics studies using tumor cell lines has been performed to systematically characterize protein complexes that may be important in disease or drug development, comparing the protein levels of cells in normal versus pathological situations [132]. Studies have shown that venoms from several snakes such as *Calloselasma rhodostoma*, *Macrovipera lebetina* and *Bothrops mattogrossensis* significantly inhibit cell viability, either with crude or fractionated venoms and in different tumor cell lines such as LS174T (colorectal adenocarcinoma), HCT116 (colorectal carcinoma), HT29 (colorectal adenocarcinoma), HEL92.1.7 (erythroleukemia), and SK-BR-3 (breast adenocarcinoma) cell lines [133,134].

To evaluate the cytotoxicity of *B. jararaca* venom, we tested two different invasive breast cancer cell lines with different phenotypic and genotypic differences, MCF7 and MDA-MB-231 cell lines. They were treated with different venom concentrations ranging from 0.32 μg/mL to 20 μg/mL. When treated with over 10 μg/mL of venom both MCF7 and MDA-MB-231 cell lines showed rounded and irregular morphologies and the MCF7 cell line detached from the plate. These results are similar to the results observed by Bernardes-Oliveira et al. (2016) when testing 50 μg/mL of venom from *B. jararaca* and *Bothrops erythromelas* for 48 h on SiHa HPV-16 and HeLa cells, both derived from cervical tumors. They observed that the cells became more rounded, with gradual size reduction and detachment from the cell monolayer [135]. In addition to the morphological analyses, we evaluated cell viability through mitochondrial cell activity using WST-1 assay for cell proliferation and viability. We observed that cell death of both MCF7 and MDA-MB-231 cell lines started with 2.5 μg/mL of *B. jararaca* venom treatment. Based on both morphological and cell viability analyses, we selected the concentrations of 0.63 μg/mL (low sub-toxic dose) and 2.5 μg/mL (high sub-toxic dose) for further proteomic analyses.

Semi-quantitative proteomics analysis of the MDA-MB-231 cell line treated with the low 0.63 μg/mL and the sub-lethal dose of 2.5 μg/mL of *B. jararaca* venom presented less proteins with FC ≥ 1.5 when compared to the MCF7 cell line. We identified 137 proteins with FC ≥ 1.5 at 2.5 μg/mL venom treatment in MCF7 while 34 proteins in MDA-MB-231 at 2.5 μg/mL venom treatment. At the low 0.63 μg/mL venom treatment, 25 proteins in MCF7 presented FC ≥ 1.5 and 16 proteins in MDA-MB-231. However, MDA-MB-231 showed more proteins with FC ≤ 0.67 compared to MCF7 cell lines. MDA-MB-231 showed 28 proteins presenting FC ≤ 0.67 at 0.63 μg/mL venom treatment and 19 proteins in MCF7; and 41 proteins with FC ≤ 0.5 at 2.5 μg/mL venom treatment in MDA-MB-231 cell lines compared to 23 proteins in MCF7 cell line. Among these proteins, our data showed that *B. jararaca* venom was able to modulate, up or down, the abundance of several proteins playing essential roles in tumorigenicity. For example, the venom treatment increased the abundance of SNX3, HEL-S-156, MCCC2, and GSTM3 in the MCF7 cell line, and H3C15/HIST2H3 and MTCH2 in the MDA-MB-231 cell line whose overexpression has been related to increase in tumorigenicity (Table 1). Furthermore, among the proteins that decreased the abundance presenting FC ≤ 0.67, we highlight PCBD, PSMD5, RPS29, H3F3B, VDAC1 and VDAC2, ATP5PD, and HIST1H4J in MCF7 cell line and IGF2BP1, THBS1, and CYR61 in MDA-MB-231 cell line. These proteins have also been described to be related to several mechanisms and pathways in the onset of different types of cancer (Table 1). Additionally, cytotoxicity analysis performed in our laboratory (data not shown) evaluated the effects of *B. jararaca* venom on non-tumor HEK293 and HUVEC cell lines, which showed relatively less sensitivity to cell-death induction by snake venoms compared to MCF7 and MDA-MB-231 cancer cells. These data suggest that cancer cells could be more sensitive to snake venom components than non-cancer cells in certain situations.

Proteomic analysis also revealed exclusive proteins, i.e., proteins that were only identified in one or two of the three conditions analyzed. Among these proteins, we highlight the UQCRC1, identified in the MCF7 cell line only when these cells were treated with 0.63 μg/mL venom. High expression of this protein was observed in 74% of cases of breast cancer and 34% of ovarian cancer [104] (Table 1). Among proteins identified exclusively in the MCF7 cell line we highlight ATR (serine/threonine-protein kinase ATR) which plays important roles for cell survival and is considered a major mediator of DNA response in human cells, preventing cells with damaged or incompletely replicated DNA from entering mitosis when cells are damaged by radiotherapy or chemotherapy during cancer treatment [53]. In the MDA-MB-231 cell line, we highlight AMOT (angiomotin) protein identified only when cells were treated with either low or high concentration venom treatment and is known to play a critical role in angiogenesis, proliferation, and migration

of cancer cells and is also known to promote proliferation and invasion of several types of tumor cells, including breast, prostate, colon, cervical, and liver cancer (Table 1).

Semi-quantitative proteomics analysis of proteins with FC ≥ 1.5 after addition of venom in both MCF7 and MDA-MB-231 cell lines also identified enrichment of proteins related to protein metabolism pathways as well as cellular stress response pathway. The protein metabolism pathway ranges from protein synthesis to post-translational protein modification and degradation [136]. It is likely that the set of modifications undergone in the protein metabolism pathway undermine the tumorigenic activity of the cell line MCF7 and MDA-MB-231. Therefore, the activation of the apoptotic pathways using snake venom may be a potential treatment approach aiming at tumor degradation preferentially over the normal cells. The ribosome pathway enrichment was also observed in both cell lines. The lack of ribosomes in the cell impairs cell growth even under optimal conditions for it to happen [137]. Protein enrichment in ribosome pathway suggests increased protein synthesis, which may be related to damage repair mechanisms and cellular responses to external stimuli. Alterations in ribosomal pathways are related to cellular alterations and susceptibility to cancer. However, there is still no consensus if deregulation of ribosomes alteration is a cancer consequence or cause [138]. In addition, downregulation of cell maintenance-related proteins may also be related to tumorigenicity, thus opening the door for debate on the role of ribosomes in tumor cell lines [139].

The increase of terms involved in cellular stress response pathway, the metabolic pathway, the proteasome pathway, and the spliceosome pathway in MCF7 cell line suggests that the cells activated the metabolic pathways in response to stress, mRNA transcription and spliceosome activation, and ultimately activation of the proteasome. The proteasome is a cell apparatus that has several cellular functions, such as cell cycle regulation, differentiation, signal transduction pathway, antigen processing for immune responses, stress signaling, inflammatory responses and apoptosis, the latter being mediated by ubiquitination [140–142]. Since the inhibition of proteasome is related to cancer onset (i.e., p53 stability), proteolytic activity of the proteasome and its role in both biology and cancer treatment suggest an effective way to treat cancer [143,144].

In the MCF7 cell line treated with *B. jararaca* snake venom, a higher number of proteins changed in abundance when compared to the MDA-MB-231 cell line. Some of our observations showed that *B. jararaca* venom was able to up or down modulate the abundance of proteins that plays essential roles in tumorigenicity, conversely to what has been frequently described in the literature to decrease or increase cell tumorigenicity [113,145]. For example, the venom treatment was able to increase abundance of SNX3, HEL-S-156, MCCC2, and GSTM3 in the MCF7 cell line and H3C15/HIST2H3 and MTCH2 in the MDA-MB-231 cell line whose overexpression is related to increase in tumorigenicity. Although we cannot explain the mechanism on how the increase in abundance of these already overexpressed proteins is contributing to cell death of MCF7 and MDA-MB-231 cells, we have clues based on a recent published manuscript by Dias and colleagues (2019). The authors demonstrated that overloading the cell stress pathway in Y1 adrenocortical mouse tumor cell line under the effect of FGF-2 growth factor disrupted the cell homeostasis and sensitized the Y1 tumor cells to stress-oriented therapeutic inhibitors. Dias concludes that further stimulation of the same signaling pathways may further increase mobilization and dependence on stress response pathways in tumor cells, improving the efficacy and selectivity of therapeutic interventions [146]. This data suggests that *B. jararaca* snake venom or some of its content may be used to down-modulate already known overexpressed proteins in cancer or overexpress oncogenic proteins that lead cells to stress, by disrupting the tumor homeostasis leading cells to death. This is particularly interesting for cancer types that are more resistant to the chemotherapeutic agents, such as the MCF7 cells. In this case, *B. jararaca* snake venom would work as an adjuvant treatment of cancer, making cells more sensitive to the cytotoxic agents.

Moreover, due to the complexity of *B. jararaca* venom and the complex response of different cell lines, this snake venom is also a promising candidate in the prognostic aid of different tumors, assisting in the assessment of tumor level, as highlighted for the several overexpressed proteins. For example, targeted-knockdown of some of these proteins may result in tumor growth inhibition. In addition, *B. jararaca* venom has induced overexpression of proteins related to pathways that impairs antitumor activity such as the protein metabolism pathways, proteasome, and cellular stress response pathways.

It is possible that many responses observed here were due to specific or multiple venom components. Nevertheless, the current approach of applying nLC-MS/MS proteomics to cancer cells treated with crude snake venom is a promising strategy for the identification of proteins with potential application in cancer cell therapy. Overall, this study was able to identify several cancer-related proteins that undergo significant changes upon venom treatment of MCF7 and MDA-MB-231 cells. Future studies should address specific mechanisms by which the snake venom and some of its content may contribute to MCF7 and MDA-MB-231 cell death and survival.
