br The complex and multifaceted process that
The complex and multifaceted process that defines EMT has attracted much attention in cancer studies and is considered an essen-tial step in the formation of metastases. For these reasons, the search for new drugs capable of inhibiting this process is an essential field of biomedical research. In this context, the phospholipases A2 (PLA2) of snake venoms have been the subject of several studies demonstrating their antitumor potential. In particular, BthTX-I, a Lys49 PLA2 homo-logue of Bothrops jararacussu, was able to induce cell death in different tumor cell lines  and was able of interfering in the Ethylmalonyl Coenzyme A of a mouse melanoma cell line (B16F10), rat adrenal gland cells (PC-12) and human promyelocytic leukemia cells (HL-60) promoting a delay in the G0/G1 phase. BnSP-6, a Lys49 homologue PLA2 from Bothrops pauloensis, inhibited the migration and proliferation of the TNBC cells, MDA-MB-231, and induced cell death by apoptosis and autophagy . BnSP-6 resulted to be cytotoxic and genotoxic in MDA-MB-231, leading to DNA damage . MVL-PLA2 from Macrovipera lebetina inhibited, in a dose-dependent manner, the adhesion and migration properties of human microvascular-endothelial cells (HMEC-1) and also inhibited the angiogenesis process both in vitro and in vivo .
Although several studies have investigated the molecular mecha-nisms of snake toxins, including the snake venom phospholipases A2, on metastasis, more studies are needed to draw a more complete sce-nario . Therefore, this work aimed to evaluate the antitumor and antimetastatic effects of BthTX-II, an Asp49-PLA2 isolated from Bothrops jararacussu venom, on a TNBC cell subtype (MDA-MB-231). Here we presented the effects of promoting apoptosis, cell cycle arrest and inhi-bition of 3D growth of BthTX-II. We also verified the inhibitory effect of BthTX-II on cellular adhesion, migration, invasion and EMT process.
2. Experimental procedures
2.1. Venom collection
Bothrops jararacussu crude venom was collected from snakes kept at the Ceta serpentarium, Animal Toxin Extraction Center, Ltda. – CNPJ: 08.972.260/0001-30, Morungaba, SP, Brazil. This serpentarium has ob-tained proof of IBAMA registration and use of renewable natural re-sources (n° 2087163).
BthTX-II was purified from Bothrops jararacussu venom as previously described by Homsi-Brandeburgo (1988)  with minor modifica-tions. The crude venom (200 mg) resuspended in 2 ml of 50 mM ammo-nium bicarbonate NH4HCO3 buffer (pH 7.8) and centrifuged at 3000g for 10 min at 4 °C. The recovered supernatant was applied to previously equilibrated CM-Sepharose Fast Flow resin (Amersham Bioscience, Brazil) previously equilibrated and fractions of 1 ml/tube were collected at 6.5 ml/h (flow rate by collector Radifrac fraction Amersham Biosci-ence, Brazil), and monitored by Ultrospec 1000 spectrophotometer (Amersham Pharmacia Biotech, Brazil) by absorbance at 280 nm. After this, were verified the homogeneity of fractions containing BthTX-II PLA2 by reverse-phase chromatography RP-HPLC C18 column (GE Healthcare-Life Sciences, Brazil). The samples were stored −20 °C .
The human breast cell lines MCF10A (non-tumorigenic breast epi-thelial cell subtype) and MDA-MB-231 (Triple-Negative breast cancer subtype) cells were obtained from the American Type Culture Collection
(ATCC® HTB-26) (Manassas, VA, USA). The MCF10A cells (ATCC®− CRL10317) were grown in DMEM:F12 medium (Sigma, Brazil) supple-mented with 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 10 ng/ml EGF (Invitrogen, Brazil) and 100 ng/ml Ctx (Sigma, Brazil) whereas the MDA-MB-231 cells were grown in IMDM (Sigma, Brazil). All media con-taining 10% heat-inactivated bovine serum (Cultilab, Brazil) and 10 μg/ ml of gentamicin. Cell cultures were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 and all the strains used were kept free of mycoplasma by checking possible contaminations by PCR.
2.4. Cell viability by MTT assay
2.5. Cell proliferation assay
The cells (2 × 105 cells/well) were seeded in a 24-well plate and treated with BthTX-II (1, 10, 25 and 50 μg/ml), for 24, 48 and 72 h. After-wards, was added 30 μl MTT in each well and incubate (5 mg/ml, Sigma M2128, USA) for 4 h, at 37 °C in 5% CO2. To stop the process was added 150 μl DMSO (Sigma-Aldrich) to each well and the absorbance at 570 mm was measured by using a microplate reader (Molecular De-vices, Menlo Park, CA, USA). The experiment was carried out in triplicate and for each time, the treatments were performed in quintuplicate. The mean value of white was subtracted from the mean value of the treated cells. As positive control, cells cultivated in IMDM medium with 10% FBS were used, while as negative control cells cultivated in IMDM serum-free were used.
2.6. LDH cytotoxicity assay
Cytotoxicity induced by BthTX-II was assessed by LDH release into the culture medium. MDA-MB-231 cells were seeded in 96-well plates (3x104cells/well) and incubated with BthTX-II (50 μg/ml) for 24 h. After incubation, released LDH in the medium was measured with the LDH Cytotoxicity Assay Kit (Cayman Chemical Company) according to the manufacturer's instructions. The absorbances were read at 490 nm (Multiskan GO Thermo Scientific, USA). LDH activity was reported as 1 Unit (U) of LDH activity defined as the amount of enzyme that catalyzes the conversion of lactate into pyruvate to generate 1.0 mol of NADH and H+. This reaction catalyzes the reduction of the tetrazolium salt at 37 °C (μmol/min/ml). The formazan produced is proportional to the quantity of LDH released into the culture medium resulting from cytotoxicity.