br a downregulation of CCND CCNE CDC A E F
a downregulation of CCND1, CCNE1, CDC25A, E2F1, AKT1 and AKT3 genes. In contrast, a tumor suppressor gene responsible for initiation of cell-cycle arrest and DNA repair, ATM, was upregulated (1.34-fold) (Fig. 2D).
3.4. BthTX-II inhibits cellular adhesion, migration, invasion and 3D growth and modulates the expression of integrins of MDA-MB-231 cells
We next tested the ability of BthTX-II to inhibit the adhesion of MDA-MB-231 PF-06424439 on different matrixes. We observed that different concentrations of BthTX-II (2.5 μg/mL to 50 μg/mL) inhibited by approx-imately 45% to 60% on different substrates. When wells were coated with matrigel, there was 57% of inhibition, while when wells were coated with fibronectin or collagen there was, respectively, an inhibi-tion of 52% and 53% at 50 μg/mL, when compared to the untreated cells (Fig. 3A). BthTX-II at 10 μg/mL and 50 μg/mL significantly inhibited the 24-hour cell migration in the wound healing assay compared to cells incubated only medium (control) (Fig. 3B). The transwell migration assay confirmed the results of BthTX-II (10 and 50 μg/mL) to reduce MDA-MB-231 migration through the transwell (Fig. 3C) by approxi-mately 57% and 50%, respectively, in relation to the control cells. By using the Matrigel-transwell assay we further observed that BthTX-II inhibited 49%, 60% and 92% the invasiveness of MDA-MB-231 cells (Fig. 3D) at 1, 10 and 50 μg/mL, respectively. BthTX-II (1, 10 and 50 μg/mL) inhibited the spheroid cell formation in breast cancer cells (MDA-MB-231) by inhibiting 3D growth and tumorsphere formation when compared to non-tumorigenic MCF10A cells (Fig. 3E).
It is important to report that BthTX-II treatment not only decreased the protein expression of three important integrins – namely α2, β1, αvβ3 (Fig. 4A) – but also downregulated their respective genes (ITGα2, ITGα3, ITGα4, ITGαV, ITGβ1 and ITGβ3) (Fig. 4B).
3.5. Modulation of epithelial and mesenchymal markers in MDA-MB-231 cells
We investigated some genes and proteins expression involved in the invasion and metastasis process, mainly those involved in epithelial-mesenchymal transition (Fig. 5). BthTX-II (50 μg/mL) decreased MCAM, CTNNB1 and TWIST1 gene expression by 0.33, 2.22 and 0.000000104 fold respectively (Fig. 5A). Conversely, CDH1 gene expres-sion was upregulated 1.43 fold by BthTX-II (Fig. 5A).
BthTX-II (10 and 50 μg/mL) induced an increase of E-cadherin (Fig. 5B) and significantly decreased the expression of cytokeratin (CK-5) and vimentin expression in MDA-MB-231 cells (Fig. 5B and C re-spectively). The same inhibitory effect on vimentin and E-cadherin ex-pression was observed when EMT was previously stimulated in MDA-MB-231 with EGF and subsequently treated with BthTX-II at the same concentrations (Supplementary Fig. 2B and C, respectively).
TNBC is the most aggressive breast cancer subtype that lacks an effi-cient therapy and is one of the principal public health concerns. For three reasons this work aimed to describe the effects of BthTX-II, a basic Asp49-PLA2 from Bothrops jararacussu venom, on this type of breast cancer. Although previous works demonstrated that another PLA2 from B. jararacussu, BthTX-I, induced cytotoxicity and cell cycle ar-rest in various cancer cells, including human breast adenocarcinoma cells (SKBR3) [19,32–35], much work is still needed to explore the ef-fects of PLA2s from snake venom on breast cancer .
BthTX-II is a 14 kDa-PLA2 that displays a weak catalytic activity . Functional studies of this protein have shown diverse activities such as myotoxicity, inflammatory activities [32,38] and edematogenicity . This work showed that BthTX-II was cytotoxic to MDA-MB-231 cells in a dose-dependent manner similarly to another homologue of PLA2 (BnSP-6) from B. pauloensis venom . The cytotoxic potential of BthTX-II was significantly lower against MCF10A, a non-tumorigenic breast cell line, indicating that BthTX-II possesses specific antitumor effects.
To evaluate the cytotoxic mechanism induced by the BthTX-II, we first investigated the autophagy mechanism. Autophagy is a conserved catabolic process responsible for the degradation of organelles and pro-teins and can be the first response to stress stimuli ; not surpris-ingly, differentiation and developmental events that require autophagic stimulus can initiate death by apoptosis and activate the ex-pression of anti-apoptotic genes . In this context, BthTX-II induced autophagy in MDA-MB-231cells but did not induce this effect in control breast cells (MCF10A), showing selectivity for TNBC.
Multiple interactions have been described suggesting a link between the apoptosis and autophagy processes . Previous works showed that snake venom toxins activate autophagy and apoptosis in breast cancer [20,40,43]. Therefore, we investigated the involvement of the ap-optosis pathway in cell death. BthTX-II induced early and late apoptosis in MDA-MB-231 cells when compared to the control, as well as modu-lated the expression of apoptosis markers that characterize the extrinsic and intrinsic apoptosis pathways, TNF, TNFRSF1A receptor, tumor sup-pressor genes BRCA1 and BRCA2, CASP8 BIRC5, MDM2 and TP53 genes. The gene TP53 that encodes the P53 protein, a tumor suppressor, has been associated with the expression of pro-apoptotic genes in cancer cells . BnSP-6 also induced early and late apoptosis and upregulated different genes related to the apoptosis pathway, such as TNF, TNFRSF10B, TNFRSF1A and CASP8, and decreased expression of the anti-apoptotic genes BCL2 and BCL2L .