br Fig Signaling pathways A representative model of

Fig. 6. Signaling pathways. A representative model of antimetastatic and antitumoral effects of BthTX-II on a human TNBC (MDA-MB-231). The BthTX-II interferes on different genes and proteins involved in apoptosis through the activation and inhibition of genes TP53 gene, tumor protein p53; PI3K, phosphoinositide-3-kinase; AKT, serine/threonine kinase, Casp8, caspase 8; BAD/BAX. Associated agonist of cell death; BCL2/BCL2-L Bcl-2-like protein 1; MAP2K, mitogen-activated protein kinase cascade; metastasis and epithelial-mesenchymal transition by the activation of CDH-1, E-cadherin; CDC25A gene1, Class III Cys-based CDC25 phosphatases, CCNDI, Cyclin D1, BRCA1/2, breast cancer 1, early onset of Pladienolide B response. Represents a BthTX-II suggested target, blue arrows means up-regulation and orange arrows means down-regulation.
invasion and metastasis and the stimulation of activity of E-cadherin on the cell surface acting to inhibit metastatic progression [57,58].
Moreover, treatment with BthTX-II reduced two important proteins for the metastatic process in TNBC cells: vimentin (a type III filament that's expressed in mesenchymal cells) and CK-5, responsible for the worse prognosis of TNBC [59–61]. Previous works showed that the treatment of MDA-MB-231 cells with CTX III, a basic polypeptide iso-lated from Naja-naja atra reduces mesenchymal biomarkers (vimentin and N-cadherin), decreased SNAIL and TWIST1 expression and restores the level of epithelial biomarker (E-cadherin) in EGF-induced MDA-MB-231 cells. BthTX-II also altered these biomarkers in EGF-induced MDA-MB-231 cells as well as in cells treated free EGF (Supplementary data). Another study associated these effects to downstream activation of phosphatidylinositol 3-kinase (PI3K)/AKT and ERK1/2 [62], suggesting that CTX III also potentially reverses the EMT process in MDA-MB-231 cells [63]. The finding that BthTX-II decreased the expression of AKT1 and AKT3 in MDA-MB-231 cells, suggests that this PLA2 could inhibit the metastatic potential and EMT process by interfering in (PI3K)/AKT pathway.
Fig. 6 summarizes the expression of different key genes and proteins involved in cell cycle signaling pathways, apoptosis and metastasis in MDA-MB-231 cells. Briefly, we suggested that these effects might be triggered upon binding of BthTX-II to some cell receptors such as the epidermal growth receptor, or to membrane phospholipids or integrins, in agreement to what previously reported [22,64,65]. Such binding on cell surface of TNBC cells would interfere PI3K-AKT pathway, responsible for cell survival and escape from apoptosis. The down-regulation of im-portant genes such as CDC25A, CCND1, CCNE1, E2F1 and TWIST1 as well as CK-5 and vimentin proteins could also provoke the inhibition of cell cycle, proliferation, invasion and metastasis, and the expression of the CDH-1 epithelial gene (E-cadherin) emphasizes the inhibition of the 
metastatic phenotype, reducing the aggressiveness of TNBC. However, despite the relevance of our findings, the discovery of the BthTX-II tar-get in MDA-MB-231 cells and full elucidation of the BthTX-II mecha-nisms require further investigations that will be the focus of future studies.
5. Conclusion
In summary, the present work shed light on BthTX-II antitumor and antimetastatic effects on MDA-MB-231. BthTX-II induced cell death, in-hibition cell proliferation, adhesion, migration, invasion and 3D growth, reducing the aggressiveness of TNBC cell line. Besides, BthTX-II de-creased vimentin, TWIST1, CK-5 and increased E-cadherin (CDH-1) ex-pression leading MDA-MB-231 cells to assume a more epithelial phenotype. Finally, since BthTX-II showed a remarkable efficacy against the EMT and metastatic processes, we propose that this PLA2 may be a potential model for drug design that aims to develop innovative antitu-moral therapies against TNBC.