br also promoted a significant Sub G cell cycle arrest
also promoted a significant Sub-G1 PF 06424439 arrest and diminution in the G1 phase and augmentation in the G2-M phase at 24 h post-treatment of MDA-MB-231 cells . The antimetastatic effect of BthTX-II on MDA-MB-231 cells was de-lineated by means of the cellular adhesion, migration, invasion and 3D growth, which are important events in the metastatic process. Previous reports described that human breast cancer invasion and progression correlate with higher levels of the components of the extracellular ma-trix (ECM), such as collagen [49–51]. The cellular cytoskeleton is con-nected with the ECM through protein embedded into the cell
membrane, called integrins . Thus, integrins are key components during cell adhesion, migration and dissemination processes. Interest-ingly, BthTX-II treatment decreased the protein and gene expression of important integrins (α2, β1, αvβ3). Integrin β1 supports cell migra-tion whereas αV-β3 seems the most important during tumor angiogen-esis [50,51]. Remarkably, as seen in previous experiments, BthTX-II was able to inhibit cell adhesion both on collagen and fibronectin, corrobo-rating with the finding that the expression levels of β1 and α2 integrins decreased upon treatment [53,54].
MVL-PLA2, a phospholipase A2 from Macrovipera lebetina, efficiently inhibits tumor cell adhesion and migration by affecting the function of adhesion receptors on substrates such as fibrin and fibronectin through integrins αvβ3 and α5β1 respectively . Another study proved that PLA2 CC-PLA2-1 and CC-PLA2-2 from Cerastes cerastes Tunisian snake venom inhibited in a dose-dependent manner adhesion of IGR39 mela-noma and HT108lk0 fibrosarcoma cells to fibrinogen and fibronectin .
EMT is an essential event in the acquisition of the invasive and met-astatic potential by cancer cells, and it is considered a hallmark of cancer . For this reason, we investigate the modulation of some epithelial and mesenchymal markers in MDA-MB-231 cells. The treatment de-creased MCAM gene expression, a cell adhesion molecule, present on the surface of cells identified as EMT inducer and of mesenchymal stemness. The up regulation of MCAM is associated with cancer progres-sion . BthTX-II decreased significantly TWIST1 and CTNNB1 genes expression; these molecules contribute to tumor metastasis and are overexpressed in a variety of tumors . CDH1 gene expression was upregulated and E-cadherin protein level increased by BthTX-II. E-cadherin is a key molecule to establish the adherent junctions of epithe-lial cells acting to decrease the aggressiveness of TNBC cells, i.e., the EMT. E-cadherin downregulation is associated with certain malignant characteristics, including tumor progression, loss of differentiation,
Fig. 5. Effect of BthTX-II on MDA-MB-231 cells and analysis of EMT marker expression (A) BthTX-II treatment promoted an up-regulation of CDH-1 gene (E-cadherin) and a significantly down-regulation of MCAM, CTNNB1 and TWIST1, compared to the control group. (B) Analysis by flow cytometry of vimentin, E-cadherin and CK-5 after treatment BthTX-II (10 and 50 μg/ ml). (C) Western blot analysis of vimentin and actin protein after treatment BthTX-II (10 and 50 μg/ml). The bands, were quantified by ImageJ software and represented in the histogram on the side. A representative scan is shown at the top of each mean (n = 3); bars, SE, all data are expressed as mean ± S.E.M. and all experiments were carried out in triplicate; differences between treatments and controls were analyzed by Unpaired t-test. Statistically significant: **p b 0.001.
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 cell cycle 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 , suggesting that CTX III also potentially reverses the EMT process in MDA-MB-231 cells . 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.