br Results br For assessing the antitumor and antimetastatic
For assessing the antitumor and antimetastatic effects of BthTX-II on MDA-MB-231 cells, BthTX-II was firstly purified from B. jararacussu venom by CM Sepharose chromatography, resulting in 6 fractions (Sup-plementary Fig. 1A), and its homogeneity (CM-5 fraction) was con-firmed by reverse-phase chromatography (RP-HPLC) C18 (Supplementary Fig. 1B) and SDS-PAGE (Supplementary Fig. 1C).
BthTX-II was cytotoxic to MDA-MB-231 CL 316243 in a dose-dependent manner (Fig. 1A) and showed cytotoxicity of around 50% at the highest doses. Interestingly, in the same concentrations, BthTX-II did not signif-icantly affect the viability of non-tumorigenic MCF10A cells. Next, we investigated whether the cytotoxicity induced by BthTX-II in MDA-MB-231 cells could be explained by the activation of autophagy. Inter-estingly, by evaluating autophagy through the fluorescent reagent, MDC, we observed that BthTX-II (50 μg/ml) was capable of inducing au-tophagy in approximately 70% of MDA-MB-231 cells and in about 30% of control breast cells (MCF10A) (Fig. 1B).
We proceed evaluating the effects of BthTX-II (50 μg/ml) treatment on gene expression. BthTX-II was capable of modulating the expression of genes involved in the signaling apoptosis pathway (Fig. 1D). The re-sults showed that TNF and its receptor TNFRSF1A genes were upregu-lated by 6.26-fold and 8.42-fold, respectively. The transcriptional levels of the tumor suppressor genes BRCA1 and BRCA2 were respec-tively 3.79 and 1.42-fold increased upon treatment with BthTX-II. The BthTX-II stimulus induces the activation of apoptosis through upregula-tion of CASP8/TP53 of 1.91-fold and 2.42-fold, respectively. The MDM2 (E3 ubiquitin-protein ligase) and ANGPT1 (pro-angiogenic factor) were downregulated by 1.56 and 5.88-fold respectively. However, when compared to the control, BthTX-II treatment did not change sig-nificantly the expression of some genes involved in the apoptosis-signaling pathway, namely BAD, BAX, BCL2, BCL2L1, BIRC5, TNFRSF10B. The LDH activity was observed in MDA-MB-231 cells after treatment with BthTX-II (50 μg/ml) for 24 h, when compared to cells treated with Triton X-100 and with Control (untreated cells) (Fig. 1E).
3.3. BthTX-II inhibits cell proliferation and promotes G2-M cell-cycle arrest in MDA-MB-231 cells
We also verified the effect of BthTX-II at different concentrations (1, 10, 25 and 50 μg/ml) on the proliferation of MDA-MB-231 cells (Fig. 2A). Even the lowest dose significantly inhibited cell proliferation after 72 h of treatment, when compared to the positive control. In addition, we accessed the cell cycle. Once treated with BthTX-II (10 and 50 μg/ml),
Fig. 1. BthTX-II induces cell deaths in MDA-MB-231cells (A) Cytotoxicity by MTT assay. MDA-MB-231 and MCF10A cells treated with BthTX-II (100, 50, 25, 12.5, 6.25, 3.125 and 1.56 μg/ ml). (B) Autophagic vacuoles on MDA-MB-231 and MCF10A cells stained with MDC after exposure to BthTX-II (50 μg/ml) for 24 h. The autophagosomes (blue) detected in MDA-MB-231 cells after treatment and the percentage of cells that present autophagic vacuoles were analyzed by ImageJ software. The white bars represent 200 μm as a reference scale. (C) Analysis of apoptosis by flow cytometry. Cells MDA-MB-231 treated with BthTX-II (10 and 50 μg/ml) or control for 24 h incubated with Annexin V/FITC and propidium iodide. The representative dot-plot acquisitions show the distribution of MDA-MB-231 cells presenting necrosis (upper left quadrant), early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant).
(D) Expression of apoptosis pathway genes by real-time PCR compared to control; these genes presented no difference (BAD, BAX, BCL2, BCL2L1, BIRC5, TNFRSF10B) while TNF, TNFRSF1A
and CASP8 were upregulated and ANGPT1 was downregulated. (A) Effects of BthTX-II (50 μg/ml) on lactate dehydrogenase (LDH) levels in MDA-MB-231 cells after 24 h. All of the experiments were performed in triplicate, the data were expressed as mean ± S.E.M., differences between treatments and controls were analyzed by One-way, Two-way ANOVA and Unpaired t-test. Statistically significant, statistically significant difference (p b 0.001) compared with MCF10A treatment in Cytotoxicity assay or control.
MDA-MB-231 cells displayed a significant inhibition of cell cycle pro-gression. BthTX-II caused a relevant Sub-G0 cell cycle in MDA-MB-231cells (19.9% and 38%, respectively), when compared to control cells (5.35%) (Fig. 2B and C). Additionally, BthTX-II promoted a signifi-cant diminution of cells in the G1 phase and cell cycle arrest in the G2-M phase at 24 h post-treatment (Fig. 2B and C).
The expression of some genes involved in the cell cycle was also evaluated after treatment of TNBC with BthTX-II and this protein induce