br BthTX II inhibits cell proliferation and promotes G M
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 Anthranilic Acid (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
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 cells 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).