br Cytosolic lactate dehydrogenase LDH is

Cytosolic lactate dehydrogenase (LDH) is used as a marker of cell death for in vitro models since LDH is released through the compro-mised cell membrane. The finding that MDA-MB-231 cells treated with BthTX-II display a decreased LDH activity, may justify the presence of necrotic or late apoptotic cells observed in the present study. These results corroborate the data obtained for BthTX-I, which promoted cell death in various cancer cell lines by apoptosis and necrosis [19].
In the current study, BthTX-II inhibited cell proliferation and pro-motes G2-M Nicotinamide arrest in MDA-MB-231 cells. These data are con-sistent with that showed that BnSP-6 inhibits MDA-MB-231 cells proliferation at 24, 48 and 72 h [21]. Different components of snake venom showed a similar behavior, Macrovipecetin, a lectin from Macrovipera lebetina venom, inhibits proliferation, migration and inva-sion of SK-MEL-28 human melanoma cells [45].
Fig. 2. BthTX-II inhibits cell proliferation and promotes G2-M cell-cycle arrest in MDA-MB-231 cells (A) Proliferation assay by MTT. BthTX-II inhibited MDA-MB-231 proliferation at 24, 48 and 72 h (B) Cell cycle analysis. BthTX-II caused a significant G2-M cell-cycle arrest phase compared to control (C) Represents the percentage of cells in the various populations of the cell cycle after BthTX-II treatment. (D) qPCR genes expressed in signaling of molecules involved in cell cycle and DNA repair assessed after treatment with BthTX-II. The genes (CCND1, CCNE1, CDC25A, E2F1, AKT1, AKT3), were downregulated while the ATM gene was upregulated. The values represent the mean ± SD of three independent experiments, analyzed by Two-way ANOVA and Tukey's posttest statistically significant difference p b 0.001 compared with control.
Fig. 3. BthTX-II inhibits cellular adhesion, migration, invasion and 3D growth of MDA-MB-231cells (A) Interference of BthTX-II in adhesion of MDA-MB-231 cells, which were pre-incubated with BthTX-II (50, 25, 10, 5, 2.5 μg/mL) for 30 min and after treatment were seeded over collagen-, fibronectin-, matrigel-coated and uncoated wells. (B). 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). Using ImageJ we quantified the filled nude area for a 24-hour period in comparison to the control, a result represented by the scratch wound coverage. (C). Cell migration was determined by Transwell assay, representative images of the (stained with hematoxylin-panotic) migrated cells were counted under a microscope showing almost 100% inhibition of migration at 50 μg/mL, selected from three independent experiments. ***p b 0.001 compared with the control. (D). using the Matrigel-transwell assay we further observed that BthTX-II inhibited 49%, 60% and 92% the invasiveness of MDA-MB-231 cells at 1, 10 and 50 μg/mL, respectively. Values represent mean ± S.D. in five different fields of triplicate membranes, analyzed by One-way ANOVA,
***p b 0.001 and R-squared 0.92 compared with control. (E) BthTX-II (1, 10 and 50 μg/mL) was also capable of inhibiting 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. 4. BthTX-II modulates the expression of integrins. (A) Graphic representation of median values of plasma membrane-integrin expression in MDA-MB-231 cells. BthTX-II promoted a reduction in median fluorescence intensity of α2 αVβ3 and β1 integrins. (B) BthTX-II treatment promoted a downregulation of ITGα2, ITGα3, ITGα4, ITGβ2, ITGαV and ITGβ1 compared to the control group. The values represent the mean ± SD of three independent experiments, analyzed by Two-way ANOVA and Bonferroni posttest statistically significant difference (p b 0.001) compared with control.
We demonstrated that BthTX-II acts on the survival cells, prolifera-tion, and cell cycle progression and on the expression of genes involved in the cell cycle of MDA-MB-231cells. Several cyclin-dependent kinases (Cdks) and their cyclin partners control cycle progression. D-type cyclins, in association with CDK4 and CDK6, promote the G1-to-S-phase transition by phosphorylating the retinoblastoma protein (pRB), thereby releasing the transcription factor E2F, leading to the expression of a subset of proliferation-associated E2F target genes [46]. Cyclin D1 is overexpressed in a subset of human cancers including breast cancer [47]. According to our data, the analysis of cell cycle mechanism showed that BthTX-II-induced G2-M phase arrest mediated mainly by the downregulation of the cell cycle regulatory proteins CDC25A, Cyclin D1, Cyclin E and E2F-1. These results corroborated the effects of BthTX-