ERK1/2 activity was measured using Muse? Cell Analyzer and Muse? MAPK Activation Dual Detection Kit (Merck Millipore). Taken together, we have shown for the first time that UA at low micromolar range may promote its anticancer action by Leuprorelin Acetate targeting glycolysis in phenotypically unique breast malignancy cells. indicate SD, n?=?5, *** test). b The percentage of cells in the G0/G1, S and G2/M phases of cell cycle was assessed using Muse? Cell Analyzer and Muse? Cell Cycle Kit. Representative histograms are shown. c Western blot analysis of the levels of p21, p27 and p53 cell cycle inhibitors. Anti–actin antibody was used as a loading control. The data represent the relative density normalized to -actin. d Senescence-associated -galactosidase (SA–gal) activity. indicate SD, n?=?3, *** test). ursolic acid, betulinic acid Both triterpenotids significantly affected breast malignancy cell metabolic activity when used at the concentration as low as 2.5?M and 24?h treatment (Fig.?1a). UA was found to be more harmful than BA and calculated IC50 values were 20.44, 22.9 and 14.58?M for MCF-7, MDA-MB-231 and SK-BR-3 cells and UA treatment, and 29.02, 30.58 and 24.47?M for MCF-7, MDA-MB-231 and SK-BR-3 cells and BA treatment, respectively (Fig.?1a). Three concentrations of UA and BA, namely 5, 10 and 20?M were selected for further analysis (Fig.?1a). UA also affected the cell cycle of breast malignancy cells more than BA (Fig.?1b). UA and Rabbit polyclonal to AMPK gamma1 BA (10?M) promoted accumulation of cells in the G0/G1 phase of the cell cycle (Fig.?1b). An increase of 11.8, 6.5 and 11% in the levels of MCF-7, MDA-MB-231 and SK-BR-3 cells in the G0/G1 phase of the cell cycle and an increase of 6.5, 4.2 and 3.3% in the levels of MCF-7, MDA-MB-231 and SK-BR-3 cells in the G0/G1 phase of the cell cycle were observed after 10?M UA and 10?M BA treatments, respectively (Fig.?1b). UA- and BA-mediated effects on selected cell cycle inhibitors, namely p53, p21 and p27 were then analyzed (Fig.?1c). Both triterpenotids caused an increase in p53 levels in MCF-7 cells (wild type p53) and exerted minimal effect on p53 levels in MDA-MB-231 and SK-BR-3 cells (mutant p53) (Fig.?1c). In contrast, an increase in p21 levels in all cells examined was noticed (Fig.?1c). Except of UA-treated Leuprorelin Acetate MCF-7 and SK-BR-3 cells, treatments with Leuprorelin Acetate UA and BA did not significantly impact the levels of p27 (Fig.?1c). 24?h stimulation with 5 and 10?M UA and BA also resulted in senescence-associated beta-galactosidase (SA–gal) activity after 7 days of UA and BA removal (Fig.?1d). Higher levels of SA–gal positive cells were observed after UA treatment than after BA treatment (Fig.?1d). The most potent effect was observed after treatment with 5?M UA that resulted in 2.77-, 2.76- and 4.29-fold increase in SA–gal positive MCF-7, MDA-MB-231 and SK-BR-3 cells compared to untreated controls, respectively (Fig.?1d). UA induces apoptosis in breast malignancy cells We also evaluated if UA and BA may induce apoptotic cell death in breast malignancy cells (Figs.?2, ?,3).3). Three markers of apoptosis were considered, namely phosphatidylserine externalization (Fig.?2) and multicaspase and mitopotential assays (Fig.?3). Open in a separate windows Fig. 2 UA- and BA-induced apoptosis in breast malignancy cells (part I). Annexin V staining. Muse? Cell Analyzer and Muse? Annexin V and Dead Cell Assay Kit (Merck Millipore) were used. UA and BA did not promote apoptosis in normal human mammary epithelial cells (HMEC). ursolic acid, betulinic acid Open in a separate windows Fig. 3 UA- and BA-induced apoptosis in breast malignancy cells (part II). Multicaspase assay Leuprorelin Acetate (a) and mitopotential assay (b). a Muse? Cell Analyzer and Muse? Multi-caspase Assay Kit were used. 30?min treatment with 10?mM hydrogen peroxide (HP) served as a positive control (C+). b Muse? Cell Analyzer and Muse? Mitopotential Assay Kit were used. ursolic acid, betulinic acid UA (20?M) promoted phosphatidylserine externalization that was more accented in MDA-MB-231 (45.48% of cells) and SK-BR-3 cells (44.86% of cells) than.