Figure 1.
Structure, scheme synthesis of α-Cyano-4-Hydroxy-3-Methoxycinnamic Acid (ACCA) and expression of MCTs in immortal human epithelial cells and human breast cancer cells.
(A) Structure and scheme of ACCA synthesis. (B) Expression of MCT1 in immortal normal human breast epithelial cell line, HBL100, and breast cancer cell lines MCF-7, T47D, and MDA-231. Lysates of the indicated cell type were analyzed by western blotting and stained with MCT1 antibody as described in «materials and method». Membranes were reprobed with EF-1α antibody to confirm equal loading.
Figure 2.
Effect of ACCA on the in vitro growth and proliferation rate of immortal human epithelial cells and human breast cancer cells.
(A) The indicated cell type were either untreated (control) or treated with 50 uM of ACCA and cell growth was assessed for 1, 2, 3, 6, and 10 days. The trypan blue exclusion test is used to determine the number of viable cells. Values represent number of cells x105. (B) The indicated cell type either untreated or treated with different doses of ACCA were seeded in 96 wells, and a standard MTT viability test was performed 24h and 48h. postreatment as described in «materials and methods». Columns, mean±SD, n = 3.
Figure 3.
Effect of ACCA on colony formation of human breast cancer cells.
The indicated cell type either untreated (UNTR) or treated with 25 or 200 uM of ACCA were allowed to grow for 3–4 weeks and the number of colonies formed was measured as «described in materials and methods». (A) Representative images of the cloning wells (A) and quantification (B) are shown. Columns, mean±SD, n = 3.
Figure 4.
Effect of ACCA in breast cancer cells.
The indicated cell type either untreated (UNTR) or treated with 200 uM of ACCA for 48h. were stained with FITC-labeled Annexin-V and propidium iodide (PI) and immediately analysed by flow cytometry. (A) Data from a representative of four experiments are shown. Cells in the bottom left quadrant 1 represent viable cells (low Annexin V and PI staining, AnV−/PI-); cells in the the bottom right quadrant 2 represent early apoptotic cells (high annexin V staining but low PI staining, AnV+/PI-);); cells in the top right quadrant 3 represent late apoptotic cells (low annexin V and high PI staining, AnV−/PI+);), and cells in the top right quadrant 4 represent necrotic cells (high annexin V and PI staining, AnV+/PI+). Shown are representative data from one of three independent experiments with samples in triplicate. (B) The percentage of early apoptotic cells (only Annexin-V stained), late apoptotic and necrotic cells was calculated using the CellQuest software (Becton Dickinson, San Jose, CA, USA). Columns, mean±SD, n = 3.
Figure 5.
Effect of ACCA on the levels of Bcl-2 family proteins in breast cancer cells.
Lysates of the indicated cell type either untreated (UNTR) or treated with 200 uM of ACCA for 48h. were analyzed by western blotting and stained with Bcl-2 and Bax antibodies as described in «materials and method». Membranes were reprobed with EF-1α antibody to confirm equal loading.
Table 1.
Effect of ACCA on the induction of apoptosis and necrosis in breast cancer cells.
Figure 6.
Effect of ACCA migration and invasion in vitro, and breast cancer tumor growth in vivo.
(A) For invasion, MDA-231 cells (105) were seeded in the upper compartment of a Matrigel chamber in serum-free DMEM containing 0.1% BSA and allowed to invade for 48 h at 37°C in the presence of the indicated concentrations of ACCA. The lower compartment contained 0.5 ml of DMEM and 10% NuSerum. For migration, MDA-231 cells (5×104) were seeded in the upper compartment in serum-free DMEM containg 0.1% BSA and allowed to migrate for 15–18 h at 37°C in the presence of the indicated concentrations of ACCA. The lower compartment contained 0.5 ml of DMEM and 10% NuSerum. At the end of the invasion and migration assays, the filters were removed, fixed, and stained as indicated in “Materials and Methods.” Invasion and migration were determined by counting of the cells that had migrated to the lower side of the filter with a microscope. Ten fields/cell line were counted. Bars, SD of triplicate samples from three independent experiments. The migration and invasion of control cells was used as a positive control (set at 100%). «UNT» refers to untreated cells. (B) MDA-231 cells (2×106) mixed in a 1∶1 ration with Matrigel were inoculated s.c. on the right flank of each nude mouse above the hind limb. One week after tumor inoculation, the mice were treated five times/week begining one week after subcutaneous tumor cell injection with vehicle (control) or ACCA (25 µmol in 200 µl). Tumor sizes at different times are expressed as the mean of the sum of two perpendicular diameters. Bars, SD of tumor volumes from five mice.