rminations, or alternatively data are presented as mean6SEM of OD. Statistical significance was determined using Student’s t test. Results ACCA Inhibits Proliferation of Human Breast Cancer Cells in a Dose and Time-Dependent Manner Because ACCA is a known inhibitor of monocarboxylate transporters with about a tenfold higher afficacy for MCT1 as compared with others MCTs, we first determined by western blot analysis the expression of the MCT1 protein in immortal normal human breast epithelial cells, HBL100, and breast cancer lines, including MCF-7, MDA-231, and T47D. Consistent with previous studies, we found that MCT1 protein is elevated in all three breast cell lines compared to HBL100 immortal breast cell line. We next investigated the effect of ACCA on cell viability. MCF-7, MDA-231, and T47D breast cancer cells were treated with vehicle or 50 mM of ACCA ACCA Affects Breast Cancer Cell Growth for different time intervals and cell viability was evaluated by trypan blue dye exclusion method. A shown in Fig. 2A, ACCA exhibited a significant get PD-1/PD-L1 inhibitor 2 reduction in cell viability across MCF-7, MDA-231 and T47D cell lines treated with 50 mM of ACCA for 1, 2, 3, 6 or 10 days as compared 10336542 to control cells. In contrast to malignant breast 2173565 tumor cells, treatment of immortal normal human breast epithelial cells, HBL-100, with vehicle or the same concentration of ACCA did not influence the cell growth of HBL100 cells. To confirm these data, MCF-7, MDA-231, and T47D breast cancer cells were treated with various concentrations of ACCA, ranging from 25 to 200 mmol/L for 24 and 48h. and cell growth was evaluated by MTT reduction assay. As shown in Fig. 2B, there is no significant difference in growth inhibition in all tumor cell lines treated with a 25 mM ACCA for 24h compared with untreated cells. However, and consistent with the data obtained above, on treatment of cells for 48 h. with increasing concentrations of ACA, tumor cell growth was strongly inhibited in a dose-dependent manner. This suppressive effect of ACCA on tumor cells was most remarkable at the concentration of 200 mM, 150 mM and 50 mM for MCF-7, T47D, and MDA-231 cells, respectively. In contrast, treatment of HBL100 immortal breast cell line, with increasing concentrations of ACCA, do not affect the growth of MDA-231, T47D and MCF-7 
cells. In total, these results suggest that ACCA acting through MCT1 selectively inhibits the growth of breast cancer cells in vitro. We next questioned whether the growth-inhibitory effect of breast cancer cell proliferation persists on removal of ACCA. MDA-231 cells were treated with medium alone or containing 50 mM of ACCA for 48 h., and proliferation was assessed by MTT assay. Our data show that, consistent with our previous results, ACCA significantly inhibited tumor cell proliferation. Plating medium was removed and replaced with growth medium containing 10% of FBS, and cell growth was monitored for a further 4 days. Our data show that the suppressive effects of ACCA at day 4 were similar to that observed at d0,. These results clearly demonstrate that the inhibitory effect of ACCA on cell proliferation persists, even on removal of the drug. Induction of Apoptosis in Human Breast Cancer Cell Lines Correlates with an Elevation in Bax Levels We next determined whether ACCA decreases cell viability of breast cancer cells through the induction of apoptosis. Thus, cells were treated for 48h with 200 uM of ACCA and phosphatidylserine translocation was measured by f