Fig 1.
LCL521 represents an acute and potent inhibitor of ACDase.
(A) MCF7 cells were treated with vehicle, or with 0.1, 0.25, 0.5, 1, 1.5, 2.5, 5, and 10μM LCL521 for 1h. Cer, Sph and S1P were then extracted and quantified by LC-MS/MS. (n = 4, three times experiments with one time duplicates and two times single experiment). The actual amounts of Cer, Sph and S1P after treatment with LCL521 are presented in S1B–S1D Fig (B). MCF7 cells were treated with vehicle or 1μM LCL521 for 15min, 30min, 1, 2, and 5h. Sph was then extracted and quantified by LC-MS/MS. (*p<0.05, n = 7, 3 times experiments with 2 times duplicates and 1 time triplicates).
Fig 2.
Synergistic effect of LCL521 and single dose of ionizing radiation.
MCF7 cells were irradiated with 0 or 2Gy using 137 Cesium irradiator. 1h after IR, each replicated treatment were further treated with vehicle or 5x 1μM LCL521. For the 5-time treatment, media were replaced every 24h with fresh media that contained either vehicle or 1μM LCL521. After that, cells were cultured for 4 weeks and then stained with crystal violet (1g/500ml formalin). Representative crystal staining is shown. Colonies count is presented in S2 Fig.
Table 1.
IC50 values for B13 and LCL521.
Fig 3.
LCL521 demonstrates significant effects on MCF7 cells cytotoxicity and proliferation.
(A) Viable cell analysis. MCF7 cells were treated with vehicles, 0.78, 1.56, 3.125, 6.25,12.5, 25, 50, and 100μM of either B13 or LCL521 for 48h and then MTT assays were performed. The results are expressed as a percentage relative to untreated cells and are presented as means ± st dev. of single experiment with 4 time replicates. (B) Effect of LCL521 on MCF7 cell cycle. MCF7 cells were treated with vehicle or with 1, 2.5, 5, 7.5 and 10 μM LCL521 for 24h. Cells were fixed with 70% ethanol overnight before adding 500μL RNase and PI solution. Samples were kept in the dark for another 45min before the FACS analysis. (n = 4, two times experiments with duplicates in each. * p<0.01). Representative flow cytometric analyses are shown in S3 Fig.
Fig 4.
Synergistic effects of LCL521 and Tamoxifen on Tamoxifen resistant MCF7 cells.
Tamoxifen resistant MCF7 cells (TamR) were plated in 96 well plate overnight before treatment with 10μM Tamoxifen (final concentration is 5μM) for 1h, then treated with LCL521 with the final concentration at 0, 1, 2.5 and 5μM. To compare LCL521 its own killing effect, wild MCF7 cells were plated in 96 well plate at same time and varies dose of LCL521 were treated at the same time. Cells were kept for 48h before MTT assay was preformed. The results are expressed as a percentage of vehicle samples and presented as means ± st dev. of triplicates. * p<0.05 (vs 0); ** p<0.01 (vs 0); *** p<0.001 (TamR vs MCF7).