Fig. 1
Analysis of 1,25(OH)2D3’s metabolism-regulating effects in BCa cells. a Extracellular acidification, respiration, and impedance rates were monitored in real-time in response to 1,25(OH)2D3 (100 nM) over the course of 3 days, followed by a 20-h recovery period, in which cells were exposed to running medium (RM) not containing the molecule. 1,25(OH)2D3 clearly induces glycolytic rate in luminal (MCF-7 and T-47D) BCa cells and reduces respiration rate to varying degrees in TNBC (MDA-MB-231, MDA-MB-468, and HCC-1143) cells. Values of each cell line are normalized to measurements obtained from the respective DMSO-treated cells. Data presented are representative of 2 biological replicates. b GC/MS analysis of TCA cycle intermediates and amino acids in select BCa cell lines treated for 72 h with 1,25(OH)2D3 (n = 4). A strong accumulation in serine is observed with treatment in MCF-7 cells, whereas levels of citrate were differentially regulated by 1,25(OH)2D3 in both cell lines. PEP phosphoenolpyruvate, α-KG alpha-ketoglutarate. c mRNA expression analysis of metabolism-related genes in MCF-7 and MDA-MB-231 cells in response to a 72 h treatment with 1,25(OH)2D3. G6PD was found to be induced by 1,25(OH)2D3 in both cell lines. Relative expression was calculated using the ∆∆Ct method, with vinculin as the housekeeping gene. Data presented are the average of 2 biological replicates. G6PD protein expression is significantly induced in MCF-7 (d) and MDA-MB-231 (e) cells by 1,25(OH)2D3 (72 h), as demonstrated by western blot and densitometric analysis. Statistical comparison between DMSO- and 1,25(OH)2D3-treated cells was made using a two-tailed Student’s t test. P values less than or equal to 0.05, 0.01 and 0.001 are depicted by *, **, and ***, respectively. Error bars ± SD; n = 3. G6PD mRNA (f), protein (g), and activity (h) levels are induced by 1,25(OH)2D3 (72 h) in all BCa cell lines. i Intracellular ATP levels were assessed in BCa cells in response to 1, 2, and 3 days of treatment with 1,25(OH)2D3. Treatment significantly induced ATP levels in MDA-MB-231 cells in 48 and 72 h, however reduced ATP levels in MCF-7 at the latest time point