1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell

Abstract

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23 ± 2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-13C5] glutamine tracer kinetics, into the TCA cycle by 31 ± 0.2% and 17 ± 0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast cancer.

Metabolomic Analysis Reveals Vitamin D-induced Decrease in Polyol Pathway and Subtle Modulation of Glycolysis in HEK293T Cells

“…We observed a slight increase in intracellular lactate concentration in vitamin D-treated cells (Fig. 2 , Table S2 ), corroborating the subtle effect of vitamin D on glycolysis, which was already described for other cell models 10 , 11 , 31 . To better understand this effect, we measured the fate of glucose carbons by incubating HEK293T cells with 13 C-glucose and measuring 13 C-lactate accumulation in the culture medium.…”

“…It has been shown that vitamin D treatment inhibits glycolytic flux in metastatic ras transformed MCF10A cells, as shown by a decrease in 3-phosphoglycerate and lactate contents and a reduced activity of lactate dehydrogenase 10 . In the same cells, vitamin D inhibits glutamine metabolism, reducing glutamate and glutamine intracellular contents 11 . Additionally, vitamin D treatment increased insulin secretion in polycystic ovary syndrome (PCOS) patients 12 , and suppressed the expression of angiotensinogen induced by hyperglycemia by blocking NF-kB-mediated pathway 13 .…”

Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis

“…However, the metabolic homeostasis of individuals with oncogenic germline mutations (RASopathies) remains unclear. Recent studies have shown that cancer cells with RAS mutations exhibit altered metabolism characterized by enhanced glucose uptake, glycolysis, lipid synthesis and glutamine catabolism ( Bryant et al, 2014 , Zheng et al, 2015 , Zhou et al, 2016 ). To gain an understanding of the effects of RAS activation on energy homeostasis in vivo , we generated knock-in mice expressing the Costello syndrome-associated Hras G12S mutation and examined energy homoeostasis in the liver.…”

Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

“…Treatment of MCF10A- ras cells with 1,25(OH) 2 D resulted in decreased glucose metabolism in the glycolytic pathway as well as its flux into the TCA cycle [20]. Additionally, 1,25(OH) 2 D inhibited glutamine uptake and its downstream flux into the TCA cycle in both MCF10A- ras and MCF10A HER2/neu (MCF10A- ErbB2 ) overexpressing cells, the latter being one of the most commonly amplified genes in breast cancer [22,23]. Given the importance of glucose and glutamine metabolism in redox balance, and the effect of 1,25(OH) 2 D on both of these pathways, the purpose of the following studies was to investigate the effect of 1,25(OH) 2 D on endogenous antioxidant defenses in early stages of breast cancer.…”