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Fig. 3. Extracellular L-amino Fer-1 profiles. Changes of (A) essential amino acid, (B) non-essential amino acid levels in the media with MCF-7 cells (red: high glucose medium; green: normal glucose medium) and MCF-10A cells (white: MEGM). Values are shown as percent change of L-amino acid in the medium after 72-hours incubation from the uncultured medium, with negative bars indicating cellular consumption and positive bars indicating production. * indicates non-chiral amino acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Cellular uptake of all essential amino acids was anticipated, as essential amino acids cannot be synthesized in mammalian cells and must be acquired from the growth media (Fig. 3). On the other hand, the net flux of nonessential amino acids was unknown. Although they can be produced in mammalian cells from glycol-ysis, glutaminolysis, or the TCA cycle, extracellular nonessential amino acids can be readily utilized by the cells to reduce the biosyn-thetic burden on the cells [18]. In this study, cellular uptake of Gly and l-Asp, two nonessential amino acids, was detected for MCF-7 cells. In contrast, release of Gly and l-Asp were observed for MCF-10A cells, suggesting metabolic differences between MCF-7 and MCF-10A cells. Gly can be synthesized from l-Ser, and both are involved in one-carbon metabolism providing methyl groups for the biosynthesis of nucleotides and cofactors [19]. Although the uptake of l-Ser was seen in both MCF-7 and MCF-10A cell lines, recent evidence suggested that cancer cells were more reliant upon the uptake of extracellular Ser. It has been reported that depletion of exogenous l-Ser reduced cancer cell proliferation by affecting nucleotide synthesis [20]. Additionally, cancer cells have shown upregulated activity of the enzymes involved in l-Ser and Gly syn-thesis [21]. Downregulation of argininosuccinate synthase 1 (ASS1), using l-Asp as a substrate, has been reported in cancer cells [22]. Decreased ASS1 activity leads to the elevated l-Asp levels in can-cer cells, allowing l-Asp to be used for nucleotide biosynthesis and to support cancerous proliferation [22]. Nonessential amino acids, specifically Gly, l-Ser, and l-Asp could be promising targets in can- 
cer therapy, as they become necessary for cancer cell proliferation [23].
3.2.1. Intracellular and extracellular free d-amino acid levels Intracellular free d-amino acid profiles were determined in both
MCF-7 and MCF-10A cells (Fig. 4 and Tables S4–S12). d-Asp, d-Ser, and d-Glu had the highest levels in both cell lines, although they were 2–22 fold higher in MCF-7 breast cancer cells. Unlike the trend of l-amino acid levels observed between the two cell lines, MCF-7 did not always exhibit higher levels of d-amino acids com-pared with MCF-10A cells. Some d-amino acid levels were higher in MCF-7 cells, i.e., d-Asp, d-Ser, d-Asn, d-Ala, d-Thr, and d-Tyr. Oth-ers showed higher levels in MCF-10A cells, i.e., d-Val, d-Leu, d-Pro, d-Lys, and d-Trp, while a few had similar levels in both cell lines (Fig. 4). It is noteworthy that d-Pro, d-Lys, and d-Trp were found only in MCF-10A cells and only after 72-hour growth. MCF-10A cells showed higher percentages of d-enantiomers for almost all the amino acids, except d-Asp and d-Asn (Fig. 5).
Extracellular (i.e., growth media) free d-amino acid profiles were determined for MCF-7 and MCF-10A cells as shown in Fig. 6, and the absolute extracellular d-amino acid levels are shown in Supple-mentary material Tables S13–S24. Cellular uptake of all d-amino acids was found in MCF-10A cells except for d-Ser and d-Lys. Con-cerning MCF-7 breast cancer cells, cellular uptake was observed
Fig. 4. Intracellular D-amino acid profiles. D-Amino acid levels in MCF-7 and MCF-10A cells after (A) 24-hours, (B) 48-hours, and (C) 72-hours growth in the associated medium. Red bars represent MCF-7 cells grown in high glucose medium, green bars represent MCF-7 cells grown in normal glucose medium, and white bars represent MCF-10A cells grown in the MEGM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for d-Leu, d-Asp, d-Gln, d-Ala, d-Tyr, and d-Val, while cellular release was observed for d-Asn, d-Arg, d-Thr, and d-Ser. Surpris-ingly, MCF-7 cells exhibited net uptake of d-Ile, d-Glu, d-Phe, and d-Lys when grown at high glucose condition, but these amino acids were released from the MCF-7 cells at the normal glucose condition.
3.2.2. Altered d-amino acid profiles and metabolism for MCF-7 breast cancer cells