br Low percentages of most
Low percentages of most d-amino acids were observed in MCF-7 compared to MCF-10A cells. It was mostly due to the high lev-els of l-amino acids in MCF-7 cells, as the corresponding d-amino acid levels were not much different between these two cell lines (Figs. 4 and 5). However, significantly high amounts of d-Asp and d-Ser were determined in MCF-7 breast cancer cells, i.e., 3 to 22 times higher than in MCF-10A cells. High levels of intracellular d-Asp may be caused by the Protease Inhibitor Cocktail of d-Asp from extracellular sources (i.e., uptake of d-Asp was observed, Fig. 6) or the biosynthe-sis of d-Asp by aspartate racemase or a combination of both . Given the fact that d-Ser levels were increased in the growth media after cell incubation (Fig. 6), biosynthesis of d-Ser by serine race-mase may occur in the cultured cells and then released into the growth medium .
Questions arise as to whether the higher intracellular levels indi-cate an important or unique function of d-Asp and d-Ser for MCF-7 breast cancer cells. Besides the essential roles in the CNS, NMDA receptors have been implicated in regulating cancer cell growth
and division . It has been demonstrated that MCF-7 breast can-cer cells expressed functional NMDA receptors with NR1 and NR2 subunits, and blockage of NMDA receptors with antagonists inhib-ited proliferation and reduced viability of cultured MCF-7 cells . Interestingly, it has been determined that d-Ser binds to NMDA receptor NR1 subunits at the glycine binding site, and d-Asp binds to NMDA receptor NR2 subunits at the glutamate binding site [5–7]. As co-agonists of NMDA receptors, high levels of d-Asp and d-Ser as shown in our work may be required or at least beneficial for MCF-7 breast cancer cell proliferation since activation of NMDA receptors in breast cancer cells is important for maintaining cell growth and viability. As they have shown increased availability and have selectively accumulated in MCF-7 breast cancer cells, d-Asp and d-Ala could be potential oncometabolites for breast cancer. Fur-ther, accumulation of specific d-amino acids in cancer cells may be the consequences of upregulated racemases, as the presence of Ser racemase and Asp racemase has been reported in mammals [24,25]. Although further investigation and validation are needed for the proposed potential oncometabolites and the possible upregulated racemases for breast cancer, this study provides a new approach for breast cancer diagnosis.
d-Amino acids were found in MCF-7 and MCF-10A cells as well as in the cultured and uncultured media. Changes in extracellular d-amino acid levels were detected, indicating the release or uptake of d-amino acids by MCF-7 and MCF-10A cells (Fig. 6). Our results demonstrated that the transfer of d-amino acids occurred between
Fig. 5. Intracellular percent D-amino acid levels. Intracellular % D-Amino acids in MCF-7 and MCF-10A cells after (A) 24-hours, (B) 48-hours, and (C) 72-hours growth in their 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.)
Fig. 6. Extracellular D-amino acid profiles. Percent changes of D-amino acids 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 d-amino acid in the medium after 72-h incubation from the uncultured medium, with negative bars indicating cellular consumption and positive bars indicating production.
cells and growth media. Amino acids are hydrophilic molecules and cannot cross the cell membrane without the aid of amino acid transporters. It has been reported that most of the trans-porters show high stereoselectivity, and only a few transporters have been shown to transport d-amino acids, e.g., LAT1, ASCT1, ASCT2, ATB0,+, and EAAT [27–29]. They have shown selectivity to d-Leu, d-Ser, d-Met, d-Phe, and d-Asp. Cellular release of d-Ser (observed in our study) through the less stereoselective amino acid transporters is likely to be important in regulating extracellular lev-els of d-Ser, which activate NMDA receptors and further affects breast cancer cell proliferation. It has been determined that cancer cells express some amino acid transporters at high levels to satisfy their increased demand for amino acids . Interestingly, all the transporters mentioned above have been shown to be upregulated in cancer cells. This further supports the possibility that altered d-amino acid profiles may be a metabolic adaptation to breast cancer cell proliferation. Thus, reducing the availability of the potential oncometabolites, d-Asp and d-Ser, to breast cancer cells could be a possible anticancer strategy. Future studies could explore more on the inhibition of the enzymes/pathways that produce d-Asp and d-Ser, and interference with the upregulated amino acid trans-porters in cancer cells that show selectivity for these d-amino acids. Another notable result was that d-Thr, d-Tyr, and d-Ala also were elevated in MCF-7 cells. Although it has been reported that d-Ala can also bind to NMDA receptor, the presence of Ala racemases have not been confirmed in mammals. Concerning d-Thr and d-Tyr, there are no studies concerning their presence and functions for breast cancer cells to our knowledge. Our results suggest that it may be worthy of exploring the roles of d-Thr, d-Tyr, and d-Ala during cancer cell proliferation.