br l Asn and d Asn may both
3.3. l-Asn and d-Asn may both serve as exchange currency during breast cancer cell proliferation
Amino Protease Inhibitor Cocktail transporters carry out not only net transport of amino acids (i.e., symporters, substrates travel in the same direc-tion), but also obligatory amino acid exchange (i.e., antiporters), which means uptake of one amino acid via this transporter is obli-gatorily coupled to the export of another amino acid . A recent study indicated that depletion of intracellular and/or extracellular l-Asn in breast cancer cells impaired the uptake of extracellular amino acids, especially l-Ser, l-Arg, and l-His, and reduced cancer cell proliferation . However, the function of d-Asn for cancer cell proliferation was not investigated. In the present study, cel-lular uptake of l-Ser, l-Arg, and l-His was observed for MCF-7 breast cancer cells (Fig. 3). In addition, cellular release of both l-Asn and d-Asn was observed for MCF-7 breast cancer cells, but not for MCF-10A cells (Figs. 3 and 6). Our results support and further sug-gest that intracellular l-Asn, together with d-Asn, exchanges with extracellular amino acids, especially l-Ser, l-Arg, and l-His, to pro-mote cancer cell proliferation . Asn, l- and d-enantiomers, may serve as exchange currency for the uptake of essential amino acids and/or low abundance nonessential amino acids that are required by cancer cells during proliferation.
3.4. Malignancy indicators may be used to indicate the presence of cancer
The question arises, as to whether the substantially altered lev-els of d- and l-amino acids in cancer cells can be used to provide a sensitive and reliable index to identify malignancy? A relatively simple malignancy indicator (MI) comparing the ratio of each l-amino acid in cancer vs. noncancerous cell line and divided by the analogous ratio of d-amino acid, are given in Table 1 (see Materi-als and methods for calculations). Clearly, there are three notable features which are: 1) increased demands of specific l-amino acids
Malignancy indicators for breast cancer.
Results in this table were obtained from MCF-7 breast cancer cells grown in nor-mal glucose condition vs. MCF-10A cells after 72-hours incubation. Calculations for: l-amino acids a , d-amino acidsb , and Malignancy Indicatorc are given in the Mate-rials and methods. Exact levels for each amino acid are given in Supplementary material (Tables S8 and S9). d indicateing that this d-amino acid was detected only in MCF-10A cells, but not in MCF-7 breast cancer cells. In these cases, the limit of detection (LOD) for these d-amino acids was used to estimate a MI value.
contribute to a high MI, 2) increased demands of specific d-amino acids contributes to low MIs, and 3) designated cellular release of specific l- and d-amino acids contribute to low MI. Gln, Phe, Ile, Val, and Leu show high MI due the significant elevated levels of L-enantiomers but decreased levels of d-enantiomers in MCF-7 breast cancer cells. Low MI values are determined for Asn, Asp, and Ser, and how these elevated d-amino acids contribute to low MIs were discussed above.
3.5. Effect of glucose concentration on MCF-7 cell proliferation and amino acid levels
Cell media with different glucose concentrations (i.e., 5 mM and 25 mM) were examined for the effect of glucose level on cancer cell proliferation. Cell number and free amino acid levels were mea-sured for MCF-7 cells with different growth times, i.e., 24-hours, 48-hours, and 72-hours (Tables S4, S5, S7, S8, S10, S11, and S25). As expected, the proliferation of MCF-7 cells increased when the glu-cose concentration in the medium was increased from 5.5 mM to 25 mM at 72 h (Fig. S3). MCF-7 cells grown in high glucose medium showed higher levels of free l-amino acids compared to those grown in normal glucose medium (Fig. 2). For instance, l-Gln levels in MCF-7 cells grown in high glucose and normal glucose condi-tion were 681 nmol/106 cells and 377 nmol/106 cells, respectively, after 24-hour incubation (Fig. 2A). However, the trends of intracel-lular l-amino acid levels for MCF-7 cells were similar despite the glucose concentration in the medium (Fig. 2). l-Gln, Gly, and l-Glu were the three amino acids with the highest levels, and the three l-amino acids with the lowest levels were always GABA, l-Cys, and l-Hyp in MCF-7 cells regardless of the growth media. Concerning d-amino acids, most of them did not show significant changes in MCF-7 cells when the glucose levels were changed in the medium (Fig. 4). The exceptions were d-Thr and d-Ser, which were higher in MCF-7 cells grown in high glucose conditions, especially after 24-hour growth.