Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

Abstract

L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP’s anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC–MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells. When we examined the response of OVCAR-8 ovarian cancer cells in culture to L-ASP, asparagine was depleted from the medium within seconds. Interestingly, intracellular asparagine was also depleted rapidly, and the mechanism was suggested to involve rapid export of intracellular asparagine followed by rapid conversion to aspartic acid by L-ASP. We also found that L-ASP-induced cell death was more closely associated with glutamine concentration than with asparagine concentration. Time-course analysis revealed the dynamics of amino acid metabolism after feeding cells with fresh medium. Overall, this study provides new insight into L-ASP’s mechanism of action, and the optimized analytical method can be extended, with only slight modification, to other metabolically active amino acids, related compounds, and a range of cultured cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0634-1) contains supplementary material, which is available to authorized users.

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231

“…With all the applied treatments no recovery for alkanes and ketones was observed from MDA-MB-231 cells. This finding is in contrast to a previously published report, 28 using an ovarian cancer cell-line (OVCAR-8) where similar recoveries were obtained for amino acids using both methanol/HEPES or methanol/AMBIC treatments but this was analysed using HPLC which may account for the differences.…”

“…13,16,25 For adherent cell cultures, there is little information in the published literature evaluating metabolite leakage in response to quenching solvent apart from a recent report assessing the use of buffer additives for ovarian cancer cells. 28 Even here, the evaluation was based only on HPLC analysis of amino acids; there was no evaluation of washing steps, or any consideration for leakage from quenching solvent. In addition, the use of quenching solvent to wash could have resulted in more leakage.…”

“…The use of buffer additives such as AMBIC, HEPES and NaCl have been evaluated with methanol/AMBIC identified as an effective quenching solvent for OVCAR-8 cells (ovarian cancer cells). 28 Another study, explored three different quenching protocols (flash freezing (−80°C), cold methanol (−20°C) and cold methanol (−40°C)) without any buffer additives to quench NSC-34 cell metabolism, and reported methanol quenching at −40°C to be reliable. 29 A further study evaluated the washing protocols with the use of phosphate buffered saline (PBS), water and no washing step for the colorectal cancer cell line HCT116 and reported extraction of metabolites with cold methanol without any washing to be satisfactory.…”

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

“…Asparaginase and glutaminase activities were also determined using a highly sensitive liquid chromatography-mass spectrometry (LC-MS)/MS assay developed by our laboratory. 22 Limits of detection for asparagine, aspartic acid, glutamine, and glutamic acid were 250, 150, 16, and 22 nM, respectively.…”

“…Asparaginase and glutaminase activities were also determined using a highly sensitive liquid chromatography-mass spectrometry (LC-MS)/MS assay developed by our laboratory. 22 Limits of detection for asparagine, aspartic acid, glutamine, and glutamic acid were 250, 150, 16, and 22 nM, respectively.Mutagenesis, expression, purification, and screening of L-ASP recombinant enzymes L-ASP expression vectors with a pelB leader sequence 23 to facilitate secretion of recombinant L-ASP into the culture supernatant were transformed into the E coli BLR (DE3) strain (Novagen) (supplemental Figure 1). After using isopropyl b-D-1-thiogalactopyranoside (IPTG) to induce transformed cells to express and secrete L-ASP, we then screened for asparaginase and glutaminase activities using the aforementioned colorimetric assays.…”