Abstract 3421: An in vitro study of the cytotoxic and cellular biochemical effects of L-glutamine moderation in pediatric sarcomas

Abstract

Abstract Background L-glutamine (gln) deprivation is being explored as a clinically relevant method of chemosensitizing cancer cells to chemotherapeutic agents. Gln plays a major role in cellular energy generation, oxidative stress and protein biosynthesis making its deprivation detrimental to cell proliferation. In the clinical setting, significant glutamine depletion is found with various preparations of L-asparaginase and with glutaminase [1]. On this basis, we hypothesize that if gln deprivation can be combined with front line anticancer agents in clinical practice currently, it may chemopotentiate their actions leading to the potential development of a new chemotherapeutic regimen for clinical use. Aims To investigate the cytotoxic and cellular biochemical effects of gln and asparagine (asn) deprivation in vitro, to explore the potential of using gln deprivation clinically. To investigate synergistic effects of gln and asn deprivation in combination with clinically used anti cancer agents in vitro. Methods and results The Ewing's Sarcoma TC32, Osteosarcoma HOS and Rhabdomyosarcoma A204 cell lines were cultured in varying concentrations of gln (0-2mM) and asn (0-200µM) for 96 hours to monitor cell proliferation and viability. Cells were stained with Propidium Iodide (PI) and analyzed using flow cytometry. Gln deprivation produced a 44% increase in growth inhibition (GI) compared to asn deprivation. 98% GI was achieved in all cell lines following simultaneous gln and asn deprivation. Cell viability was >85% under all conditions. Apoptosis analysis using FITC Annexin V and PI staining showed no induction of apoptosis or necrosis upon gln and asn deprivation, apoptosis was <10% and cell death was <5%. Cell cycle analysis showed no significant difference in cell cycle phase distribution between cells grown in complete and deprived conditions. Cell recovery was investigated using clonogenic survival assays. Clonogenic ability following asn deprivation over 96 hours was 93%. Cells could not regain their proliferative capacity following 72 hours of gln and 48 hours of simultaneous gln and asn deprivation with Clonogenic survival <25%. Synergy studies using deprivation in combination with Doxorubicin, Temozolomide, Methotrexate, Cisplatin, Vincristine and Topotecan are being performed to assess the effect of deprivation on cytotoxicity. Conclusions For the sarcoma cell lines investigated, asn deprivation alone has no effect on doubling time, cell cycle distribution or survival. However, at least 48 hours of gln deprivation is associated with both growth inhibition and loss of clonogenic survival for these cells. The potential for gln deprivation as a chemosensitizing effect in this situation is currently being investigated.