The synergism of 6-mercaptopurine plus cytosine arabinoside followed by PEG-asparaginase in human leukemia cell lines (CCRF/CEM/0 and (CCRF/CEM/ara-C/7A) is due to increased cellular apoptosis

Abstract

Background: The only effective drug against ALL that inhibits protein synthesis is Asparaginase (ASNase). The drug depletes asparagine (Asn) in serum and cells and since the leukemic T-cells (thymic origin cells) lack asparagine synthetase, the amino acid starvation leads to apoptosis. When PEG-ASNase is combined with antimetabolite drugs such as ara-C, or combinations of 6-MP followed by ara-C, it augments the cytotoxic effect synergistically against human T-leukemia cells.

Materials and methods: Synergism studies with two- or three-drug combination regimens in the human leukemia cell lines, CEM/0 and CEM/ara-C/7A have been investigated along with its effect in inducing apoptosis.

Results: The IC50 (approximately Dm) values of ara-C were 0.032 microM and 0.11 microM, and that of PEG-ASNase were 0.002 IU/ml and 1.52 IU/ml against CEM/0 and CEM/ara-C/7A cells, respectively. Thus, CEM/ara-C/7A cell line that is partially resistant to ara-C exhibited 681-fold cross-resistant to PEG-ASNase as compared to CEM/0. The concurrent drug exposure of ara-C and PEG-ASNase for 48 hours resulted in IC50 values of 0.56 nM for ara-C and 0.56 mIU/ml for PEG-ASNase respectively, in CEM/0 cells which represents a 57.4-fold synergism compared to ara-C alone. In the CEM/ara-C/7A cell line, the co-incubation with these two drugs resulted in IC50 value of 0.015 microM for ara-C and 0.015 IU/ml for PEG-ASNase respectively, or a 7.25-fold synergism as compared to ara-C and 101.1-fold synergism in comparison with PEG-ASNase alone. Pre-clinical studies involving three-drug combination consisting of 6-MP, ara-C and PEG-ASNase in a sequence-specific manner showed a 15.6-fold synergism against CEM/0 cell line over the two-drug combination of 6-MP followed by ara-C or approximately 160-fold syneryism over ara-C alone.

Conclusion: The two-drug combination of ara-C and PEG-ASNase or the three-drug combination of 6-MP, ara-C and PEG-ASNase in the ara-C sensitive and resistant cell line showed significant drug synergism and CEM/ara-C/7A cells exhibited collateral sensitivity to PEG-ASNase. The three-drug combination also induced dose-dependent apoptotic DNA fragmentation which was higher than the two-drug combination of 6-MP and ara-C. We also conclude that the sequence specific use of PEG-ASNase in combination with the nucleoside analog drugs may benefit leukemia patients in early relapse.