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Cancer Chemother Pharmacol (2008) 62:215226
DOI 10.1007/s00280-007-0593-6
ORIGINAL ARTICLE
The depletion of cellular ATP by AG2034 mediates cell death or cytostasis in a hypoxanthine-dependent manner in human prostate cancer cells
Oluwakemi Obajimi Peter W. Melera
Received: 24 April 2007 / Accepted: 1 September 2007 / Published online: 25 September 2007 Springer-Verlag 2007
Abstract
Purpose 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoyl-amino-l-glutamic acid (AG2034), is a classical antifolate, an analog of folic acid that has been shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting the de novo synthesis of purines. We examined the eVect of this drug on cell proliferation, steady-state ATP levels, de novo and hypoxanthine salvage ATP synthesis, and on the phosphorylation of AMP kinase, in two diVerent androgen independent prostate cancer cell lines, DU145 and PC-3.
Methods Cells were maintained in culture medium containing 10 nM 5-methyl tetrahydrofolate supplemented with or without 1.7 [afii9839]M hypoxanthine and 1.5 [afii9839]M thymi-dine. Cytotoxicity of AG2034 was determined by clonogenic assays. AG2034-induced inhibition of cell proliferation was determined by electronic counting of cells over varying periods of time. Total cellular AMP and ATP pre- and post-drug treatment was quantiWed by reverse-phase HPLC. [14C]-Glycine incorporation and [3H]-hypoxanthine conversion into ATP were determined by liquid scintillation counting of HPLC isolated ATP fractions. The
phosphorylation of AMP kinase (AMPK) was detected by western blotting.
Results In the absence of 1.7 [afii9839]M hypoxanthine, AG2034 was cytotoxic to both DU145 and PC-3 cells. In its presence, the cells remained cytostatic for 14 days after which time DU145 but not PC-3 re-initiated growth that was maintained for 35 days even though steady-state levels of ATP in both cell lines remained depleted and [14C]-glycine incorporation into ATP was inhibited by >95%. Salvage purine synthesis as measured by incorporation of [3H]-hypoxanthine into ATP was maintained in both cell lines albeit to diVerent levels. When AG2034 was added to the culture medium in the presence or absence of 1.7 [afii9839]M hypoxanthine, cellular ATP levels were reduced by 80% within 24 h in both the cell lines. In the absence of hypoxanthine, the AMP/ATP ratio in PC-3 cells increased by 38% and was accompanied by a modest increase in the level of phosphorylated AMPK; no increase was observed in the presence of hypoxanthine where the AMP/ATP ratio...