Oluwakemi Obajimi1, Judith Clancy Keen, Peter W Melera. 1. Department of Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 401 Haddon Avenue, Camden, NJ 08103, USA. obajimol@umdnj.edu
Abstract
BACKGROUND: 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-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non-tumorigenic prostatic epithelial cells, RWPE-1. METHODS AND RESULTS: Cells were cultured in medium containing 10 nM 5-methyl-tetrahydrofolate supplemented with/without 1.7 microM hypoxanthine/1.5 microM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. Total ATP was quantified by reverse-phase HPLC and [(14)C]-glycine incorporation and [(3)H]-hypoxanthine conversion into ATP by liquid scintillation counting. Protein expression levels were determined by Western blotting, cell cycle analysis by propidium iodide staining and cell-senescence by beta-galactosidase staining. AG2034 inhibited LNCaP cell proliferation causing death in the absence of hypoxanthine and cytostasis in its presence. However, RWPE-1 cells were resistant to AG2034 when hypoxanthine was present. AG2034 elevates AMP/ATP ratios but is unable to activate AMPK in RWPE-1 when hypoxanthine is present. Drug exposure increased expression levels of p53, p21, p27, and p16 in both cell lines and increased senescence-associated-beta-gal staining in LNCaP with/without hypoxanthine, but primarily in its absence in RWPE-1. CONCLUSIONS: LNCaP cells primarily depend upon de novo while RWPE-1 cells largely favor salvage synthesis for maintenance of their ATP pools. With AG2034 treatment, ATP synthesis via hypoxanthine salvage is insufficient to support growth of LNCaP but enough to restore ATP levels and support RWPE-1 growth. The anti-proliferative effect of AG2034 involves increasing phosphorylation of AMPK. These results indicate that AG2034 activates p53 and AMPK mediating the induction of signaling pathways leading to senescence.
BACKGROUND: 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-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non-tumorigenic prostatic epithelial cells, RWPE-1. METHODS AND RESULTS: Cells were cultured in medium containing 10 nM 5-methyl-tetrahydrofolate supplemented with/without 1.7 microM hypoxanthine/1.5 microM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. Total ATP was quantified by reverse-phase HPLC and [(14)C]-glycine incorporation and [(3)H]-hypoxanthine conversion into ATP by liquid scintillation counting. Protein expression levels were determined by Western blotting, cell cycle analysis by propidium iodide staining and cell-senescence by beta-galactosidase staining. AG2034 inhibited LNCaP cell proliferation causing death in the absence of hypoxanthine and cytostasis in its presence. However, RWPE-1 cells were resistant to AG2034 when hypoxanthine was present. AG2034 elevates AMP/ATP ratios but is unable to activate AMPK in RWPE-1 when hypoxanthine is present. Drug exposure increased expression levels of p53, p21, p27, and p16 in both cell lines and increased senescence-associated-beta-gal staining in LNCaP with/without hypoxanthine, but primarily in its absence in RWPE-1. CONCLUSIONS: LNCaP cells primarily depend upon de novo while RWPE-1 cells largely favor salvage synthesis for maintenance of their ATP pools. With AG2034 treatment, ATP synthesis via hypoxanthine salvage is insufficient to support growth of LNCaP but enough to restore ATP levels and support RWPE-1 growth. The anti-proliferative effect of AG2034 involves increasing phosphorylation of AMPK. These results indicate that AG2034 activates p53 and AMPK mediating the induction of signaling pathways leading to senescence.