Proceedings of the British Pharmacological Society at http://www.pA2online.org/abstracts/Vol10Issue4abst118P.pdf Spermidine/Spermine N1-Acetyltransferase (SSAT): a Key Contributor to the Cytotoxicity of Antineoplatic Drugs
Li J, Wallace HM. Division of Applied Medicine, University of Aberdeen, AB25 2ZD, Aberdeen, UK
Prostate cancer is the second most diagnosed cancer and the sixth leading course of cancer death in men worldwide with a high morbidity in Europe and in the US. In tumour cells polyamine (putrescine, spermidine, and spermine) syntheses are upregulated during rapid growth. Spermidine/Spermine N1-Acetyltransferase (SSAT), the first catabolic enzyme in polyamine catabolic pathway, is induced by a range of anticancer drugs. This results in polyamine depletion and subsequent inhibition of tumour cell growth (Wallace, 2007). However, the mechanism of SSAT related antineoplastic effect is not fully understood in terms of polyamine homeostasis. The aim of this study was to characterise SSAT in polyamine homeostasis and its role in the cytotoxicity of antineoplastic drugs.
LNCaP prostate cancer cell line transfected with human SSAT cDNA under the Tet-off Advanced Inducible Gene System [SSAT gene transcription is controlled by addition or withdraw of Tetracycline in culture medium, generating SSAT− (not induced) and SSAT+ (induced) cells] was used as the model system. Cells were grown in RPMI1640 with 10% (v/v) Tetracycline free foetal bovine serum, 0.5mg/ml G418, 1 mM aminoguanidine, 150 µg/ml hygromycin B and 1 µg/ml tetracycline. A new method was developed to measure polyamine content using Liquid Chromatography Mass Spectrometry (LC-MS). Polyamine efflux was measured using [3H]-putrescine to monitor the intracellular and extracellular polyamine content (Wallace & Mackarel, 1998). Aspirin and 5-fluorouricil (5-FU) were used as antineoplastic drugs.
SSAT+ cells showed the slowest proliferation rate compared with SSAT− cells without treatment (p < 0.001). Spermidine depletion was seen in both cell lines treated by Aspirin (2 mM) and 5-FU (50 µM) for 48 h. N1-acetylpolyamines and putrescine were increased by 5- FU in SSAT− cells. In contrast to SSAT+ cells, there was a decrease in N1-acetylpolyamines. Furthermore, polyamine efflux was increased by Aspirin and 5-FU in SSAT− cells (p < 0.001), but with little difference in SSAT+ cells.
These results indicate that increase in SSAT was associated with growth inhibition in LNCaP prostate cancer cells. The growth inhibition may be related to decreased intracellular polyamine content. The cytotoxic effects of aspirin and 5-FU may be partially attributed to the changes in polyamine homeostasis regulated by changes in SSAT activity and expression.
Wallace, H.M. (2007). Targeting polyamine metabolism: a viable therapeutic/preventative solution for cancer? Expert Opin. Pharmacother. 8, 2109-2116.
Wallace, H.M. & Mackarel, A.J. (1998). Measurement of polyamine efflux from cells in culture. Polyamine Protocols, Methods in Molecular Biology. 79, 157-165.
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