Spermidine/Spermine N1-acetyltransferase (SSAT) induction as a marker of response to therapy in prostate cancer

Jun Li, Heather Wallace. Division of Applied Medicine, University of Aberdeen, Aberdeen AB25 2ZD, UK.

Polyamines (putrescine, spermidine and spermine) are essential for cell survival. Spermidine/spermine N1-acetyltransferase (SSAT) is the rate-limiting enzyme converting spermidine and spermine into acetylpolyamines to maintain intracellular polyamine homeostasis. A number of antiproliferative agents and polyamine analogues prevent cancer cell growth via SSAT induction (Wallace, 2007). This is related to the production of hydrogen peroxide and accumulation of putrescine during polyamine back-conversion in which N1-acetylpolyamine oxidase and spermine oxidase participate (Wallace et al., 2003). Our hypothesis is that SSAT induction plays a central role in the response of cancer cells to anticancer drugs. Thus, it may act as a biomarker where SSAT activity indicates an effective drug response. The aim of this study was to determine whether altering SSAT expression in human cancer cells changes the response of the cells to chemotherapeutic or chemopreventative drugs.

Prostate cancer cell lines LNCaP wild type and LNCaP cells transfected with human SSAT cDNA responsive to tetracycline (Tet) (a kind gift from Dr Carl Porter; Roswell Park Memorial Institute, Buffalo, USA) were used as the model system. Cells were grown in RPMI1640 with 10% (v/v) foetal bovine serum ± Tet. Cells in the presence of Tet expressed basal SSAT activity, while SSAT expression was induced in the absence of Tet. MTT assay was used to determine the IC50 of cytotoxic agents. SSAT activity was determined by measuring the incorporation of radiolabelled [3H]-actyl-CoA into monoacetylspermidine. Polyamine content was quantified by mass spectrometry.

Fig 1. Effect of Aspirin and 5-FU on SSAT Activity at 48 h exposure in LNCaP cells.

Cells were seeded at a density of 2.4 x 104 cells/cm2 in duplicate and incubated for 48 h growth. After 48 h, media were replaced with fresh media containing drugs. Cells were incubated for further 48 h and SSAT activity was assayed (Values are mean ± SEM, n = 3 with 4 replicates per experiment. Statistical analysis was performed using One-way ANOVA with Dunnett’s post-test). p††† < 0.001; p** < 0.01p*** < 0.001.

Aspirin (2 mM) and 5-FU (50 µM) had little effect on SSAT-suppressed (Tet+) cells but did decrease SSAT activity in the SSAT-overexpressed (Tet-) cells. This correlated with a higher cell number in these cells (results not shown). Aspirin slightly increased SSAT activity in WT. Thus it may be that while increased SSAT is necessary for a decrease in cell growth decreased SSAT may be a prerequisite for increased cell growth.

Wallace, H.M., Fraser, A.V. & Hughes, A. (2003). A perspective of polyamine metabolism. Biochem. J. 376, 1-14.

Wallace, H.M. (2007). Targeting polyamine metabolism: a viable therapeutic/preventative solution for cancer? Expert Opin. Pharmacother. 8, 2109-2116.