Concentration-dependent effects ofN1,N11-diethylnorspermine on melanoma cell proliferation

Abstract

-Diethylnorspermine (DENSPM) is a polyamine analog that is currently under investigation as a novel anticancer drug. Although it has shown promising preclinical activity, there has been large variation in responsiveness reported between different human cancers. During our studies into the causes of this variation, we observed a consistent increase in cell proliferation at low drug concentrations (<10 lM) in human melanoma cells resistant to the drug. At higher concentrations, growth inhibition was seen in all cell lines, with IC 50 values ranging 2-180 lM. We hypothesized that DENSPM may mimic endogenous polyamines at low concentrations, supporting cell growth in resistant lines. We also observed that DENSPM downregulated polyamine transport in a manner similar to that for spermidine, a finding that confirms previous reports. Finally, DENSPM could rescue cells from growth arrest by the ornithine decarboxylase inhibitor difluoromethylornithine, which depletes intracellular polyamines. Taken together, these results suggest that DENSPM, at clinically relevant concentrations, can mimic endogenous polyamines and induce proliferation in resistant human melanoma cells. ' An early observation in the development of DENSPM was the heterogeneous cytotoxic response of different cell lines to the drug. This was observed in human melanoma cells 14 and lung carcinomas. 15 Consistently, cell lines sensitive to DENSPM overexpress spermidine/spermine N 1 -acetyltransferase (SSAT) in the presence of the drug. 5,14,16,17 SSAT induction involves the transcription factor Nrf-2 and the cofactor polyamine-modulated factor-1, which bind to a polyamine response element in the SSAT promoter. 18,19 Some cells resistant to DENSPM show no expression of Nrf-2 and little induction of SSAT. 18 The variation in SSAT induction has been demonstrated in primary tumor tissue, suggesting that variation in drug response will be evident in patients. 20 We investigated the antiproliferative activity of DENSPM in a number of human melanoma cell lines, to identify possible biomarkers for drug response. Inhibition of growth in all cell lines was DENSPM concentration-dependent. However, we observed a consistent increase in cell proliferation at low concentrations in those lines that showed the least sensitivity to the drug. The results suggested that DENSPM, and possibly other polyamine analogs, may promote growth in resistant cells, a finding that has important implications on its use in the clinical setting. Material and methods Cell linesThe human melanoma lines AO2JLO, MM96L, MM426, MM608, MM2058 and MM170 were obtained from Dr. P. Parsons (Queensland Institute for Medical Research, Brisbane, Australia). The human Lox melanoma line was kindly provided by Dr. C. Porter (Roswell Park Cancer Institute, Buffalo, NY), and the murine B16 melanoma line was obtained from the ATCC (Manassas, VA). All lines were grown in RPMI-1640 containing 10% FCS in a humidified atmosphere of 5% CO 2 . Proliferation assayCells were seeded at 10 3 /well in 96-well plat...

Polyamine-dependent Regulation of Spermidine-Spermine N1-Acetyltransferase mRNA Translation

“…However, it has been previously reported that the response of different cell types to DENSPM is quite variable. Some cells show high SSAT induction, and cytotoxicity, at concentrations as low as 1 M (27), whereas others require a drug concentration at least an order of magnitude higher for induction (28,29). SSAT expression is regulated at several levels, including translation (5,11), which requires elevated polyamine levels for optimum protein production.…”