Combination of 5-Fluorouracil and N1,N11-Diethylnorspermine Markedly Activates Spermidine/Spermine N1-Acetyltransferase Expression, Depletes Polyamines, and Synergistically Induces Apoptosis in Colon Carcinoma Cells

Woonyoung Choi,Eugene W Gerner,Latha Ramdas,Jheri Dupart,Jennifer Carew,Lynsey Proctor,Peng Huang,Wei Zhang,Stanley R Hamilton

doi:10.1074/jbc.m409930200

Woonyoung Choi, Eugene W Gerner + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m409930200

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Abstract

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N1,N11-diethylnorspermine (DENSPM) or alpha-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N1-acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma.

Highlights

From the Departments of ‡Pathology and ¶Molecular Pathology, Division of Pathology and Laboratory Medicine, The University of Texas M
We examined whether combinations of 5-FU with drugs that target polyamine metabolism, i.e. N1,N11diethylnorspermine (DENSPM) or ␣-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53
Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma

Summary

Introduction

From the Departments of ‡Pathology and ¶Molecular Pathology, Division of Pathology and Laboratory Medicine, The University of Texas M. Our recent functional genomic studies have rediscovered that 5-FU affects polyamine pathways to reduce the levels of polyamines in treated colon cancer cells [10], an observation that was originally made by Russell et al in 1974 in a liver cancer xenograft mouse model [11]. It appears that 5-FU targets two major cellular processes, i.e. polyamine metabolism as well as processing and synthesis of DNA and RNA. These two processes may have intimate inter-relationships because polyamines bind DNA and RNA and affect chromosomal structure, RNA stability, and translation [12]

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