The induction of polyamine catabolism and its production of H 2 O 2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N 1 -acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H 2 O 2 . However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N 1 ,N 1 -bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N 1 -acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H 2 O 2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.The natural polyamines, spermine, spermidine, and putrescine, are ubiquitous polycationic alkylamines that are required for normal eukaryotic cell growth and differentiation (1, 2). Neither mammalian cells lacking polyamine biosynthetic enzymes nor cells depleted of polyamines are able to replicate (3). Polyamine metabolism is frequently dysregulated in many types of cancer, including breast, prostate, and lung cancer (1, 4 -6). Consequently, the polyamine metabolic pathway has become an attractive target for the development of antineoplastic agents (5,7,8).Although early work focused on developing drugs that inhibited polyamine biosynthesis, more recent attention has been given to polyamine analogues that, in addition to down-regulating biosynthesis, also upregulate polyamine catabolism (9 -14). Until recently, mammalian intracellular polyamine catabolism was considered to be a consequence of two enzymes, the rate-limiting and inducible cytosolic spermidine/ spermine N 1 -acetyltransferase (SSAT) 3 and a relatively constitutively expressed, peroxisomal N 1 -acetylpolyamine oxidase (PAO) (1, 2). The products of SSAT/PAO activities on spermine and spermidine are the reactive oxygen species, H 2 O 2 , spermidine, and putrescine, re...

The natural polyamines are aliphatic cations with multiple functions and are essential for cell growth. Soon after the critical requirement of polyamines for cell proliferation was recognized, the metabolism of polyamines was pursued as a target for antineoplastic therapy. Initially, much attention was focused on the development of inhibitors of polyamine biosynthesis as a means to inhibit tumor growth. The best-characterized inhibitor is alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. While compensatory mechanisms in polyamine metabolism reduce the effectiveness of DFMO as a single chemotherapeutic agent, it is currently undergoing extensive testing and clinical trials for chemoprevention and other diseases. There has been increasing interest over the last two decades in the cytotoxic response to agents that target the regulation of polyamine metabolism rather than directly inhibiting the metabolic enzymes in tumor cells. This interest resulted in the development of a number of polyamine analogs that exhibit effective cytotoxicity against tumor growth in preclinical models. The analogs enter cells through a selective polyamine transport system and can be either polyamine antimetabolites that deplete the intracellular polyamines or polyamine mimetics that displace the natural polyamines from binding sites, but do not substitute in terms of growth-promoting function. Synthesis of the first generation of symmetrically substituted bis(alkyl)polyamine analogs in the mid-1980s was based on the theory that polyamines may utilize feedback mechanisms to auto-regulate their synthesis. In the 1990s, unsymmetrically substituted bis(alkyl) polyamine analogs were developed. These compounds display structure-dependent and cell type-specific cellular effects and regulation on polyamine metabolism. More recently, a novel class of analogs has been synthesized, which include conformationally restricted, cyclic and long-chain oligoamine analogs. The development and use of these analogs have provided valuable information for understanding the molecular mechanisms of targeting the polyamine pathway as a means of cancer therapy.

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor ␣ (ER␣) and ER␣ target genes in ER-positive human breast cancer cell lines, whereas neither ER␤ nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ER␣ in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ER␣ transcription rather than affect mRNA or protein stability. Polyamines are naturally occurring polycationic alkylamines that are absolutely required for cell growth. Because of their positively charged amine groups, polyamines interact with negatively charged molecules like DNA, RNA, proteins, and phospholipids to modify their structure and conformation. Rapid tumor growth is associated with significantly increased polyamine biosynthesis (1, 2). In human breast cancer, studies have demonstrated the important roles of polyamine biosynthesis and action in tumor development and metastasis, and increased polyamine levels are often associated with aggressive forms of breast tumors (3-7). Because of the requirement for polyamines in breast cell growth and the demonstration of dysregulated polyamine metabolism in breast tumor cells, polyamine metabolism has become a rational target for breast cancer therapy. Polyamine analogues were designed based upon the theory that the analogues can mimic some of the self-regulatory functions of natural polyamines but are unable to substitute for natural polyamines in their growth promoting roles (8). Recently, a novel class of polyamine analogues has been developed that includes conformationally restricted, cyclic, and long chain oligoamine analogues (9). Our recent studies showed that oligoamines effectively inhibit growth of human breast cancer cell lines in culture and mouse xenografts (10). We also demonstrated that specific oligoamines reduced ornithine decarboxylase activity and induced the activity of the polyamine catabolic enzyme, spermidine/spermine N 1 -acetyltransferase, thereby significantly decreasing the intracellular polyamine pools in several human breast cancer cell lines (10, 11).Estrogens are thought to play a major role in breast cancer development. Because of the pivotal role of the estrogen-estrogen receptor (ER) 3 axis in breast cancer progression, targeting ER or its ligands is a major strategy for breast cancer treatment. Estrogen effects are exerted through the activation of specific estrogen receptors. Unfortunately, many breast tumors develop estrogen independence, perhaps as a consequence of multiple mechanisms including altered balance of co-regulatory proteins or the emergence of other growth signaling pathways. These molecular mechanisms are still poorly understood, and t...