Spermine Oxidase SMO(PAOh1), Not N1-Acetylpolyamine Oxidase PAO, Is the Primary Source of Cytotoxic H2O2 in Polyamine Analogue-treated Human Breast Cancer Cell Lines

Abstract

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...

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis -induced colon tumorigenesis

“…S2). Taken together, these data indicate that, consistent with previous reports (21,28), SMO is the source of ROS leading to cytotoxic DNA damage in response to BFT.…”

“…The data presented in this study, combined with additional published and preliminary results, argue strongly that it is SMO that plays an important role in the production of inflammation-associated ROS and DNA damage and therefore represents a potential chemopreventive or chemotherapeutic target (21,28). Although SMO expression is highly inducible and active isoforms have been documented in both the nucleus and cytosol (46)(47)(48)(49), the second polyamine oxidase, APAO, is confined to the peroxisome and therefore its activity seems less likely to yield pathological consequences (50).…”

“…Chemiluminescent SMO and APAO enzyme activity assays of HT29/c1 cell pellets and radiographic SSAT enzyme activity assays of mouse tissue were performed as previously reported (28).…”

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