Introduction Polyamine analogues have demonstrated significant activity against human breast cancer cell lines as single agents as well as in combination with other cytotoxic drugs. This study evaluates the ability of a polyamine analogue N 1 , N 11 -bis(ethyl)norspermine (BENSpm) to synergize with six standard chemotherapeutic agents, 5-fluorouracil (FU), fluorodeoxyuridine, cis -diaminechloroplatinum(II) (C-DDP), paclitaxel, docetaxel, and vinorelbine. Materials and methods Four human breast cancer cell lines (MDA-MB-231, MCF-7, Hs578t, and T47D) and one immortalized, non-tumorigenic mammary epithelial cell line (MCF-10A) were used for in vitro combination studies with BENSpm and cytotoxic drugs. Xenograft mice models generated with MDA-MB-231 cells were used for in vivo studies with BENSpm and paclitaxel. Results and conclusion BENSpm exhibited synergistic inhibitory effect on cell proliferation in combination with 5-FU or paclitaxel in human breast cancer cell lines (MDA-MB-231 and MCF-7) and was either antagonistic or less effective in the non-tumorigenic MCF-10A cell line. Synergism was highest with 120 h concomitant treatment or pre-treatment with BENSpm for 24 h followed by concomitant treatment for 96 additional hours. Since the cytotoxic effects of many polyamine analogues and cytotoxic agents are believed to act, in part, through induction of the polyamine catabolic enzymes SSAT and SMO, the role of these enzymes on synergistic response was evaluated in MDA-MB-231 and MCF-7 treated with BENSpm and 5-FU or paclitaxel. Combination treatments of BENSpm with 5-FU or paclitaxel resulted in induction of SSAT mRNA and activity in both cell lines compared to either drug alone, while SMO mRNA and activity were increased only in MDA-MB-231 cells. Induction was greater with BENSpm/paclitaxel combination than BENSpm/5-FU. Further, RNAi studies demonstrated that both SSAT and SMO play a significant role in the response of MDA-MB-231 cells to treatment with BENSpm and 5-FU or paclitaxel. In MCF-7 cells, only SSAT appears to be involved in the response to these treatments. In an effort to translate combination studies from in vitro to in vivo, and to form a basis for clinical setting, the in vivo therapeutic efficacy of BENSpm alone and in combination with paclitaxel on tumor regression was evaluated in xenograft mice models generated with MDA-MB-231 cells. Intraperitoneal exposure to BENSpm or taxol singly and in combination for 4 weeks resulted in significant inhibition in tumor growth. These findings help elucidate the mechanisms involved in synergistic drug response and support combinations of polyamine analogues with chemotherapeutic agents which could potentially be used in the treatment of breast cancer.

PURPOSE:Polyamines are essential for normal growth; however, the requirement for, and the metabolism of, polyamines are frequently dysregulated in cancer. Polyamine analogues have demonstrated promising preclinical results in multiple model systems of cancer, but their clinical utility has been limited by apparent toxicity. A representative compound of a new generation of short chain, conformationally restricted polyamine analogues, CGC-11047 has been synthesized and ongoing phase I clinical trials indicate it to be well tolerated at weekly doses of 610 mg (dose escalation is still in progress). Therefore, studies were designed to gain a better understanding of its effects on cellular polyamine biochemistry and efficacy in the treatment of human lung cancer models in vitro and in vivo.METHODS:Human lung cancers cell lines representing non-small cell and small cell lung cancers were investigated for their growth and biochemical response to CGC-11047. Effects of in vitro treatment with CGC-11047 on cell growth, the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and the expression and activity of the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxides (SMO) were measured. Additionally, the overall effects on intracellular polyamine pools were monitored. Finally, the in vivo efficacy of CGC-11047 in the treatment of a nude mouse model of human non-small cell lung cancer was evaluated.RESULTS:CGC-11047 effectively inhibited the growth of both small cell and non-small cell lung cancer cells in vitro. The greatest biochemical effects were observed in the non-small cell lung cancer cells where in addition to a profound down regulation of ODC activity, there was a significant increase in polyamine catabolism leading to a greater degree of polyamine pool depletion and greater accumulation of CGC-11047 when compared with the changes observed for the small cell lines. Importantly, CGC-11047 was found to be highly significant (P < 0.0001) in delaying the progression of established tumors in an in vivo model of human non-small cell lung cancer.CONCLUSION:CGC-11047 represents a promising new polyamine analogue that warrants further preclinical and, potentially, clinical evaluation in lung cancer.

The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO‐produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase‐1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI‐H157 human non‐small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation‐induced carcinogenesis.

As more data emerge, the significance of polyamine catabolism in polyamine homeostasis, drug response, and disease etiology is expanding. Importantly, the regulation and function of the polyamine catabolic pathway has emerged as a rational target for drug intervention in both chemotherapeutic and chemopreventive strategies. Mammalian intracellular polyamine catabolism had long been thought to be a two-step process primarily regulated by a rate-limiting acetyltransferase, spermidine/spermine N1-acetyltransferase (SSAT), followed by the activity of a constitutively expressed acetylpolyamine oxidase (PAO). However, as recent reports have clearly demonstrated, mammalian polyamine catabolism also includes the activity of a previously unrecognized spermine oxidase (SMO/PAOh1). The production of reactive oxygen species (ROS) and other toxic products by these various polyamine catabolic enzymes can result in both useful and potentially dangerous consequences. This chapter will examine some of the most recent findings related to polyamine catabolism and will address the cloning and characterization of mammalian polyamine oxidases, including the newly discovered SMO/PAOh1. Additionally, further characterization of the highly regulated SSAT, as facilitated by many recent advances with transgenic models, will be discussed with respect to the potential role that it and the oxidases play in determining response to various drugs and stimuli. Although the polyamine catabolic pathway is well described and being studied in multiple organisms, this work will focus primarily on results directly related to mammalian systems, with special emphasis given to the relationship between polyamine catabolism and human disease. Specifically, data indicating that the induction of polyamine catabolism by specific antitumor polyamine analogs plays a direct role in determining drug response will be discussed. Also to be examined is the recent recognition that the oxidation of polyamines contributes to disease processes, and the potential targeting of polyamine catabolism as a strategy for chemoprevention.

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

The recent cloning of the mammalian gene coding for N1-acetylpolyamine oxidase (PAO) provides the opportunity to directly examine the role of human PAO (hPAO) in polyamine homeostasis as well as its potential role in determining cellular response to antitumor polyamine analogues. To facilitate the study of this enzyme, the production, purification, and characterization of the recombinant hPAO is reported. hPAO oxidizes N1-acetylspermidine (Km=2.1 μM, Kcat=15.0 s−1) and has very high affinity for N1-acetylspermine (Km=0.85 μM, Kcat=31.7 s−1). The recombinant hPAO does not efficiently oxidize spermine, thereby demonstrating a significant difference in substrate specificity from the previously described human spermine oxidase PAOh1/SMO. Importantly, hPAO demonstrates the ability to oxidize a subset of antitumor polyamine analogues, suggesting that this oxidase activity could have a significant effect on determining tumor sensitivity to these or similar agents. Transfection of A549 human lung cancer cells with an hPAO-expressing plasmid leads to a profound decrease in sensitivity to those analogues which act as substrates, confirming its potential to alter drug response. One similarity that hPAO shares with human PAOh1/SMO, is that certain oligoamine analogues are potent inhibitors of its oxidase activity. The results of these studies demonstrate how changes in polyamine catabolism may affect drug response.

5084 The critical nature of polyamines in cell growth and the frequent dysregulation of their metabolism in cancer have led to the targeting of the polyamine metabolic pathway as an antitumor strategy. Importantly, recent molecular characterization of each of the polyamine catabolic enzymes and their potential to profoundly alter drug response has propelled considerable interest into understanding their role in modulating tumor cell sensitivity to the antitumor polyamine analogues. Analogue-induced oxidation of the natural polyamines by PAO and the newly discovered spermine oxidase, SMO(PAOh1), has been implicated in the cytotoxic response of solid tumor types to specific polyamine analogues. But the potential that oxidase activity may lead to drug resistance by detoxifying the antitumor analogues has received less attention. Here we examine the substrate specificity of recombinant human PAO with respect to the natural polyamines and various antitumor analogues and examine the effects of overexpression of human PAO in the analogue-sensitive A549 human lung adenocarcinoma cell line. The results indicate that PAO, in addition to effectively oxidizing N1-acetylspermine and N1-acetylspermidine, also rapidly oxidizes several symmetrically and unsymmetrically substituted antitumor polyamine analogues, including N1, N11-bis(ethyl)norspermine (BENSpm), N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane (CPENSpm), N1-ethyl-N11-(cycloheptyl)methyl-4,8,diazaundecane (CHENSpm), and (S)-N1-(2-methyl-1-butyl)-N11 -ethyl-4,8,diazaundecane (IPENSpm). When overexpressed in A549 cells, PAO oxidizes each of the analogues, reduces their intracellular accumulation, blocks the downstream apoptotic events common in response to treatment with these agents, and confers significant resistance to each of these compounds. By contrast, the response to representative oligoamine polyamine analogues that are not PAO substrates is not altered by PAO overexpression. These results suggest a mechanism of resistance to specific antitumor polyamine analogues and provide insight into means for improving the design and use of new agents with greater clinical potential.

5307Polyamines are naturally occurring polycations that are essential for cell growth. Many forms of cancer, including breast, prostate, lung, and colon cancer, lose the ability to regulate polyamine metabolism. The activity of one enzyme involved in the degradation of polyamines, spermine oxidase (PAOh1/SMO), is decreased in invasive breast cancer. PAOh1/SMO is a FAD-dependent enzyme that directly oxidizes spermine to spermidine while releasing hydrogen peroxide. Diminished PAOh1/SMO activity may be associated with reduced levels of hydrogen peroxide and decreased apoptosis, thereby contributing to breast cancer cell growth. PAOh1/SMO mRNA and activity have recently been found to be inducible in several breast cancer cell lines with a spermine analogue, N1,N11-Bis(ethyl)norspermine (BENSpm). The estrogen receptor alpha (ER) negative cell line MDA-MB-231 exhibited a high induction of …

Purpose The polyamine analogue, N 1 , N 11 -diethylnorspermine (DENSpm), is currently being evaluated in clinical trials for the treatment of solid tumors. The response of solid tumors to this drug has been associated with superinduction of the polyamine catabolic enzyme, spermine/spermidine N 1 -acetyltransferase (SSAT). Therefore, to estimate the response of breast cancers to DENSpm, we measured induction of SSAT in breast cancer explants treated in vitro with this polyamine analogue. Experimental design Expression of SSAT protein was evaluated by immunohistochemistry in tissue explants from 38 invasive breast cancer tumors incubated in vitro in the presence (or absence) of DENSpm. In addition, SSAT enzymatic activity was measured in tissue explants from four tumors with high cellularity. Results SSAT expression was significantly increased in 30 of 38 tumor samples treated with DENSpm compared to untreated controls. This induction of SSAT protein expression was found specifically in neoplastic cells of the treated samples, and was seen in all histologic patterns (ductal, lobular, and mucinous) of breast cancer examined. In tumor samples evaluated for changes in SSAT enzymatic activity, these changes correlated closely with changes in protein expression. Conclusions Immunohistochemical staining for induction of SSAT correlates with measures of enzymatic activity in a small sample where measurements were possible and suggests that immunohistochemistry may be used for predicting response of breast cancers to DENSpm. A high proportion of breast cancers induced SSAT in response to DENSpm, supporting the continued consideration of this class of agents for treatment of breast cancer.

Several polyamine analogues have efficacy against a variety of epithelial tumor models including breast cancer. Recently, a novel class of polyamine analogues designated as oligoamines has been developed. Here, we demonstrate that several representative oligoamine compounds inhibit in vitro growth of human breast cancer MDA-MB-435 cells. The activator protein-1 (AP-1) transcriptional factor family members, c-Jun and c-Fos, are up-regulated by oligoamines in MDA-MB-435 cells, suggesting a possible AP-1-dependent induction of apoptosis. However, the use of a novel c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125, suggests that inhibition of c-Jun activity sensitized tumor cells to oligoamine-induced cell death. To directly test this hypothesis, cells were stably transfected with the dominant-negative mutant c-Jun (TAM67), which lacks the NH2-terminal transactivation domain. Cells overexpressing …

Purpose The induction of polyamine catabolism has been directly associated with the cytotoxic response of various tumor types to the antitumor polyamine analogues. Initially, human polyamine catabolism was assumed to be under the control of a rate-limiting spermidine/spermine N 1 -acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (PAO). We have recently cloned a new polyamine analogue-inducible human polyamine oxidase (PAOh1/SMO) that efficiently uses spermine as a substrate. The induction of PAOh1/SMO in response to multiple polyamine analogues was examined in representative lung tumor cell lines. Methods Representatives of three different classes of antitumor polyamine analogues were examined for their ability to induce PAOh1/SMO. Results The human adenocarcinoma line, NCI A549 was found to be the most responsive line with respect to induction of PAOh1/SMO in response to analogue exposure. Similar to previous observations with SSAT expression, PAOh1/SMO induction was found to occur primarily in non-small-cell lung cancers cell lines. Using a series of polyamine analogues, it was found that the most potent inducers of PAOh1/SMO possessed multiple three-carbon linkers between nitrogens, as typified by N 1 ,N 11 -bis(ethyl)norspermine. Conclusions Since PAOh1/SMO is an analogue-inducible enzyme that produces H 2 O 2 as a metabolic product, it may play a significant role in determining the sensitivity of various human tumors to specific polyamine analogues.