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Originally published In Press as doi:10.1074/jbc.M508177200 on October 5, 2005

J. Biol. Chem., Vol. 280, Issue 48, 39843-39851, December 2, 2005
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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*

Allison Pledgie{ddagger}, Yi Huang{ddagger}, Amy Hacker{ddagger}, Zhe Zhang{ddagger}, Patrick M. Woster§, Nancy E. Davidson{ddagger}1, and Robert A. Casero, Jr.{ddagger}2

From the {ddagger}Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21231 and the §Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, Detroit, Michigan 48202

Received for publication, July 26, 2005 , and in revised form, September 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-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 H2O2. 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 N1,N1-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 N1-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 H2O2 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 N1-acetyltransferase (SSAT)3 and a relatively constitutively expressed, peroxisomal N1-acetylpolyamine oxidase (PAO) (1, 2). The products of SSAT/PAO activities on spermine and spermidine are the reactive oxygen species, H2O2, spermidine, and putrescine, respectively (depending on the starting substrate), and 3-acetoaminopropanol. The activity of the SSAT/PAO pathway has been linked previously with the cytotoxic response of several tumor types to specific polyamine analogues (10, 15–17). However, recent studies have clearly demonstrated that an additional enzyme exists in the mammalian polyamine catabolic pathway, an inducible spermine oxidase (SMO/PAOh1) (18, 19). SMO(PAOh1) is a cytosolic protein that is selectively active on spermine producing H2O2, spermidine, and the aldehyde 3-aminopropanol (20, 21). More importantly, the expression of this enzyme is induced by some of the same agents that induce SSAT, suggesting that induction of both of the polyamine catabolic pathways can lead to the production of H2O2 (22).

Because the production of H2O2 through polyamine catabolism has been implicated in the cytotoxic response of several tumor types to multiple polyamine analogues, the purpose of this study was to determine the origin of H2O2 in response to cellular exposure to the antitumor polyamine analogue, N1,N11-bis(ethyl)norspermine (BENSpm) (an agent that has been evaluated in phase I and II clinical trials), and thereby to determine the role of each of the polyamine catabolic enzymes in the BENSpm response (23, 24). Previous studies implicating PAO in H2O2 production in response to analogue exposure were performed with the polyamine oxidase inhibitor, MDL72527, thought to be specific for PAO. With the recognition that MDL72527 is also a potent inhibitor of SMO(PAO1), the results of earlier studies may require reexamination (25–27). Also, previous attempts to examine directly the role of SSAT in cellular response through the use of siRNA strategies were limited by a transient transfection approach, thus making it difficult to assess long term effects of extended SSAT knockdown in response to analogue treatment (28). To overcome the limitations of transient knockdown, a stable transfection strategy was used to constitutively express siRNAs targeting the rate-limiting steps of polyamine catabolism, SSAT and SMO(PAOh1), either alone or in combination.

This study represents the first use of stably expressed siRNAs directed against multiple key polyamine metabolic enzymes and demonstrates that SSAT and SMO(PAOh1) induction contribute significantly to the antiproliferative effects of BENSpm in a cell type-specific manner. Furthermore, the experimental results confirm that SMO(PAOh1) enzyme activity, not PAO enzyme activity, is the source of cytotoxic H2O2 produced followed exposure to BENSpm in specific breast cancer lines, whereas SSAT induction results in a decrease in intracellular polyamine levels through the acetylation of polyamines that are then exported from the cell. With a better understanding of the relative contribution made by each of the independent polyamine catabolic pathways to the cytotoxic activity of polyamine analogues, it is hoped that more selective and effective agents can be designed for use against breast cancer.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Culture Conditions, and Reagents—The acquisition and maintenance of the breast cancer cell lines, MDA-MB-231, Hs578t, MCF-7, and T47D, have been described previously (29). BENSpm and MDL72527 were synthesized as described previously (14, 30). 5-(and -6)-Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), mixed isoforms, was purchased from Molecular Probes (Eugene, OR). Catalase and 3-amino-1,2,4-triazole (AT) were purchased from Sigma.

RNA Isolation, Reverse Transcription-PCR, and Real Time PCR—Total cellular RNA was isolated from cultured cell lines using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 3 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) primers (Invitrogen). Conventional PCR was performed using the cDNA as a template with the following primers: SSAT forward, 5'-ATCTAAGCCAGGTTGCAATGA, and SSAT reverse, 5'-GCACTCCTCACTCCTCTGTTG; SMO(PAOh1) forward, 5'-CGCAGACTTACTTCCCCGGC, and SMO(PAOh1) reverse, 5'-CGCTCAATTCCTCAACCACG; SMO(PAOh1) isoform 1 forward, 5'-CGACCACAATCACGACACTG, and SMO(PAOh1) and isoform 1 reverse, 5'-GCCGAGGGCAAGATTCGCCG; SMO(PAOh1) isoform 2 forward, 5'-GCCCCGGGGTGTGCTAAAGAG, and SMO(PAOh1) and isoform 2, reverse 5'-CGGAAAACAGCACCTGCATGG; SMO(PAOh1) isoform 3 forward, 5'-CGCAGACTTACTTCCCCGGCTCAG, and SMO(PAOh1) isoform 3 reverse, 5'-CTGCATGGGCTCGTTGTATAAATC; SMO-(PAOh1) isoform 4 forward, 5'-CCAGGCCTCAGCCCGCCCCAG, and SMO(PAOh1) isoform 4 reverse, 5'-GCTGTTCTGGGAACTTGGAAGAG; PAO forward, 5'-CCTACAGTTTGTGTGGGAGGA, and PAO reverse, 5'-ATGAATAGGAGCCACGGAAGT; actin forward, 5'-ACCATGGATGATGATGATATCGC and actin reverse, 5'-ACATGGCTGGGGTCTGAAG. PCR products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. For real time PCR, cDNA was amplified by using SYBR green (Sigma) according to the manufacturer's instructions. Real time PCR data were acquired and analyzed using Sequence Detector version 1.7 software (PerkinElmer Life Sciences) and were normalized to the GAPDH house-keeping gene.

Analysis of Intracellular Polyamine Levels and Enzyme Activities—The polyamine content of treated and untreated cells was determined by using the precolumn dansylation, reversed phase high pressure liquid chromatography method as described previously using 1, 7-diaminoheptane as the internal standard (31). SSAT and ODC activities were determined by using 14C-labeled substrates and scintillation counting of end products produced as described previously (12). The enzyme activities of SMO(PAOh1) and PAO in cell lysates were assayed as described previously by using 250 µM spermine (Sigma) or N1-acetylspermine (Sigma), respectively, as the substrate (18). Protein concentrations were determined by using the Bradford method (32).

Cell Growth and MTT Assays—Cells were plated at a cell density of 5,000 cells/well in 6-well tissue culture plates. After attachment overnight, the medium was replaced, and cells were incubated with or without 10 µM BENSpm for up to 96 h. Every 24 h, cells were detached by trypsinization and counted using a Coulter particle counter. MTT assays were performed as described previously (29). Briefly, following attachment overnight, cells were incubated with increasing concentrations of BENSpm in the presence or absence of 25 µM MDL72527 for 96 h. All of the experiments were plated in quadruplicate and were performed three times. The results from the MTT assays were validated by direct comparison to a conventional cell growth assay.

Flow Cytometry—MDA-MB-231 and MCF-7 cells were plated at a cell density of 100,000 cells in 10-cm culture dishes and were treated with 10 µM BENSpm for up to 96 h. Adherent and nonadherent cells were collected, sedimented at 200 x g for 10 min, washed with ice-cold phosphate-buffered saline, fixed with 4.44% formaldehyde (Sigma), and stained with Hoechst 33258 (Sigma). BD-LSR (BD Biosciences) was used to perform FACS, and the cell cycle was analyzed using Cell-Quest software (BD Biosciences).

RNA Interference and Transfections—The SMO(PAOh1) stable siRNA clones were generated by annealing and inserting the following oligonucleotides (Invitrogen) into the pSilencer 2.1-U6 neo expression vector (Ambion, Austin, TX) according to the manufacturer's instructions: SMO(PAOh1) forward, 5'-GAT CCG CAC TTC TTG AGC AGG GTT TTC AAG AGA AAC CCT GCT CAA GAA GTG CTT TTT TGG AAA, and SMO(PAOh1) reverse, 5'-AGC TTT TCC AAA AAA GCA CTT CTT GAG CAG GGT TTC TCT TGA AAA CCC TGC TCA AGA AGT GCG. The following oligonucleotides (Invitrogen) targeting the SSAT gene were annealed to form the hairpin siRNA template insert that was then ligated into the pSilencer 2.1-U6 hygro expression vector (Ambion) according to the manufacturer's instructions: SSAT forward, 5'-GAT CCG TGA TCC TCC CAC CTC AGC TTC AAG AGA GCT GAG GTG GGA GGA TCA CTT TTT TGG AAA, and SSAT reverse, 5'-AGC TTT TCC AAA AAA GTG ATC CTC CCA CCT CAG CTC TCT TGA AGC TGA GGT GGG AGG ATC ACG.

Lipofectamine was used to transfect 4 µg of the targeting plasmid or provided nonsense control plasmid (Ambion) into MDA-MB-231 and MCF-7 cells. Single clones representing MDA-MB-231 nonsense vector control, MDA-MB-231 {Delta}SMO(PAOh1) (SMO(PAOh1) stably knocked down alone), MDA-MB-231 {Delta}SSAT (SSAT stably knocked down), MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) (both SMO(PAOh1) and SSAT stably knocked down), MCF-7 nonsense vector control, MCF-7 {Delta}SMO(PAOh1) (SMO(PAOh1) stably knocked down), MCF-7 {Delta}SSAT (SSAT stably knocked down), and MCF-7 {Delta}SSAT/{Delta}SMO(PAOh1) (both SMO(PAOh1) and SSAT stably knocked down) were chosen. Clones were selected and maintained in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) supplemented with 5% fetal bovine serum (Mediatech), 1% glutamine (Mediatech), and 500 µg/ml G418 (Sigma) or 500 µg/ml hygromycin (Roche Applied Science) as required. All data presented here are the average of multiple, independent experiments performed using at least three clones for each cell type.



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  		FIGURE 1.
BENSpm induces SSAT and SMO(PAOh1) mRNA and activity in multiple human breast cancer cell lines. MDA-MB-231, Hs578t, MCF-7, and T47D breast cancer cell lines were treated with 10 µM BENSpm for 24 h. A, real time PCR for SSAT mRNA (black bars), PAO mRNA (white bars), and SMO(PAOh1) mRNA (gray bars) was performed as described under "Experimental Procedures"; all values were normalized to the GAPDH housekeeping gene. Values are the means ± S.D. of four independent experiments performed in duplicate. B, SSAT activity was determined as described under "Experimental Procedures." C, SMO(PAOh1) activity was assayed as described under "Experimental Procedures" using 250 µM spermine as the substrate. D, PAO activity was determined as described under "Experimental Procedures" using 250 µM N1-acetylspermine as the substrate. Enzyme activity values are the means ± S.D. of three independent experiments performed in triplicate.

 
Measurement of H2O2—Cells were treated for 24 h with 10 µM BENSpm with or without co-treatment of 25 µM MDL72527 or 500 units/ml catalase, and intracellular H2O2 was detected by FACS. Following treatment, adherent cells were harvested with trypsin and were combined with nonadherent cells. Cells were washed with 1x phosphate-buffered saline (Mediatech), and 1 x 106 cells were treated with 10 µM CM-H2DCFDA for 30 min at 37 °C. Ten thousand cells were then analyzed by FACS on a BD-LSR (BD Biosciences) as reported previously (33).

Statistical Methods—For cell growth assays, the longitudinal data were analyzed using a mixed effects model that accounts for the correlation among repeated measurements (Fig. 2). An exchangeable covariance structure was assumed in the mixed effects model, and cell growth data were fit with a quadratic growth curve model (Fig. 2). Analysis of variance was used to examine the changes in SSAT and SMO(PAOh1) mRNA and activity in BENSpm-treated cell lines (Figs. 3 and 4). Bonferroni adjustment was applied for multiple comparisons. Analysis of covariance was used to examine the difference in the sensitivity to BENSpm among the treated cell lines while controlling for treatment concentration effect (Fig. 5). Pairwise least square means were compared when the overall difference among cell lines was observed. p values were not adjusted for multiple comparisons in the analysis of this experiment (Fig. 5). A p value of ≤0.05 was considered a statistically significant difference between compared groups. All analyses were conducted with SAS System software (version 9.1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
BENSpm Induces SSAT and SMO(PAOh1) mRNA and Activity in Multiple Human Breast Cancer Cell Lines—The induction of SSAT, PAO, and SMO(PAOh1) mRNA and activity by BENSpm was examined in four breast cancer cell lines that represent a wide range of breast cancer phenotypes, MDA-MB-231, Hs578t, MCF-7, and T47D cells. Following treatment with 10 µM BENSpm for 24 h, real time PCR was used to examine changes in SSAT, PAO, and SMO(PAOh1) mRNA (Fig. 1A). SSAT mRNA was induced in all four cell lines following BENSpm exposure, although induction of SMO(PAOh1) mRNA was only seen in MDA-MB-231 and HS578t cells; no PAO mRNA induction was detected in any cell line. Changes in enzyme activity for each of the catabolic enzymes were then examined in each cell line following the same treatment. SSAT enzyme activity was induced in each cell line with the greatest induction observed in BENSpm-treated MDA-MB-231 and Hs578t cells (Fig. 1B). The induction of SMO(PAOh1) enzyme activity closely correlated with the induction of mRNA with induction only seen in MDA-MB-231 and Hs578t cells (Fig. 1C), although no induction of PAO enzyme activity was detected in any of the breast cancer cell lines examined (Fig. 1D). No significant expression or induction of SMO(PAOh1) or SSAT was seen in an immortalized nontumorigenic mammary epithelial cell line, MCF-10A, suggestive of a behavior similar to MCF-7 cells (data not shown).



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  		FIGURE 2.
BENSpm inhibits the growth of multiple breast cancer cell lines. MDA-MB-231, Hs578t, MCF-7, and T47D breast cancer cell lines were treated with 10 µM BENSpm for 96 h. Cells were detached with trypsinization and were counted every 24 h as described under "Experimental Procedures." The results are the means ± S.D. of three independent experiments performed in triplicate with p < 0.001 after 48 h of BENSpm treatment in each cell line as determined using a mixed effects model.

 
BENSpm Inhibits Cell Growth, Reduces Intracellular Polyamine Levels, and Reduces ODC Enzyme Activity in Several Human Breast Cancer Cell Lines—The effect of BENSpm on breast cancer cell growth was examined by treating MDA-MB-231, Hs578t, MCF-7, and T47D cells with 10 µM BENSpm for 96 h. Treatment of each cell line with 10 µM BENSpm for ≥48 h significantly inhibited cell growth (Fig. 2). Cell growth in each cell line was inhibited similarly by BENSpm through 96 h of exposure. FACS analysis showed no difference in cell cycle staging in BENSpm treated MDA-MB-231 and MCF-7 cells; both cell lines arrested in G1 phase after 48 h of BENSpm treatment and remained in a G1 block through 96 h (data not shown). All four breast cancer cell lines treated with 10 µM BENSpm for 24 h exhibited a similar decrease of ~50% in the levels of spermine, spermidine, and putrescine upon BENSpm treatment with a similar level of BENSpm accumulation in each cell line (TABLE ONE). BENSpm treatment also reduced ODC enzyme activity between 5- and 16-fold in each cell line (TABLE ONE).


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  		TABLE ONE
Effects of BENSpm treatment on intracellular polyamine levels and ODC activity

MDA-MB-231, Hs578t, MCF-7, and T47D cells were treated with 10 µM BENSpm for 24 h. Intracellular polyamine levels and ODC enzyme activity were assayed as described under "Experimental Procedures." Values are the means ± S.D. of three independent experiments in duplicate for the polyamines and three independent experiments in triplicate for ODC enzyme activity.

 
Selection and Validation of {Delta}SSAT, {Delta}SMO(PAOh1), and {Delta}SSAT/ {Delta}SMO(PAOh1) Clones—To assess the relative role played by SSAT and SMO(PAOh1) in determining BENSpm response, RNA interference was used to knock down the expression of each enzyme, alone and in combination, in MDA-MB-231 and MCF-7 cells. These cell lines were chosen as they are representative of both hormone-insensitive and hormone-sensitive breast cancers, respectively, and display different enzyme responses to BENSpm. All data presented here are averages of multiple, independent experiments performed using three clones for each cell type. The knockdown of SSAT and SMO(PAOh1) mRNA and enzyme activity in MDA-MB-231 and MCF-7 cells was confirmed by real time PCR and activity assays (Fig. 3). The SSAT and SMO(PAOh1) siRNAs used were both specific and efficient in reducing the related mRNA and activity levels. Furthermore, the knockdown of SMO(PAOh1) in MDA-MB-231 and MCF-7 cells did not significantly alter the induction of SSAT mRNA or activity in either cell line. Similarly, knocking down SSAT in MDA-MB-231 cells did not significantly affect the induction of SMO(PAOh1) mRNA or activity. In the absence of drug treatment, the growth of MDA-MB-231 and MCF-7 cells was not altered by the knockdown of either enzyme alone or in combination (data not shown).

The Knockdown of SSAT Reduces BENSpm-induced Intracellular Polyamine Depletion but Has No Effect on BENSpm-induced Down-regulation of ODC Enzyme Activity—Intracellular and extracellular polyamines were then measured to examine the effects of the knockdown of SSAT and SMO(PAOh1) on the polyamine content within each cell line. Knocking down SSAT, SMO(PAOh1), or the combination did not significantly alter the intracellular polyamine levels in untreated MDA-MB-231 or MCF-7 cells, suggesting that basal polyamine homeostasis in untreated breast cancer cell lines is not dependent on either SSAT or SMO(PAOh1) activity (TABLE TWO). BENSpm treatment of MDA-MB-231 {Delta}SMO(PAOh1) and MCF-7 {Delta}SMO(PAOh1) cells reduced the intracellular polyamine levels to a similar extent (~70–90%) as in BENSpm-treated vector control cells or BENSpm-treated wild-type parental cell lines (TABLE ONE). However, the knockdown of SSAT, either alone or in combination with SMO(PAOh1), in both MDA-MB-231 and MCF-7 cells reduced BENSpm-induced polyamine depletion such that spermine and spermidine levels were only lowered by ~50% (TABLE TWO). Furthermore, although both acetylspermine and acetylspermidine were detected intracellularly in BENSpm-treated vector and {Delta}SMO(PAOh1) cells, neither acetylated polyamine was detected in BENSpm-treated {Delta}SSAT or {Delta}SSAT/{Delta}SMO(PAOh1) cells. In addition, acetylspermine was detected in the media in BENSpm-treated vector and {Delta}SMO(PAOh1) cells but not in the media from BENSpm-treated {Delta}SSAT and {Delta}SSAT/{Delta}SMO(PAOh1) MDA-MB-231 cells (data not shown). BENSpm accumulation was similar in each of the cell lines examined. To determine whether the knockdown of SMO(PAOh1) or SSAT, either alone or in combination, affected other parts of the metabolic pathway, we chose to examine one of the rate-limiting steps in polyamine biosynthesis, ODC. The basal activity level of ODC was similar among all MDA-MB-231- and MCF-7-transfected cell lines (data not shown). ODC enzyme activity significantly decreased in all cell types regardless of SSAT or SMO(PAOh1) knockdown (ODC enzyme activity decreased significantly from 574.1 to 41.7 pmol of CO2/mg of protein/h in BENSpm-treated MDA-MB-231 vector-transfected cells and from 520.4 to 25.4 pmol/CO2/mg of protein/h in BENSpm-treated MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells (p <0.001); ODC enzyme activity decreased significantly from 1194.8 to 75.2 pmol CO2/mg of protein/h in BENSpm-treated MCF-7 vector-transfected cells and from 1221.8 to 61.8 pmol CO2/mg of protein/h in BENSpm-treated MCF-7 {Delta}SSAT/{Delta}SMO(PAOh1) cells (p < 0.001)).


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  		TABLE TWO
Effects of BENSpm treatment on intracellular and extracellular polyamine levels

Cells were treated with 10 µM BENSpm for 24 h. Intracellular polyamine levels in MDA-MB-231 and MCF-7 cells were assayed as described under "Experimental Procedures." Values are the means ± S.D. of three independent experiments performed in duplicate. ND indicates not detected.

 



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  		FIGURE 3.
siRNA directed against SMO(PAOh1) and SSAT specifically and efficiently reduces respective SSAT and SMO(PAOh1) mRNA and activity induction by BENSpm. Transfected MDA-MB-231 cells (black bars) and MCF-7 cells (white bars) were treated with 10 µM BENSpm for 24 h. Real time PCR for SSAT mRNA (A) and real time PCR for SMO(PAOh1) mRNA (C) was performed as described under "Experimental Procedures"; all values were normalized to the GAPDH housekeeping gene. Values are the means ± S.D. of four independent experiments performed in duplicate. B, SSAT enzyme activity was assayed as described under "Experimental Procedures." D, SMO(PAOh1) enzyme activity was assayed as described under "Experimental Procedures" using 250 µM spermine as the substrate. Enzyme activity values are the means ± S.D. of three independent experiments performed in triplicate. Analysis of variance demonstrated that the induction of SSAT mRNA and activity with BENSpm in MDA-MB-231 vector and MDA-MB-231 {Delta}SMO(PAOh1) cells was significantly different from BENSpm-treated MDA-MB-231 {Delta}SSAT cells and MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively). BENSpm-induced SSAT mRNA and activity in MCF-7 vector and MCF-7 {Delta}SMO(PAOh1) cells were significantly different from BENSpm-treated MCF-7 {Delta}SSAT and MCF-7 {Delta}SSAT/{Delta}SMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively). BENSpm-treated MDA-MB-231 vector and MDA-MB-231 {Delta}SSAT cells had a similar induction of SMO(PAOh1) mRNA and activity (p =0.069 and p = 0.114, respectively) that was significantly different from BENSpm-treated MDA-MB-231 {Delta}SMO(PAOh1) and MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively).

 
SSAT, SMO(PAOh1), and SSAT/SMO(PAOh1) Knockdowns Differentially Reduce the Sensitivity of MDA-MB-231 Cells to BENSpm Treatment—MTT assays were used to examine the effects of blocking SSAT and/or SMO(PAOh1) induction on the response of MDA-MB-231 and MCF-7 cells to BENSpm treatment (Fig. 4). The knockdown of SMO(PAOh1) significantly reduced the sensitivity of MDA-MB-231 cells to BENSpm with concentrations greater than 5 µM BENSpm but had no effect on the response of MCF-7 cells to BENSpm. Co-treatment of MDA-MB-231 {Delta}SMO(PAOh1) cells with BENSpm and MDL72527, the polyamine oxidase inhibitor, did not further alter their sensitivity to BENSpm, providing further evidence that the induction of PAO does not play a role in their response to BENSpm (data not shown). Knockdown of SSAT alone significantly reduced the sensitivity of MDA-MB-231 cells to BENSpm with concentrations greater than 1 µM and modestly reduced the sensitivity of MCF-7 cells to BENSpm (Fig. 4). However, MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells were significantly less sensitive to BENSpm than either of the single knockdown cells. As expected, the combined knockdown of these two enzymes in MCF-7 cells did not change their response to BENSpm as compared with the knockdown of SSAT alone. Standard cell growth assays were performed to confirm these results using 1 and 10 µM BENSpm treatments for 96 h in each cell line, and similar results were obtained (data not shown). Flow cytometry analysis demonstrated that the knockdown of either SSAT or SMO(PAOh1) in MDA-MB-231 cells reduced the accumulation of cells in G1 after BENSpm treatment, although the knockdown of both enzymes nearly prevented the BENSpm-induced G1 block (data not shown). In MCF-7 cells, the knockdown of SSAT alone reduced the accumulation of cells in G1, although dual knockdown did not further alter their response to BENSpm (data not shown).

Effects of SSAT and SMO(PAOh1) Knockdown on Hydrogen Peroxide Production—To determine whether the production of hydrogen peroxide by polyamine catabolism plays a role in the antiproliferative effects of BENSpm and to determine which catabolic pathway is responsible for any H2O2 production, CM-H2DCFDA, an oxidation-sensitive fluorescent probe, was used to detect H2O2 production in BENSpm-treated MDA-MB-231 and MCF-7 cells. Treatment of MDA-MB-231 vector-transfected cells for 24 h with 10 µM BENSpm produced a significant increase in fluorescence comparable with that seen for treated wild-type cells (Fig. 5). However, co-treatment with either MDL72527, the polyamine oxidase inhibitor that inhibits both SMO(PAOh1) and PAO enzyme activity, or catalase, which catalyzes the breakdown of H2O2, prevented the increase in fluorescence over control (untreated) cells (data not shown). It is important to note that 25 µM MDL72527 was used to inhibit all oxidase activity in this study, significantly less than 250 µM, which was previously reported in other cell lines (22). To test the possibility that PAO was producing H2O2 but the H2O2 was rapidly detoxified by peroxisomal catalase, an inhibitor of catalase (AT) was used. Co-treatment of MDA-MB-231 cells with BENSpm and AT, or with BENSpm, AT, and catalase, still resulted in increased fluorescence, although co-treatment with BENSpm, AT, and MDL72527 did not increase fluorescence (data not shown). Furthermore, no change in fluorescence was seen in MDA-MB-231 {Delta}SMO(PAOh1) or MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells upon any treatment schedule. Exposure of MDA-MB-231 {Delta}SSAT cells to BENSpm increased fluorescence similar to BENSpm-treated MDA-MB-231 vector-transfected cells, indicating that SSAT induction by BENSpm does not lead to the production of H2O2 through PAO activity; rather the generation of H2O2 in BENSpm-treated MDA-MB-231 cells results primarily from the induction of SMO(PAOh1) activity. All of the controls examined were consistent with SMO(PAOh1) activity being the source of H2O2. No evidence of H2O2 production was seen in MCF-7 wild-type, vector, {Delta}SSAT, {Delta}SMO(PAOh1), or {Delta}SSAT/{Delta}SMO(PAOh1) cells with BENSpm (data not shown).



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  		FIGURE 4.
Effects of SMO(PAOh1) and SSAT knockdown on the sensitivity of breast cancer cell lines to BENSpm. Transfected MDA-MB-231 (A) and transfected MCF-7 (B) cells were exposed to increasing concentrations (0.1–25 µM) of BENSpm for 96 h. The effect on cell growth was assayed using the MTT assay as described under "Experimental Procedures." The results are the means ± S.D. of three independent experiments performed in quadruplicate. Analysis of covariance demonstrated that for BENSpm ≥5 µM, MDA-MB-231 {Delta}SMO(PAOh1) and MDA-MB-231 {Delta}SSAT cells were statistically less sensitive to BENSpm than MDA-MB-231 vector cells (p = 0.020 and p =0.005, respectively). MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) cells were statistically less sensitive to BENSpm than either of the single knockdowns (p < 0.001 and p < 0.001). There was no statistically significant difference in the growth of cells between any of the MCF-7 cell lines.

 



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  		FIGURE 5.
Effects of BENSpm-induced fluorescence in MDA-MB-231 cells detected by CM-H2DCFDA. MDA-MB-231 vector-transfected (A), MDA-MB-231 {Delta}SMO(PAOh1) (B), MDA-MB-231 {Delta}SSAT (C), and MDA-MB-231 {Delta}SSAT/{Delta}SMO(PAOh1) (D) cells were treated with 10 µM BENSpm for 24 h, harvested, and treated with 10 µM CM-H2DCFDA for 30 min. 1 x 105 cells were analyzed by flow cytometry as described under "Experimental Procedures." The x axis represents F1 fluorescence intensity, and the y axis represents cell number. Shown are representative results from one of three experiments that gave similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Induction of polyamine catabolic enzymes has been studied as a potential strategy for anti-cancer therapy (7, 9, 15, 16). The recent cloning of SMO(PAOh1) has enhanced our ability to examine the role that polyamine catabolism may play in cancer (18). SMO(PAOh1) oxidizes spermine to spermidine and produces the toxic reactive oxygen species, H2O2. However, the reactive oxygen species production through induced polyamine catabolism had been hypothesized previously to occur through the classical two-step catalysis by SSAT/PAO (15–17). Reactive oxygen species are known to contribute to a number of destructive processes within cells, including DNA damage, and have been implicated in the cytotoxic effects of some polyamine analogues (11, 14, 16, 34, 35). It was hypothesized that the H2O2 produced by polyamine catabolism contributes to cell growth inhibition, and pharmacologic manipulation of its activity could be used as a strategy to target tumor cells. However, the source or sources of H2O2 and the exact role that each polyamine catabolic enzyme plays in the response to polyamine analogue exposure have not been conclusively determined.

The results presented here demonstrate that RNA interference can be used effectively to knock down the mRNA expression and enzyme activity of two key polyamine catabolic enzymes in human breast cancer cell lines and report the first use of stable siRNAs directed against multiple polyamine metabolic enzymes simultaneously. The stable knockdown of SSAT in this study prevented BENSpm-induced SSAT activity by ~95% and also reduced the BENSpm-induced depletion of polyamine levels in both cell lines. These results are consistent with studies from Chen et al. (28) who reported the use of transient siRNA suppression of SSAT in SK-MEL-28 human melanoma cells. By using a transient knockdown approach, they demonstrated the role of SSAT in spermine depletion and caspase-mediated apoptosis. However, the stable nature of the cell lines generated here provides a more useful system for the examination of long term effects than that afforded by the transient knockdown system reported previously.

The data presented here also demonstrate that SMO(PAOh1) mRNA and activity are differentially induced by BENSpm in several breast cancer cell lines. No induction of PAO mRNA or activity was seen in any of the BENSpm-treated breast cancer cell lines examined (Fig. 1), suggesting that in the breast cancer cell lines examined, PAO is constitutively expressed at low levels. In addition, no significant expression or induction by BENSpm of SMO(PAOh1) was seen in an immortalized nontumorigenic mammary epithelial cell line, MCF-10A (data not shown). The BENSpm-induced oxidase activity utilized only spermine, the preferred SMO(PAOh1) substrate, as a substrate in MDA-MB-231 and Hs578t cells and showed no activity with either N1-acetylspermine, the preferred PAO substrate, or spermidine (data not shown). Vujcic et al. (19) recently reported that BENSpm can induce both PAO and SMO(PAOh1) mRNA in HEK-293 cells. However, the real time PCR and enzyme activity data shown here suggest that PAO is not induced by BENSpm treatment in these breast cancer cell lines.

A key question was whether the antiproliferative effects of BENSpm are due, in part, to the induction of either of the polyamine catabolic pathways and the associated H2O2 production. BENSpm treatment of MDA-MB-231 vector cells resulted in increased fluorescence (detected using the oxidation-sensitive fluorescent probe CM-H2DCFDA), indicative of an increase in reactive oxygen species. Co-treatment of these cells with MDL72527 or catalase prevented the increase in fluorescence, and furthermore, the specific knockdown of SMO(PAOh1) prevented the BENSpm-induced shift in fluorescence. Because the siRNA targeting SSAT had no effect on measurable H2O2, the H2O2 produced in BENSpm-exposed MDA-MB-231 cells must originate from SMO(PAOh1) activity induction, not from PAO activity as proposed previously (11). Furthermore, results obtained using AT demonstrate that inhibition of catalase activity does not alter the FACS profile of either untreated or BENSpm-treated cells, indicating that endogenous catalase is not involved in detoxifying PAO-produced H2O2 and confirming that the primary enzyme involved in the production of H2O2 in response to BENSpm in the human breast cancer cell lines examined is SMO(PAOh1).

It was initially hypothesized that the induction of SSAT enzyme activity contributed to the antiproliferative effects of BENSpm through production of the substrate for PAO activity, the acetylated polyamines, and that the cell growth inhibition resulted from PAO-produced H2O2. However, no PAO enzyme activity was detected in any BENSpm exposed cell line, and furthermore, no H2O2 was detected in MDA-MB-231 {Delta}SMO(PAOh1) cells. After polyamines are acetylated by SSAT, they can either be oxidized by PAO or be exported from the cell (1–3). Because no PAO activity was observed, the induction of SSAT was hypothesized to acetylate the polyamines, which were then exported from the cell. Therefore, the main effect of SSAT induction was the reduction of intracellular polyamine levels. The knockdown of SMO(PAOh1) in MDA-MB-231 and MCF-7 cells had no significant effect on BENSpm-induced reduction in intracellular polyamine levels. However, BENSpm treatment of SSAT knockdown cells resulted in a smaller reduction of intracellular polyamine levels as did BENSpm treatment of vector or {Delta}SMO(PAOh1) cells. Furthermore, although the acetylated polyamines were detected intracellularly and in the media of BENSpm-treated vector and {Delta}SMO(PAOh1) MDA-MB-231 and MCF-7 cells, neither of the acetylated polyamines were detected intracellularly or in the media from either of the SSAT knockdown cells. These results indicate that BENSpm-induced SSAT enzyme activity results in the acetylation of spermine and spermidine, which are subsequently exported from the cell rather than serving as a substrate for PAO, thus resulting in decreased intracellular polyamine levels. The down-regulation of ODC enzyme activity frequently coincides with cell growth inhibition and is likely involved in the antiproliferative effects of BENSpm found in this study (36–38). More importantly, the knockdown of SSAT and/or SMO(PAOh1) did not affect the down-regulation of ODC enzyme activity by BENSpm treatment in MDA-MB-231 or MCF-7 cells, suggesting that other parts of the polyamine metabolic pathway are not affected in the knockdown cell lines.

The results of this study provide further evidence that the effects of polyamine analogues are cell line-specific. In MDA-MB-231 cells, blocking the induction of either SMO(PAOh1) or SSAT individually reduces the sensitivity to BENSpm. When the induction of both SSAT and SMO(PAOh1) is prevented, MDA-MB-231 cells become significantly more resistant to the growth inhibitory effects of BENSpm than the vector controls or the individual knockdowns alone. These results demonstrate that the antiproliferative effects of BENSpm can be mediated through both SSAT and SMO(PAOh1) induction. Thus, agents that target the induction of both of these catabolic enzymes may possess greater antiproliferative activity as compared with agents that target only one of the enzymes. Targeting specific tumor types with agents that induce both SSAT and SMO(PAOh1) may be a rational approach to improve the design of antitumor polyamine analogues.

In summary, this study demonstrates that SMO(PAOh1) and SSAT are major targets of BENSpm in specific human breast cancer cell lines and, furthermore, that these catabolic enzymes act together in determining the response of some breast cancer cell lines to BENSpm. The antiproliferative effects of BENSpm in MDA-MB-231 cells are mediated in part through the production of H2O2 by SMO(PAOh1) and by the export of acetylated polyamines formed by the activity of SSAT. These results demonstrate the independent effects of the polyamine catabolic enzymes in response to polyamine analogue treatment and provide insight for the development of more specific anticancer agents for the treatment of breast cancer.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health and the Department of Defense. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, 1650 Orleans St., CRB 409, Baltimore, MD 21231. Tel.: 410-955-8489; Fax: 410-614-4073; E-mail: davidna{at}jhmi.edu. 2 To whom correspondence may be addressed: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, 1650 Orleans St., CRB 551, Baltimore, MD 21231. Tel.: 410-955-8580; Fax: 410-614-9884; E-mail: rcasero{at}jhmi.edu.

3 The abbreviations used are: SSAT, spermidine/spermine N1-acetyltransferase; SMO(PAOh1), spermine oxidase; BENSpm, N1,N11-bis(ethyl)norspermine; ODC, ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase; PAO, N1-acetylpolyamine oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorter; CM-H2DCFDA, 5-(and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; AT, 3-amino-1,2,4-triazole;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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