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
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m508177200
ISSN1083-351X
AutoresAllison Pledgie, Yi Huang, Amy Hacker, Zhe Zhang, Patrick M. Woster, Nancy E. Davidson, Robert A. Casero,
Tópico(s)Cannabis and Cannabinoid Research
ResumoThe 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. 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. The natural polyamines, spermine, spermidine, and putrescine, are ubiquitous polycationic alkylamines that are required for normal eukaryotic cell growth and differentiation (1Huang Y. Pledgie A. Casero Jr., R.A. Davidson N.E. Anti-Cancer Drugs. 2005; 16: 229-241Crossref PubMed Scopus (78) Google Scholar, 2Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (769) Google Scholar). Neither mammalian cells lacking polyamine biosynthetic enzymes nor cells depleted of polyamines are able to replicate (3Thomas T. Thomas T.J. Cell. Mol. Life Sci. 2001; 58: 244-258Crossref PubMed Scopus (759) Google Scholar). Polyamine metabolism is frequently dysregulated in many types of cancer, including breast, prostate, and lung cancer (1Huang Y. Pledgie A. Casero Jr., R.A. Davidson N.E. Anti-Cancer Drugs. 2005; 16: 229-241Crossref PubMed Scopus (78) Google Scholar, 4Kingsnorth A.N. Wallace H.M. Bundred N.J. Dixon J.M. Br. J. 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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 (9Gabrielson E.W. Pegg A.E. Casero Jr., R.A. Clin. Cancer Res. 1999; 5: 1638-1641PubMed Google Scholar, 10Casero R.A. Wang Y. Stewart T.M. Devereux W. Hacker A. Smith R. Woster P.M. Biochem. Soc. Trans. 2003; 31: 361-365Crossref PubMed Google Scholar, 11Casero Jr., R.A. Pegg A.E. FASEB J. 1993; 7: 653-661Crossref PubMed Scopus (392) Google Scholar, 12Casero Jr., R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P.R. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar, 13Fogel-Petrovic M. Kramer D.L. Vujcic S. Miller J. McManis J.S. Bergeron R.J. Porter C.W. Mol. Pharmacol. 1997; 52: 69-74Crossref PubMed Scopus (43) Google Scholar, 14Bergeron R.J. Neims A.H. McManis J.S. Hawthorne T.R. Vinson J.R. Bortell R. Ingeno M.J. J. Med. 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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) 3The abbreviations used are: SSATspermidine/spermine N1-acetyltransferaseSMO(PAOh1)spermine oxidaseBENSpmN1,N11-bis(ethyl)norspermineODCornithine decarboxylaseAdoMetDCS-adenosylmethionine decarboxylasePAON1-acetylpolyamine oxidaseGAPDHglyceraldehyde-3-phosphate dehydrogenasesiRNAsmall interfering RNAFACSfluorescence-activated cell sorterCM-H2DCFDA5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetateAT3-amino-1,2,4-triazoleMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 3The abbreviations used are: SSATspermidine/spermine N1-acetyltransferaseSMO(PAOh1)spermine oxidaseBENSpmN1,N11-bis(ethyl)norspermineODCornithine decarboxylaseAdoMetDCS-adenosylmethionine decarboxylasePAON1-acetylpolyamine oxidaseGAPDHglyceraldehyde-3-phosphate dehydrogenasesiRNAsmall interfering RNAFACSfluorescence-activated cell sorterCM-H2DCFDA5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetateAT3-amino-1,2,4-triazoleMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. and a relatively constitutively expressed, peroxisomal N1-acetylpolyamine oxidase (PAO) (1Huang Y. Pledgie A. Casero Jr., R.A. Davidson N.E. Anti-Cancer Drugs. 2005; 16: 229-241Crossref PubMed Scopus (78) Google Scholar, 2Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (769) Google Scholar). 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 (10Casero R.A. Wang Y. Stewart T.M. Devereux W. Hacker A. Smith R. Woster P.M. Biochem. Soc. Trans. 2003; 31: 361-365Crossref PubMed Google Scholar, 15McCloskey D.E. Yang J. Woster P.M. Davidson N.E. Casero Jr., R.A. Clin. Cancer Res. 1996; 2: 441-446PubMed Google Scholar, 16Ha H.C. Woster P.M. Yager J.D. Casero Jr., R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11557-11562Crossref PubMed Scopus (271) Google Scholar, 17Marton L.J. Pegg A.E. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 55-91Crossref PubMed Scopus (708) Google Scholar). However, recent studies have clearly demonstrated that an additional enzyme exists in the mammalian polyamine catabolic pathway, an inducible spermine oxidase (SMO/PAOh1) (18Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr., R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar, 19Vujcic S. Diegelman P. Bacchi C.J. Kramer D.L. Porter C.W. Biochem. J. 2002; 367: 665-675Crossref PubMed Scopus (188) Google Scholar). SMO(PAOh1) is a cytosolic protein that is selectively active on spermine producing H2O2, spermidine, and the aldehyde 3-aminopropanol (20Wang Y. Murray-Stewart T. Devereux W. Hacker A. Frydman B. Woster P.M. Casero R.A. Biochem. Biophys. Res. Commun. 2003; 304: 605-611Crossref PubMed Scopus (113) Google Scholar, 21Bellelli A. Cavallo S. Nicolini L. Cervelli M. Bianchi M. Mariottini P. Zelli M. Federico R. Biochem. Biophys. Res. Commun. 2004; 322: 1-8Crossref PubMed Scopus (37) Google Scholar). 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 (22Devereux W. Wang Y. Stewart T.M. Hacker A. Smith R. Frydman B. Valasinas A.L. Reddy V.K. Marton L.J. Ward T.D. Woster P.M. Casero R.A. Cancer Chemother. Pharmacol. 2003; 52: 383-390Crossref PubMed Scopus (58) Google Scholar). spermidine/spermine N1-acetyltransferase spermine oxidase N1,N11-bis(ethyl)norspermine ornithine decarboxylase S-adenosylmethionine decarboxylase N1-acetylpolyamine oxidase glyceraldehyde-3-phosphate dehydrogenase small interfering RNA fluorescence-activated cell sorter 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate 3-amino-1,2,4-triazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. spermidine/spermine N1-acetyltransferase spermine oxidase N1,N11-bis(ethyl)norspermine ornithine decarboxylase S-adenosylmethionine decarboxylase N1-acetylpolyamine oxidase glyceraldehyde-3-phosphate dehydrogenase small interfering RNA fluorescence-activated cell sorter 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate 3-amino-1,2,4-triazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 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 (23Hahm H.A. Ettinger D.S. Bowling K. Hoker B. Chen T.L. Zabelina Y. Casero Jr., R.A. Clin. Cancer Res. 2002; 8: 684-690PubMed Google Scholar, 24Wolff A.C. Armstrong D.K. Fetting J.H. Carducci M.K. Riley C.D. Bender J.F. Casero Jr., R.A. Davidson N.E. Clin. Cancer Res. 2003; 9: 5922-5928PubMed Google Scholar). 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 (25Wallace H.M. Fraser A.V. Amino Acids (Vienna). 2004; 26: 353-365Crossref PubMed Scopus (137) Google Scholar, 26Seiler N. Amino Acids (Vienna). 2004; 26: 317-319PubMed Google Scholar, 27Seiler N. Duranton B. Raul F. Prog. Drug Res. 2002; 59: 1-40PubMed Google Scholar). 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 (28Chen Y. Kramer D.L. Jell J. Vujcic S. Porter C.W. Mol. Pharmacol. 2003; 64: 1153-1159Crossref PubMed Scopus (33) Google Scholar). 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. 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 (29Hahm H.A. Dunn V.R. Butash K.A. Devereux W.L. Woster P.M. Casero R.A. Davidson N.E. Clin. Cancer Res. 2001; 7: 391-399PubMed Google Scholar). BENSpm and MDL72527 were synthesized as described previously (14Bergeron R.J. Neims A.H. McManis J.S. Hawthorne T.R. Vinson J.R. Bortell R. Ingeno M.J. J. Med. Chem. 1988; 31: 1183-1190Crossref PubMed Scopus (184) Google Scholar, 30Bey P. Bolkenius F.N. Seiler N. Casara P. J. Med. Chem. 1985; 28: 1-2Crossref PubMed Scopus (115) Google Scholar). 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 (31Kabra P.M. Lee H.K. Lubich W.P. Marton L.J. J. Chromatogr. 1986; 380: 19-32Crossref PubMed Scopus (238) Google Scholar). SSAT and ODC activities were determined by using 14C-labeled substrates and scintillation counting of end products produced as described previously (12Casero Jr., R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P.R. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar). 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 (18Wang Y. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero Jr., R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar). Protein concentrations were determined by using the Bradford method (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). 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 (29Hahm H.A. Dunn V.R. Butash K.A. Devereux W.L. Woster P.M. Casero R.A. Davidson N.E. Clin. Cancer Res. 2001; 7: 391-399PubMed Google Scholar). 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 × 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 ΔSMO(PAOh1) (SMO(PAOh1) stably knocked down alone), MDA-MB-231 ΔSSAT (SSAT stably knocked down), MDA-MB-231 ΔSSAT/ΔSMO(PAOh1) (both SMO(PAOh1) and SSAT stably knocked down), MCF-7 nonsense vector control, MCF-7 ΔSMO(PAOh1) (SMO(PAOh1) stably knocked down), MCF-7 ΔSSAT (SSAT stably knocked down), and MCF-7 ΔSSAT/ΔSMO(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. 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 1× phosphate-buffered saline (Mediatech), and 1 × 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 (33Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliman C.L. Korsmeyer S.J. Cell. Mol. Life Sci. 2000; 75: 241-251Google Scholar). 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).FIGURE 3siRNA 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 ΔSMO(PAOh1) cells was significantly different from BENSpm-treated MDA-MB-231 ΔSSAT cells and MDA-MB-231 ΔSSAT/ΔSMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively). BENSpm-induced SSAT mRNA and activity in MCF-7 vector and MCF-7 ΔSMO(PAOh1) cells were significantly different from BENSpm-treated MCF-7 ΔSSAT and MCF-7 ΔSSAT/ΔSMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively). BENSpm-treated MDA-MB-231 vector and MDA-MB-231 ΔSSAT 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 ΔSMO(PAOh1) and MDA-MB-231 ΔSSAT/ΔSMO(PAOh1) cells (p < 0.0001 and p < 0.0001, respectively).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Effects 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 ΔSMO(PAOh1) and MDA-MB-231 ΔSSAT cells were statistically less sensitive to BENSpm than MDA-MB-231 vector cells (p = 0.020 and p =0.005, respectively). MDA-MB-231 ΔSSAT/ΔSMO(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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Effects of BENSpm-induced fluorescence in MDA-MB-231 cells detected by CM-H2DCFDA. MDA-MB-231 vector-transfected (A), MDA-MB-231 ΔSMO(PAOh1) (B), MDA-MB-231 ΔSSAT (C), and MDA-MB-231 ΔSSAT/ΔSMO(PAOh1) (D) cells were treated with 10 μm BENSpm for 24 h, harvested, and treated with 10 μm CM-H2DCFDA for 30 min. 1 × 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). 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).TABLE ONEEffects of BENSpm treatment on intracellular polyamine levels and ODC activityCell lineTreatmentPolyaminesBENSpmODC activityPutrescineSpermidineSperminenmol/mg proteinnmol/mg proteinpmol CO2/mg protein/hMDA-MB-231Control3.9 ± 0.236.9 ± 4.647.0 ± 8.2ND577.9 ± 26.1BENSpm2.3 ± 0.310.9 ± 1.220.6 ± 3.438.5 ± 4.476.7 ± 4.8Hs578tControl2.6 ± 0.746.6 ± 3.245.3 ± 2.9ND480.6 ± 19.5BENSpm1.2 ± 0.215.7 ± 1.622.2 ± 1.145.2 ± 4.793.4 ± 9.2MCF-7Control6.2 ± 0.365.8 ± 6.952.2 ± 7.8ND1525.7 ± 116.6BENSpm2.7 ± 0.226.8 ± 5.426.4 ± 4.752.3 ± 5.993.4 ± 12.4T47DControl4.3 ± 0.560.7 ± 4.447.0 ± 2.8ND1264.6 ± 65.3BENSpm2.2 ± 0.426.2 ± 1.714.7 ± 0.942.6 ± 2.4103.1 ± 6.5 Open table in a new tab
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