Induction of Programmed Cell Death in Human ... - Cancer Research

[CANCER RESEARCH 55, 3233-3236,

August 1, 1995]

Advances in Brief

Induction of Programmed Cell Death in Human Breast Cancer Cells by an Unsymmetrically Alkylated Polyamine Analogue1 Diane E. McCloskey, Robert A. Casero, Jr., Patrick M. Woster, and Nancy E. Davidson2 The Johns Hopkins Oncolog\ Center, Johns Hopkins University School of Medicine. Baltimore, Maryland 21231 ID. E. M., R. A. C, N. E. D.], and Wavne Stale University, Detroit. Michigan 48202 IP. M. W./

Abstract The need for antineoplastic compounds with novel mechanisms of ac tion is great. One such agent is the recently synthesized polyamine analogue /V'-ethyl-A'n-((cyclopropyl)methyl)-4,8-diazaundecane (CPENSpm). Expo sure of hormone-dependent and -independent human breast cancer cells to 0.1-10 UM CPENSpm led to both growth inhibition and induction of pro grammed cell death. Fragmentation of DNA to high molecular weight frag ments and oligonucleosomal-sized fragments, both characteristic of pro grammed cell death, was determined to be time and concentration dependent Depletion of natural polyamine pools and accumulation of the analogue was also demonstrated. These data provide the first evidence that a polyamine analogue induces programmed cell death.

that hormone-dependent MCF7 human breast cancer cells, growing in vivo, undergo programmed cell death upon estrogen withdrawal (12). Furthermore, even hormone-independent breast cancer cells retain the ability to undergo programmed cell death in response to diverse agents including fluoropyrimidines (13) and paclitaxel.4 The evidence presented here suggests that the polyamine analogue CPENSpm, in addition to significantly reducing the growth of both hormone-depen dent and hormone-independent breast cancer cells, can initiate the cell death program. Materials and Methods Compounds, Cell Lines, and Culture Conditions. CPENSpm was syn thesized as described previously (4). For all experiments, a concentrated solution (10 mM in water, stored at -20°) was diluted with medium to the

Introduction The polyamine metabolic pathway is a potential chemotherapeutic target because polyamines are essential for cellular growth and dif ferentiation (1, 2). We have reported previously that Ar',A'12-bis(eth-

desired concentration.

yl)spermine inhibits growth and depletes intracellular polyamine pools in several breast cancer cell lines (3). Polyamine analogues that are unsymmetrically alkylated but structurally similar to the bis(ethyl)polyamines have been synthesized as potential antitumor agents (4, 5). We have now investigated the ability of one of these analogues, CPENSpm,3 to deplete intracellular polyamines, inhibit

231, and Hs578t cells were maintained in DMEM supplemented with 5% fetal bovine serum and 2 mM glutamine. Cultures were incubated at 37°Cin a

growth, and activate programmed cell death pathways in human breast cancer cell lines. Programmed cell death is an active, energy-depen dent process in which the cell is an active participant in its own destruction (6, 7). Initiation by specific signals activates a cascade of biochemical and morphological events that results in the irreversible degradation of genomic DNA and subsequent elimination of the cell. Fragmentation of genomic DNA is considered the committed step of programmed cell death (8), and any compound that effects DNA structure could potentially be involved in the activation or inhibition of this pathway. A relationship between polyamines and programmed cell death is therefore suggested since spermidine and spermine have been reported to stabilize chromatin (9, 10), polyamine-depleted cells have been shown to undergo changes in chromatin and DNA structure (9, 10), and spermine can protect against programmed cell death in thymocytes (11). Programmed cell death pathways are of specific interest as a therapeutic target in breast cancer. Previously, we have demonstrated Received 5/31/95; accepted 6/21/95. 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. 1This work was supported by NIH grants CA09071, CA57545, CA/ES66204, CA63552, and CA51085, and the Susan G. Komen Foundation. 2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology

MDA-MB-468

and ZR-75-1 cells were maintained in

improved minimal essential medium supplemented with 5% fetal bovine scrum (Biofluids, Rockville, MD) and 2 mM glutamine. MCF7, T47D, MDA-MB-

humidified testing was Growth triplicate at

5% CO, atmosphere and passaged every 5 days. Hycopla&na routinely negative. Inhibition Assay. Exponentially growing cells were plated in 1-5 x IO4 cells/cm2 in 24-well plates. After attachment, medium

was changed, and cells were incubated in the absence or presence of at least six CPENSpm concentrations. After 120 h, the cells were detached by trypsinization and counted using a Coulter Counter. Initial experiments demonstrated that cell numbers determined using a Coulter counter were equivalent to numbers of trypan blue excluding cells (viable cells) determined by cell counting using a hemocytometer. IC5ns were determined from plots of the percentage of untreated control cell number versus the logarithm of the drug concentration. All experiments were carried out at least twice, and values reported are mean ±SD of individual points from all experiments. DNA Fragmentation Assays. Exponentially growing cells were plated at 1-5 X IO4 cells/cm2. After attachment, the medium was changed, and cells were incubated with or without drug for the desired exposure time. At harvest, medium and trypsinized cells were combined, and cells were pelleted by centrifugation. For analysis of oligonucleosomal DNA fragmentation, DNA was isolated as described previously (13). Equivalent amounts of DNA (15—20 fig) were loaded into wells of a 1.6% agarose gel and electrophoresed in TBE. For analysis of high molecular weight DNA fragmentation, the cell pellet was resuspended in 1% low melting point agarose and cast in a plug mold. Following digestion with proteinase K and RNase A, plugs were loaded to a 0.8% agarose gel and electrophoresed in 0.5 x TBE for 12-14 h at 6 V/cm using program 6 of a PPI-200 field inversion timer (MJ Research, Watertown, MA). Analysis of Intracellular Polyamine Pools and SSAT Activity. The polyamine content of treated and untreated cells was determined by precolumn dansylation and reversed-phase HPLC using 1,7-diaminoheptane as the inter nal standard (14). SSAT activity was measured using cellular extracts as reported previously (15). Enzyme activity is expressed as pmol /V'-[14C]ace-

Center, Johns Hopkins University School of Medicine, 422 N. Bond Street, Baltimore, MD 21231. 3 The abbreviations used are: CPENSpm, /V'-ethyl-Af"-((cyclopropyl)methyl)-4,8-diazaundecane; SSAT, spermidine/spermine W'-acetyltransferase; IC50, concentration required to inhibit cell growth 50%; TBE, 8.9 mM Tris, 89 IHMboric acid, and 2 mM EDTA.

4 D. E. McCloskey, S. H. Kaufmann, L. J. Prestigiacomo, and N. E. Davidson. Paclitaxel induces programmed cell death in MDA-MB-468 human breast cancer cells, submitted for publication.

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POLYAMINE ANALOGUE-INDUCED CELL DEATH

120

110

110

MCF7

100

MDA-MB-468

100

90

90

Fig. 1. Concentration dependence of growth in hibition of MCF7 and MDA-MB-468 breast cancer cells by CPENSpm. Exponentially growing MCF7 and MDA-MB-468 cells were incubated in the pres ence or absence of CPENSpm for 120 h as described in "Materials and Methods." Values reported are the

SO

i 1 3 t

mean of at least six individual data points deter mined in at least two separate experiments; bars, SD.

70 60 50 40 30 20 10

io-1

io-'

io-*

10-'

io-1

[CPENSpm] (M)

tylspermidine formed/mg protein/min. Protein concentrations were determined by the method of Bradford (16). Northern Blot Analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (17). Northern blot analysis was carried out as described previously (3) using the full-length human SSAT cDNA clone

io-'

io*

io-'

[CPENSpm] (M)

MDA-MB-468

MCF7 U C, C2 C5 C1C 12345

UTOUâ„¢ C, C2 C5 C10 123456

AP3/F7 as a probe (3, 18).

Results Inhibition of Growth of Human Breast Cancer Cell Lines by CPENSpm. The sensitivity of six breast cancer cell lines to CPENSpm was assessed by measurement of growth inhibition result ing from continuous exposure to 0.1-30 JMMCPENSpm. The concen tration dependence of growth inhibition in a representative hormonedependent ceil line (MCF7) and hormone-independent cell line (MDA-MB-468) is shown in Fig. 1. All cell lines exhibited concen tration-dependent growth inhibition from 0.1 to 10 /XMCPENSpm, with growth inhibition of 75 to 98% resulting from exposure to 10 p.M CPENSpm. The IC5()s for CPENSpm ranged from 0.2 to 1.3 JJ.M,and there was no correlation between the estrogen receptor status of the cell lines and sensitivity to CPENSpm (Table 1). Induction of Programmed Cell Death by CPENSpm. The sig nificant growth inhibitory activity of CPENSpm led us to investigate whether part of this effect was a result of programmed cell death induction. Fragmentation of genomic DNA to high molecular weight (a50 kb) fragments is characteristic of the occurrence of programmed cell death and may represent the committed step of the process (8). Field inversion gel electrophoresis was used to assess whether 96 h exposure to CPENSpm resulted in high molecular weight DNA frag mentation (Fig. 2A). CPENSpm concentrations ar5 JIM for MCF7 cells and s2 /XMfor MDA-MB-468 cells were sufficient to induce DNA fragmentation in a concentration-dependent manner. DNA frag mentation was not detected in untreated control cells of either cell line. Similar concentration-dependent high molecular weight DNA fragmentation was also detected in T47D, ZR-75-1, MDA-231, and Hs578t cells (data not shown). Table 1 Sensitivity of breast cancer cell lines to CPENSpm Exponentially growing cells were incubated in the presence or absence of CPENSpm for 120 h, harvested, and counted. IC50s were determined from plots of the percentage of control cell number versus the logarithm of the CPENSpm concentration. Values reported are averages from two experiments. lineMCF7T47DZR-75-1MDA-MB-468MDA-MB-231Hs578tEstrogen Cell receptor (/¿M)+status

IC50

1.1+ 0.4+ 1.10.61.30.2

3234

23.69.46.6-

MCF7

MDA-MB-468

U 1

Fig. 2. DNA fragmentation resulting from CPENSpm exposure of MCF7 and MDAMB-468 cells. A. MCF7 and MDA-MB-468 cells were incubated in the presence or absence of CPENSpm for 96 h and then analyzed by field inversion gel electrophoresis to assess high molecular weight (s50 kb) DNA fragmentation. Lanes in the MCF7 gel: Lane I. untreated control; Lanes 2-5, 1, 2, 5, and 10 /¿MCPENSpm, respectively, 96 h. Lanes in the MDA-MB-468 gel: Lane I, untreated control, time zero; Lane 2, untreated control, 96 h; Lanes 3-6, 1, 2, 5, and 10 /J.MCPENSpm, 96 h. The numbers represent the position of A W/ndlll markers. B, isolated DNA from MCF7 and MDA-MB-468 cells incubated in the presence or absence of CPENSpm was analyzed by agarose gel electrophoresis to assess oligonucleosomal DNA fragmentation. Lanes in the MCF7 gel: Lane I, untreated control, 96 h; Lane 2. 10 JIM CPENSpm, 96 h. Lanes in the MDA-MB-468 gel represent: Lane /. 123-bp DNA ladder marker; Lane 2. untreated control, 96 h; Lane 3, 10 /IM CPENSpm, 96 h.

POLYAMINE

ANALOGUE-INDUCED

CELL DEATH

Table 2 Effects of CPENSpm on polyamine pools, SSAT activity, and SSAT mRNA expression in MDA-MB-468 cells Following incubation of exponentially growing MDA-MB-468 cells in the presence or absence of 10 (¿M CPENSpm for 24 h, intracellular polyamine levels and SSAT activity were determined as described in "Materials and Methods." Values are averages ±SD of ^6 determinations. SSAT mRNA expression was determined by Northern analysis, and the value reported was determined by densitometric analysis. protein)TreatmentControl

RNAexpression

Polyamines (nmol/mg activity (pmol/mgprotein/min)57 ±0.4

1.9 + 0.7Spm21.115.8 1.4 ±0.5Spd7.2 CPENSpmPut"2.0 ' Put, putrescine; Spd, spermidine; Spm, spermine; ND, not detected.

(fold induction)14

±22

±2.0 8.3 ±3.4CPENSpmND31.0 ±7.6SSAT

The above data suggested that programmed cell death pathways had been activated. Because fragmentation of DNA into oligonucleosomal-sized fragments is also associated with programmed cell death in many cell systems, DNA isolated from CPENSpm-treated cells was examined for evidence of such fragmentation. The hormone-indepen dent MDA-MB-468 cell line was chosen as the main focus of these studies because we have previously characterized fluoropyrimidine, paclitaxel,4 and epidermal growth factor induction of programmed

17566 ±5308SSAT

polyamine analogue treatment can induce programmed cell death. High molecular weight DNA fragmentation, which may represent the committed step of this cell death process, resulted from continuous exposure of six breast cancer cell lines to CPENSpm. Oligonucleosomal-sized DNA fragmentation, which is associated with pro grammed cell death in many cell systems, was also observed. Intra cellular depletion of natural polyamine pools and accumulation of the analogue were both associated with CPENSpm treatment. No rela cell death in this cell line (13, 19). MCF7 cells were included as a tionship was found between the steroid hormone receptor status of the representative hormone-dependent cell line. Oligonucleosomal DNA cells and the effects of CPENSpm because programmed cell death fragmentation was detected in both MCF7 and MDA-MB-468 cells was detected in both hormone-dependent and hormone-independent after exposure to 10 /XMCPENSpm for 96 h but not in untreated cell lines and sensitivity to CPENSpm was similar for all cell lines. control cells (Fig. 2B). Induction of MDA-MB-468 DNA fragmenta This suggests that CPENSpm has potential for use even against tion was both time and concentration dependent. Detectable oligonuhormone-independent breast cancers. cleosomal fragmentation resulted from 72 h exposure to 5 or 10 JUM It is currently not known how polyamine analogues exert their CPENSpm, while CPENSpm concentrations as low as l JU.Mwere effects, but several possible mechanisms are consistent with this sufficient to induce detectable fragmentation after 96 h (data not demonstrated ability to induce programmed cell death. Polyamines shown). have been shown to interact with and stabilize DNA and direct Effects of CPENSpm on Intracellular Polyamines, SSAT Activ interaction of polyamine analogues with DNA, and consequent struc ity, and SSAT mRNA Expression. The detection of DNA fragmen tural changes have been postulated as one site of action (22, 23). The tation in these breast cancer cell lines suggested that CPENSpm accumulation of CPENSpm and the depletion of the natural poly activity was cytotoxic to at least a portion of the cell population rather amines by down-regulation of the biosynthetic enzymes and the than merely antiproliferative. As exposure to polyamine analogues up-regulation of the catabolic pathway produce a natural polyaminecan result in polyamine depletion (3, 20, 21), it was of interest to depleted environment that could favor the analogue/DNA interaction. determine whether polyamine depletion could be involved in the Such analogue/DNA interactions may result in greater accessibility observed CPENSpm-induced DNA fragmentation. Exposure of and susceptibility of DNA to the fragmentation that is an essential part MDA-MB-468 cells to 10 JU.MCPENSpm for 24 h resulted in a of programmed cell death. Similarly, loss of natural polyamine/DNA significant decrease in the natural polyamines and intracellular accu interactions, which could interfere with normal gene expression and mulation of the analogue (Table 2). Polyamine depletion can result cellular function, may result and trigger cell death as depletion of from increased activity of SSAT, and induction of SSAT activity by natural polyamines occurs concurrently or subsequently to accumu symmetrically substituted polyamine analogues has been correlated lation of the analogue. These possibilities are consistent with reports with the cytotoxicity of these compounds in non-small cell lung that spermine can protect against programmed cell death in thymocancer and melanoma cell lines (20, 21). Thus, the effect of cytes (11), and polyamine-depleted cells can undergo changes in CPENSpm on SSAT activity was examined. SSAT activity was chromatin structure (9, 10). highly induced («300-fold induction over control) in MDA-MB-468 Recent reports have detailed complex regulation of polyamine cells after 24 h exposure to 10 /XMCPENSpm (Table 2), and this transport to maintain intracellular polyamine content at levels that are induction was associated with a 4-fold increase in SSAT mRNA sufficient for, but not toxic to, normal cellular function (24, 25). This expression (Table 2). SSAT activity was not significantly induced in suggests another mechanism by which polyamine analogues could any of the other five breast cancer cell lines tested (data not shown). exert cytotoxic effects. Interference with regulatory mechanisms of polyamine transport could result in the accumulation of the analogue Discussion to a level that results in disruption of the balance of positive and negative charges normally maintained intracellularly and could lead to The search for better cancer chemotherapeutic agents has included cell death. agents that target polyamine pathways because polyamines are essen tial for tumor cell growth (1, 2). 7V,Af'-bis(ethyl) analogues of sperm The up-regulation of the polyamine catabolic enzyme SSAT by polyamine analogues provides another potential link to programmed ine have been of particular interest, and one of the members of this family, Wl,./V11-bis(ethyl)norspermine, is now in Phase 1 clinical trials. cell death induction. The action of SSAT produces N-acetyl poly amine derivatives, which are then oxidized by polyamine oxidase with Unsymmetrically substituted polyamine analogues that are structur the consequent production of hydrogen peroxide. It has been postu ally similar to the bis(ethyl)polyamine analogues have been synthe lated that, in the developing embryo, oxidation of polyamines by sized recently for evaluation as potential antitumor agents (4, 5). The polyamine oxidase results in production of hydrogen peroxide, which studies presented here demonstrate that one of these unsymmetrically alkylated analogues, CPENSpm, has significant growth-inhibitory activates programmed cell death (26). However, it is unlikely that CPENSpm cytotoxicity is solely dependent on SSAT induction beeffects in human breast cancer cells and provide the first evidence that 3235

POLYAMINE

ANALOGUE-INDUCED

cause preliminary evidence indicates that of the breast cancer cell lines tested, MDA-MB-468 is the only one in which SSAT activity is significantly induced. Additional studies are needed to dissect the mechanisms by which CPENSpm acts to produce both antiproliferative and cytotoxic effects. However, these studies indicate that the relationship between polyamines, polyamine analogues, and cell death pathways should be explored. Additionally, the combination of CPENSpm or other poly amine analogues with other agents that act through programmed cell death induction should be considered. Acknowledgments

11. 12.

13.

14.

15.

We thank Pamela Tamez for technical assistance. References 1. Casero, Jr. R. A., and Pegg, A. E. Spermidine/spermine /V'-acetyltransferase—the turning point in polyamine metabolism. FASEB J., 7: 653-661, 1993. 2. Pegg, A. E. Polyamine metabolism and its importance in neoplastic growth as a target for chemotherapy. Cancer Res., 48: 759-774, 1988. 3. Davidson, N. E., Mank, A. R., Prestigiacomo, L. J., Bergeron, R. J., and Casero, R. A., Jr. Growth inhibition of hormone-responsive and -resistant human breast cancer cells in culture by A/'A'2-bis(ethyl)spermine. Cancer Res., 53: 2071-2075, 1993. 4. Saab, M. H., West, E. E., Bieszk, N. C, Preuss, C. V., Mank, A. R., Casero, R. A., Jr., and Woster, P. M. Synthesis and evaluation of unsymmetrically substituted polyamine analogues as modulators of human spermidine/spermine-W'-acetyltransferase (SSAT) and as potential antitumor agents. J. Med. Chem., 36: 2998-3004, 1993. 5. Casero, R. A., Jr., Mank, A. R., Saab, N. H., Ronghui, W., Dyer, W. J., and Woster, P. M. Growth and biochemical effects of unsymmetrically substituted polyamine analogues in human lung tumor cells. Cancer Chemother. Pharmacol., 36: 69-74, 1995. 6. Isaacs, J. T. Role of programmed cell death in carcinogenesis. Environ. Health Perspect. 101 (Suppl. 5): 27-34, 1993. 7. Wyllie A. H. Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview. Cancer Metastasis Rev., //: 95-103, 1992. 8. Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R., and Sikorska, M. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J., 12: 3679-3684, 1993. 9. Marlon, L. J., and Morris, D. R. Molecular and cellular functions of the polyamines. In: P. P. McCann, A. E. Pegg, and A. Sjoerdsma (eds.), Inhibition of Polyamine Metabolism: Biological Significance and Basis for New Therapies, pp. 79—105.New York: Academic Press, 1987. 10. Porter, C. W., and Janne. J. Modulation of antineoplastic drug action by inhibitors of polyamine biosynthesis. In: P. P. McCann, A. E. Pegg, and A. Sjoerdsma (eds.),

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Inhibition of Polyamine Metabolism: Biological Significance and Basis for New Therapies, pp. 203-248. New York: Academic Press, 1987. Brune, B., Hartzell, P., Nicotera, P., and Orrenius, S. Spermine prevents endonuclease activation and apoptosis in thymocytes. Exp. Cell Res., 195: 323-329, 1991. Kyprianou, N., English, H. F., Davidson, N. E., and Isaacs, J. T. Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation. Cancer Res., 51: 162-166, 1991. Armstrong, D. K., Isaacs, J. T., Ottaviano, Y. L., and Davidson, N. E. Programmed cell death in an estrogen-independent human breast cancer cell line, MDA-MB-468. Cancer Res., 52: 3418-3424, 1992. Kabra, P. M., Lee, G. K., Lubich, W. P., and Marron, L. J. Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reverse phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissues. J. Chromatogr. Biomed. Appi., 380: 19-32, 1986. Casero, R. A.. Jr., Celano, P., Ervin, S. J., Wiest, L., and Pegg, A. E. High specific induction of spermidine/spermine A^-acetyltransferase in a human large cell lung carcinoma. Biochem. J., 270.- 615-620, 1990.

16. Bradford, C. A. A rapid and sensitive method for quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248—254, 1976. 17. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156159, 1987. 18. Xiao, L., Celano, P., Mank, A. R., Pegg, A. E., Casero, R. A., Jr. Characterization of a full-length cDNA which codes for the human spermidine/spermine /V'-acetyltransferase. Biochem. Biophys. Res. Commun., 179: 407-415, 1991. 19. Armstrong, D. K., Kaufmann, S. H., Ottaviano, Y. L., Furuya, Y., Buckley, J. A., Isaacs, J. T., and Davidson, N. E. Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells. Cancer Res., 54: 5280-5283, 1994. 20. Casero, R. A., Mank, A. R., Xiao, L., Smith, J., Bergeron, R. J., and Celano, P. Steady-state messenger RNA and activity correlates with sensitivity to N',N12bis(ethyl)spermine in human cell lines representing the major forms of lung cancer. Cancer Res., 52: 5359-5363, 1992. 21. Porter, C. W., Ganis, B., Libby, P. R., and Bergeron, R. J. Correlations between polyamine analogue-induced increases in spermidine/spermine /V'-acetyltransferase

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