Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation

Jerry Moffatt1, Makiko Hashimoto1, Akiko Kojima1, David Opare Kennedy1, Akira Murakami2, Koichi Koshimizu2, Hajime Ohigashi3 and Isao Matsui-Yuasa1,4

1 Department of Food and Nutrition, Faculty of Human Life Science, Osaka, City University, Osaka 558-8585,
2 Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493 and
3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto, University, Kyoto 606-8502, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The efficacy of the antitumor activity of 1'-acetoxychavicol acetate (ACA), reported to be a suppressor of chemically induced carcinogenesis, was evaluated in Ehrlich ascites tumor cells. ACA treatment resulted in changes in morphology and a dose-dependent suppression of cell viability. Apoptosis, characterized by nuclear condensation, membrane blebbing, cell shrinkage and a significant induction of caspase-3-like protease activity at 8 h in a time-course study were observed. Formation of apoptotic bodies was preceded by lowering of intracellular polyamines, particularly putrescine, and both dose- and time-dependent inhibitory and activation effect by ACA on ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), respectively. Administration of exogenous polyamines prevented ACA-induced apoptosis represented by a reduction in the number of apoptotic bodies and also caused reduction in the induced caspase-3-like protease activity at 8 h. These findings suggest that the anticarcinogenic effects of ACA might be partly due to perturbation of the polyamine metabolic pathway and triggering of caspase-3-like activity, which result in apoptosis.

Abbreviations: ACA, 1'-acetoxychavicol acetate; AP-1, activated protein 1; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FCS, fetal calf serum; ICE, interleukin-1ß-converting enzyme; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor {kappa}B; NMBA, N-nitrosomethylbenzylamine; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SSAT, spermidine/spermine N1-acetyltransferase; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among the natural compounds screened from diverse edible plants in Thailand and other countries in south-eastern Asia for antitumor properties, 1'-acetoxychavicol acetate (ACA), obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberaceae), potently inhibits tumor promoter-induced Epstein–Barr virus activation (1). It inhibits xanthine oxidase (2), the enzyme that catalyses the hydroxylation of purine species to yield superoxide ions, and occurs in large amounts in certain brain tumors. In a series of studies on rodent models of carcinogenesis, ACA exhibited chemopreventive effects on chemically induced tumor formation in mouse skin (3) and in the mouth (4), colon (5,6) and esophagus (7) of rats. It also prevents formation of focal lesions positive for the placental form of glutathione S-transferase, elicited by a choline-deficient L-amino acid defined diet (8) and reduces 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced reactive oxygen species (ROS) in mouse skin (9). In lipopolysaccharide- and interferon-{gamma}-stimulated macrophages, ACA markedly suppresses the induction of inducible nitric oxide synthase through its inhibitory effects on transcription factors such as nuclear factor {kappa}B (NF-{kappa}B) and activated protein 1 (AP-1) (10), thereby preventing excessive production of nitric oxide in the inflammatory response. These mechanisms may contribute, at least in part, to its potential use as an anticancer drug.

The polyamines (putrescine, spermidine and spermine) are indispensable for proliferation and differentiation of cells. Depletion of polyamines is associated with reduced cell proliferation, and the levels of polyamines rapidly increase during cell growth (1115). The importance of polyamines in cell growth is further evidenced in the observations that dysregulation of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzymes in the synthesis and biodegradation, respectively, of polyamines, affects the concentration of polyamines which modulates polyamine synthesis, degradation, uptake and excretion (16,17). These biochemical and physiological modifications can eventually control vital cell events such as apoptosis (1823). Polyamines may be involved in the onset of DNA degradation, as spermidine and spermine stabilize chromatin- and polyamine-depleted cells in which chromatin and DNA structural changes occur (23,24).

Apoptosis is a fundamental biological regulatory mechanism leading to selective cell deletion. This built-in death program is required for tissue growth and homeostasis; it can be initiated by both internal physiological responses and external stimuli including oxidative stress and chemotherapeutic agents (25). Apoptotic events include DNA fragmentation, nuclear condensation, membrane blebbing and cell shrinkage (26). Caspase activation precedes the cleavage of specific proteins and is critical in apoptosis (26,27).

In the present study, we sought to evaluate whether ACA exerts its antitumor effects by inducing apoptosis in Ehrlich ascites tumor cells, and by the mediation of polyamines in the apoptotic events, as determined by morphological changes and caspase-3-like protease activity.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
ACA was isolated from Languas galanga as previously reported (1). L-[1-14C]Ornithine hydrochloride (56 mCi/mmol) and L-[acetyl-1-14C]acetyl coenzyme A (56 mCi/mmol) were obtained from Moravek Biochemicals (Brea, CA). Fluorescence caspase-3 substrate [acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide (Ac-DEVD-MCA)] was obtained from the Peptide Institute, Inc. (Osaka, Japan). Putrescine, spermidine and spermine were purchased from Sigma–Aldrich Corp. (Tokyo, Japan) and fetal calf serum (FCS) from JRH Biosciences (Lenexa, Australia). Other chemicals used in this study were special grade commercial products.

Cell culture
Ehrlich ascites tumor cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C for 3–4 days in Eagle's minimum essential medium containing 10% FCS, washed and cultured again at a concentration of 1 x 106 cells/ml in fresh medium. ACA was dissolved in dimethyl sulfoxide (DMSO) and diluted in cultured medium immediately before use (final DMSO concentration <0.25%). In all experiments control cells were incubated in medium and DMSO only.

Assay of cell viability
Cell viability was determined by Trypan Blue exclusion analysis. Cells (1 x 106) treated with ACA at various concentrations were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C overnight in Eagle's minimum essential medium containing 10% FCS. To a cell suspension was added an equal volume of 0.4% Trypan Blue (Sigma) and the number of viable cells was evaluated under a field microscope. Assays were performed in triplicate.

ODC activity assay
ODC activity was assayed as described (28). Briefly, after incubation with or without ACA at various concentrations and for various periods of time, cells were washed with phosphate-buffered saline and suspended in 0.15 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 250 µM pyridoxal phosphate, 0.1 mM EDTA and 2.5 mM dithiothreitol. The cells were disrupted by three cycles of freezing and thawing, then crude extracts were prepared for the enzyme assay by centrifugation at 30 000 x g for 20 min. ODC activity was measured by estimating the release of 14CO2 from L-[1-14C]ornithine. The protein concentration was measured by the Bradford method (29).

Assay of SSAT
The enzyme extract was prepared from cells as described (30). In brief, cells were washed with phosphate-buffered saline and suspended in 0.15 ml of 50 mM Tris–HCl buffer (pH 7.8). The cells were then disrupted by three cycles of freezing and thawing, and crude extracts were prepared for enzyme assay by centrifugation at 30 000 x g for 20 min. SSAT activity was measured by estimating the incorporation of 14C into monoacetyl-spermidine.

Determination of intracellular polyamines
Cells (2 x 106) collected by centrifugation were extracted with 0.2 ml of 0.4 N perchloric acid. The supernatant was stored at –20°C until used. The polyamines were separated on an ODS-II column (4.6 x 150 mm, particle size 5 µm, Shimadzu Techno-Research, Kyoto, Japan) using solvents A [10 mM 1-hexane sulfonic acid (sodium salt), 100 mM sodium perchloric acid] and B (one part solvent A to three parts methanol). The sample was eluted with 96% solvent A and 4% solvent B for 3 min, and then with a programmed solvent gradient using a linear gradient curve. The gradient changed from 20% to 55% solvent B from 3.1 min to 25 min at a flow rate of 0.7 ml/min. Eluted fractions were mixed with O-phthalaldehyde (0.7 ml/min) and the fluorescence was measured at excitation and emission wavelengths of 345 and 440 nm, respectively, for assay of polyamines with an RF535 fluorescence monitor (Shimadzu). The DNA content of the perchloric acid-precipitable materials was determined by the method of Schneider et al. (31) using calf thymus DNA as a standard.

Determination of apoptosis
The cells were fixed with 1% glutaraldehyde in phosphate-buffered saline (PBS) for 20 min, stained with 4',6-diamidino-2-phenylindole (DAPI) (1 µg/ml) and examined with a 40x objective under epifluorescence optics (Olympus, BX-FLA). Apoptotic cells were evaluated by scoring cells for fragmented nuclei and condensed chromatin.

Transmission electron microscopy
Transmission electron microscopy was performed to provide ultrastructural evidence of apoptosis. Cells were washed twice with 1.5% glutaraldehyde in 0.062 M cacodylate buffer, pH 7.4, plus 1% sucrose, then (at 2 x 106 cells/ml) fixed with the same solution at room temperature for 1 h. After centrifuging, the pellets were post-fixed with 1% OsO4, dehydrated in an ethanol series and embedded in Polybed (Polyscience Inc., Warrington, PA). Ultra-thin sections were stained with uranyl acetate and lead citrate and observed under a JEM 1200 EXII electron microscope (JEOL, Tokyo, Japan) at 80 kV.

Caspase-3-like protease activity assay
Cells (2 x 106) were harvested from 35 mm culture dishes, washed with ice-cold PBS, and resuspended in 300 µl of buffer A [10 mM HEPES pH 7.2, 5 mM EGTA, 0.1% CHAPS, 5 mM dithiothreitol (DTT), 1.5 mM MgCl2 and protease inhibitors, including 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin and 20 µg/ml leupeptin]. The cell suspension was lysed by three cycles of freezing and thawing. After centrifugation at 12 000 x g for 20 min at 4°C, 50 µl of supernatant (containing 10 µg protein) from cell lysates was transferred to individual wells of a 96-well plate, and incubated with 50 µM of the enzyme substrate Ac-DEVD-MCA in 50 µl of buffer B (100 mM HEPES pH 7.25, 10% sucrose, 0.1% CHAPS, 5 mM DTT and 0.0001% Nonidet P-40) at 37°C for 1 h. Levels of released AMC were measured using a spectrofluorophotometer (Wallac 1420 ARVOSX) with excitation at 380 nm and emission at 460 nm. The amounts of released AMC were calculated from a standard AMC curve. Fluorescent units were converted to nanomoles of released AMC per hour. Protein concentration was measured by the Bradford method (29).

Preparation and culture of hepatocytes
Hepatocytes were isolated by the collagenase perfusion method from Sprague–Dawley rats (150–200 g) under sodium pentobarbital anesthesia. The cells were counted using a hemocytometer and their viability (>90%) was ascertained by Trypan Blue exclusion. The cells were seeded at a density of 2.5 x 105 cells per ml of Williams' medium E in 35 mm culture dishes and maintained in a humidified incubator with 5% CO2 and 95% air at 37°C. After overnight incubation, the culture medium was changed to serum-free experimental medium and cultures were incubated with varying concentrations of ACA for 24 h. Control cultures contained DMSO only.

Analysis of viability of hepatocytes
This was evaluated by the Neutral Red assay (32). Culture medium was replaced with 500 µl of Neutral Red solution [0.4% Neutral Red in sodium bicarbonate:phenol-free Hank's solution (1:80), pH 7.0]. Hepatocytes were incubated with Neutral Red solution at 37°C and removed after 2 h. Monolayers of cultures were washed with 1% formaldehyde, 1% calcium chloride, then a mixture of 1% acetic acid and 50% ethanol was added to extract the dye from lysosomes. The amount of extracted dye was measured at 540 nm with a spectrophotometer.

Statistical analysis
All data are presented as means ± SD and statistical evaluations were done using Student's t-test. P <= 0.05 was used to indicate a statistically significant difference.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of ACA on viability of Ehrlich ascites tumor cells
The effect of ACA on the viability of Ehrlich ascites tumor cells was examined by means of the Trypan Blue exclusion method. After 3–4 days culture in Eagle's minimum essential medium containing 10% FCS, cells were diluted and incubated in fresh medium with or without ACA. Cell viability was assessed 24 h later. As shown in Figure 1Go, treatment of cells with 10–40 µM ACA resulted in significant reduction in cell viability.



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Fig. 1. Effect of ACA on viability of Ehrlich ascites tumor cells. Cells (1 x 106) were incubated with or without ACA at various concentrations in Eagle's minimum essential medium containing 10% FCS. Cell viability was determined after 24 h by the Trypan Blue exclusion method as described in Materials and methods. *P < 0.05, **P < 0.005 compared with control.

 
Effect of ACA on ODC induction
To evaluate the effect of ACA on cell proliferation, we examined the induction of ODC, an enzyme that is rate-limiting enzyme in polyamine biosynthesis and is essential for cell proliferation. ODC activity increased rapidly after changing the medium and peaked at 4 h. This increase was transient, declining to the control level after 8 h. ACA markedly inhibited the increase in ODC activity (Figure 2AGo). The dose–response course showed that ACA is a potent inhibitor of ODC induction in the cells (Figure 2BGo).



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Fig. 2. Time course (A) and dose-dependent (B) effects of ACA on the induction of ODC in Ehrlich ascites tumor cells. Cells (1 x 106) were incubated with ({blacksquare}) 40 µM ACA or without ({blacklozenge}) ACA in Eagle's minimum essential medium containing 10% FCS for the indicated times and (B) at various concentrations and harvested after 4 h. ODC activity was determined by treatments as described in Materials and methods. Results are the means ± SD from three experiments.

 
Effect of ACA on SSAT induction
To examine further the role of ACA in polyamine metabolism, the activity of SSAT, a rate-limiting enzyme in polyamine degradation, was assayed. Addition of ACA significantly enhanced SSAT activity, with a peak at 4 h as compared with control levels. This increase was transient and declined to the control level after 8 h (Figure 3AGo). In a dose–response test, cells were treated for 4 h with ACA. SSAT activity increased in a dose-dependent manner (Figure 3BGo).



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Fig. 3. Time course (A) and dose-dependent (B) effects of ACA on the induction of SSAT in Ehrlich ascites tumor cells. Cells (1 x 106) were incubated with 40 µM ACA ({blacklozenge}) or without ACA ({blacksquare}) in Eagle's minimum essential medium containing 10% FCS at 37°C for the indicated times and (B) at various concentrations and harvested after 4 h. SSAT activity was determined as described in Materials and methods. Results are the means ± SD from three experiments.

 
Effect of ACA on intracellular polyamine concentration
To determine the effect of ACA on intracellular polyamine concentration, their levels were assayed by HPLC. At 4 h the concentrations of putrescine, spermidine and spermine increased in untreated cells. Levels of putrescine in cells incubated with ACA decreased markedly and dose dependently (in the range of 10–40 µM ACA). Compared with that of putrescine, the effect of ACA on spermine and spermidine moved from insignificant at lower concentrations to significant depletion at 40 µM ACA (Figure 4Go).



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Fig. 4. Effect of ACA on intracellular polyamine content. Ehrlich ascites tumor cells (1 x 106) were incubated without ACA (white bars) or with ACA at various concentrations (hatched bars) in Eagle's minimum essential medium containing 10% FCS at 37°C for 4 h. The cells were harvested and then intracellular polyamine content was determined as described in Materials and methods. Results are mean ± SD from three experiments. *P < 0.05, **P < 0.005 compared with control.

 
Effect of exogenous polyamines on cell viability
Compared with control cells, the addition of ACA markedly reduced cell viability at 24 h. However, addition of putrescine, spermidine or spermine to 40 µM ACA-treated cells caused a significant recovery in cell viability compared with that of control cells (Figure 5Go).



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Fig. 5. Effect of exogenous polyamines on viability of ACA-treated cells. Cells (1 x 106) were preincubated with or without ACA for 4 h in Eagle's minimum essential medium containing 10% FCS. Putrescine (PUT), spermidine (SPD) or spermine (SPM) was then added. Cell viability was determined after 24 h by the Trypan Blue exclusion method as described in Materials and methods. *P < 0.05, compared with control.

 
Assessment of apoptosis
Determination of apoptosis in ACA-treated Ehrlich ascites tumor cells was also performed using fluorescence microscopy of DAPI-stained cells. At 24 h the ACA-treated cells showed morphological changes characteristic of apoptosis, such as chromatin condensation, bleb formation around the nucleus and nuclear fragmentation (Figure 6Go). Percentages of apoptotic cells counted are shown in Table IGo. Transmission electron microscopy revealed that ACA-treated cells exhibited a cellular and nuclear morphology that is classically described for apoptotic cells (Figure 7Go).



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Fig. 6. Nuclear morphological changes observed by staining with DAPI. Cells were incubated for 24 h without ACA (A and B) or in the presence of 40 µM ACA (C and D). Harvested cells were treated as described in Materials and methods. A and C, phase contrast microscopy; B and D, fluorescence microscopy (magnification: x600).

 

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Table I. Effect of exogenous polyamines on apoptotic body formation in Ehrlich ascites tumor cellsa
 


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Fig. 7. Transmission electron micrographs of control and ACA-treated cells. Cells were incubated for 24 h in the absence (A) or presence (B) of 40 µM ACA. Transmission electron microscopy was performed as described in Materials and methods. Magnification: (A) x15 700; (B) x11 300.

 
Effect of exogenous polyamines on ACA-induced caspase-3-like activity
Because of the significance of caspases in apoptosis, the role of caspase-3-like protease in the ACA-induced apoptotic cell death observed was evaluated. At 8 h, ACA markedly induced caspase-3-like protease activity in the cells. To clarify further the involvement of polyamines in ACA-induced apoptosis, the degree of caspase activation with added polyamines was assessed. In cells supplemented with polyamines, the degree of caspase activity was suppressed (Table IIGo).


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Table II. Effect of exogenous polyamines on capsase-3-like proteolytic activity in Ehrlich ascites tumor cellsa
 
Effect of ACA on viability of isolated rat hepatocytes
To evaluate the specificity of cytotoxicity of ACA in tumor cells, we studied its effect on normal cells using isolated primary rat hepatocytes. Treatment with increasing concentrations of ACA under similar conditions as the tumor cell did not result in any significant changes in cell viability. As shown in Figure 8Go, cell viability improved slightly as ACA concentrations increased after 24 h.



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Fig. 8. Effect of ACA on viability of hepatocytes. Hepatocytes were incubated with or without ACA for 24 h, then viability was determined by the Neutral Red assay as described in Materials and methods.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of apoptosis by ACA in Ehrlich ascites tumor cells, a significant induction of caspase-3-like protease activity and involvement of polyamine metabolism in the apoptotic process are the notable findings of this study. A number of observations demonstrating anticarcinogenic effects of ACA have been made (110). However, our observation that a tumor cell line responds to ACA by apoptosis is, to our knowledge, novel.

In models of animal studies that examined the chemopreventive effects of ACA on chemically induced carcinogenesis, a reduction in polyamine concentration correlated with reduced incidences of both neoplasms and tumor formation (46). For instance, it has been suggested that the reduction of polyamine pools and ODC activity contributes to the antitumor effect of ACA in azoxymethane-induced colonic aberrant crypt foci (6) and in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats (7). However, resistance to apoptosis has been suggested to be a mechanism by which aberrant crypt foci in preneoplastic lesions could escape death and sustain proliferation in carcinogenesis induced by azoxymethane or by cholic acid deficient diet (33), and apoptosis could mediate the inhibitory role of DFMO on NMBA-induced esophageal carcinogenesis in rats (34). The present in vitro observation, that ACA causes a reduction in ODC activity and increase in SSAT activity, underscores the compound's intrinsic potential to dysregulate the polyamine metabolic pathway and thereby impair the maintenance of adequate polyamine levels that is necessary for both neoplastic development and tumor growth, coupled with its partial ability to induce apoptosis to suppress cell proliferation

The exact mechanism by which ACA modulates the activities of the enzymes is not known. Regarding the suppression of ODC activity, one possible mechanism could be through the influence on protein kinase pathways. In leukemia L1210 cells, a reduction in ODC activity was observed when a p44/42 mitogen-activated protein kinase (MAPK) was inhibited by the putative inhibitor PD98059 (35). We have found in a preliminary study that ACA inhibits the activity of MAPK in Ehrlich ascites tumor cells. It is conceivable, therefore, that in the present study ACA could have reduced ODC activity by inhibiting a MAPK-dependent pathway. A reduction in ROS has also been implicated in the suppression of ODC activity (36). In HL-60 cells, ACA decreases the generation of ROS, resulting in antitumor activity (9). Hence, another possible mechanism whereby ACA suppresses ODC induction in our study may reside in this ability markedly to reduce ROS production on mitogen stimulation. The mechanism by which ACA activated SSAT in our model is not clear. However, activation of SSAT, particularly by polyamine analogs and inhibitors, has been shown to be determinant of cell death (37) and apoptosis (38,39) in tumor cells. In the event of deregulation of a cellular event such as protein synthesis, post-transcriptional mechanisms such as stabilization of SSAT protein bring about a shift in polyamine pools that can simultaneously and differentially regulate polyamine biosynthesis and catabolism (40).

The results also show that ACA treatment caused appreciable changes in cell morphology and viability whilst it had no cytotoxic effect on normal rat hepatocytes, showing the tumor cell target specificity of ACA. Also found in this study was that, when ACA treatment reduced intracellular polyamine levels, particularly that of putrescine, apoptosis was observed subsequently. Pro-apototic signals, including Ca2+ ions, glucocorticoids and polyamine inhibitors, which induce apoptosis whilst reducing polyamine levels (1822,4143), show that imbalances in polyamine content lead to apoptosis in tumor cell lines and are consistent with our results. Polyamines are known to interact with DNA and stabilize it. Perturbation of the polyamine–DNA complex is likely to affect chromatin structure with resultant interference in gene expression and cellular function (23,24). Precise mechanisms by which polyamine levels are reduced is not entirely clear. However, we suggest that the observation could be related to the impairment in ODC induction observed with ACA-treated cells. Alternatively, besides regulating polyamine levels, the induction of SSAT activity that generates acetylated derivatives could elicit apoptotic signals, which might be involved in the observed features (38,44).

Recent studies have demonstrated that interleukin-1ß-converting enzyme (ICE) or ICE-like cysteine proteases (caspases) play crucial roles in apoptosis by cleaving as yet unidentified vital cellular proteins in the induction of apoptotic cell death (26). Caspase-3 is well known to cleave poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair and maintenance of genome integrity (27), and act in association with cytosolic factors from HeLa cells to induce DNA fragmentation (45). It also elicits the degradation of DNA during apoptosis by stimulating a DNase on cleaving an inhibitor bound to the DNase (46). The significance of imbalances in intracellular polyamine content during ACA-induced apoptosis was assessed by examining the effects of polyamine replenishment on ACA-treated cells by counting apoptotic bodies and assaying caspase-3-like protease activity. In the data that indicate that the polyamines prevented the proteolytic activity induced in ACA-treated cells, there was a general correlation with the number of apoptotic cells scored in cultures also treated with exogenous polyamines. Thus, the protective effect of exogenous polyamines on cells undergoing apoptosis is probably, at least in part, due to the ability of polyamines to impair caspase-3-mediated events that lead to apoptosis. The suppressive effects of spermine and spermidine on apoptosis in thymocytes have been attributed to their prevention of endonuclease activation through modification of chromatin structure (41). Though caspase activation presumably precedes endonuclease activation, deregulation of intracellular polyamine levels by a polyamine analogue has been implicated in caspase-3-mediated programmed cell death (47), and polyamines prevent activation of CPP32-like activity in KCl-induced apoptosis (48).

In the present study, appreciable, though low, levels of apoptotic bodies were scored, demonstrated by events including chromatin condensation, cell shrinkage and caspase-3-like protease activation upon ACA treatment. This observation contrasts with previous findings that Ehrlich ascites tumor cells have a rather low susceptibility to apoptosis, a feature apparently due to the propensity of these cells to accumulate heat shock proteins on exposure to apoptosis-inducing agents (49). It is also possible that, as found in tissue culture studies, the majority of apoptotic cells are not phagocytozed, but rather enter a process termed secondary necrosis (50).

Apoptosis as a mechanism of inhibition of carcinogenesis by ACA is to be further considered and whether polyamines directly inhibit caspase activity or act in an upstream pathway of signaling would be an area of interest for further study.


    Notes
 
4 To whom correspondence should be addressed Email: yuasa{at}life.osaka-cu.ac.jp Back


    Acknowledgments
 
This investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 23, 2000; revised July 10, 2000; accepted August 22, 2000.