Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by N{omega}-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines

Rajan Singh1, Shehla Pervin1, Guoyao Wu3 and Gautam Chaudhuri1,2,4

1 Department of Obstetrics and Department of Gynecology and Molecular and Medical Pharmacology,
2 Jonsson Comprehensive Cancer Center, University of California School of Medicine, Los Angeles, CA 90095-1740 USA and
3 Department of Animal Science, Texas A and M University College Street, TX 77843, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown previously that (NOHA) an intermediate in the nitric oxide (NO) synthetic pathway and an inhibitor of arginase significantly reduced intracellular polyamines, activated caspase-3 and induced apoptosis in the human breast cancer cell line MDA-MB-468. These actions of NOHA were abolished in the presence of exogenous L-ornithine suggesting that a reduction in the intracellular polyamine content might be responsible for the activation of caspase-3 and apoptotic actions of NOHA. In order to further explore this possibility, we used SAM-486A and {alpha}-difluoromethylornithine (DFMO), which are inhibitors of S-adenosylmethionine decarboxylase (SAMDC), and ornithine decarboxylase (ODC), respectively, either alone or in combination to reduce the intracellular polyamine levels. We then assessed whether a reduction in polyamine levels by these two compounds to a similar degree to that produced by NOHA activated caspase-3 which occurs prior to the onset of apoptosis. We observed that both SAM-486A and DFMO, either alone or in combination, inhibited cell proliferation, induced p21 and arrested cells in the G0–G1 phase of the cell cycle but failed to activate caspase-3 as assessed by enzymatic assay of caspase-3, western blot analysis of the proteolytic cleavage of caspase-3 protein as well as TUNEL assay. Furthermore, pre-incubation of the cells with SAM-486A and DFMO for 4 days, either alone or in combination significantly inhibited the activation of caspase-3 and apoptosis by NOHA when compared with that observed with cells treated with NOHA alone. Our results, therefore, indicate that the activation of caspase-3 and apoptosis observed with NOHA cannot be solely explained by a reduction in intracellular polyamine levels and that other mechanisms need to be also considered.

Abbreviations: DFMO, {alpha}-difluoromethylornithine; NOHA, N{omega}-hydroxy-L-arginine; NO, nitric oxide; NOS, nitric oxide synthase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The polyamines putrescine, spermidine and spermine are intracellular cationic molecules essential for cell proliferation and regulation of the cell cycle (14). Although the precise mechanisms by which polyamines lead to cell proliferation are not known, it has been suggested that it may be a result of their ability to bind DNA and affect gene expression by bringing about structural changes in chromatin, thereby stimulating cell growth (5). On this basis, numerous inhibitors of polyamine biosynthesis have been developed for treatment of malignant tumors and have been demonstrated to have antiproliferative effects in vitro (6,7) and in vivo (810). However, recent studies indicate that excessive accumulation of polyamines may also favor either malignant transformation or apoptosis, depending on the cell type and the stimulus (11,12). Therefore, either depletion or accumulation of polyamines can lead to apoptosis depending on the cell type, although the precise mechanisms involved in producing these diametrically opposite effects are not known.

We have demonstrated previously that N{omega}-hydroxy-L-arginine (NOHA) an inhibitor of arginase initially inhibited proliferation followed by apoptosis of the human breast cancer cell line MDA-MB-468, which also expresses high arginase activity (13). This NOHA-induced apoptosis was not observed in the presence of L-ornithine. On this basis we speculated that the apoptotic actions of NOHA were most probably a result of a reduction in intracellular polyamine content. The primary objective of the present study was to elucidate whether the NOHA-induced inhibition of cell proliferation, followed by activation of caspase-3 and apoptosis was solely dependent on its ability to reduce intracellular polyamine levels or whether other factors independent of this effect were involved.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture and proliferation
MDA-MB-468 cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM (Gibco BRL, Rockville, MD) containing 20 mM D-glucose, 2 mM L-glutamine, 10 mg/ml insulin and 10% fetal bovine serum (FBS). For the experiments, the cells were maintained in 5% FBS. Cultures were treated with an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), SAM-486A (3 mM) which was obtained from Barbara Willi (Novartis Pharma AG, Basel, Switzerland), {alpha}-difluoromethylornithine (DFMO) (5 mM), an inhibitor of ornithine decarboxylase (ODC) from Calbiochem (San Diego, CA) and NOHA (1 mM) (Cayman Chemicals, Ann Arbor, MI), an inhibitor of arginase, either alone or in combination for different time periods and viable cells were counted using trypan blue exclusion method using hemocytometer.

Cell-cycle analysis by flow cytometry
Cells (1x106) were plated in 60-mm plates with or without drugs for various time periods. After cell trypsinization, the cells were resuspended in PBS containing 1% Triton X-100, 0.1 mg/ml RNaseA and propidium iodide (Sigma Chemicals, St Louis, MI) to a final concentration of 0.05 mg/ml. The cells were subjected to FACS caliber flow cytometry and the percentage of cells in each phase of cell cycle was obtained using Modfit Software (Varity Software House, Topsham, ME).

Polyamine and ornithine analysis
Polyamine and ornithine concentrations were determined from the cells treated with or without various drugs as described previously by Wu et al. (14). Briefly, 5x106 cells were acidified with 1 ml of 1.5 M HClO4 and were neutralized with 0.5 ml of 2 M K2CO3. The neutralized extracts were used for polyamine analysis by an ion-pairing HPLC method involving precolumn derivitization with o-phthaldialdehyde. The assay mixture contained 100 µl sample and 100 µl of 1–2% benzoic acid (in 40 mM sodium borate, pH 9.5) and 1 ml HPLC H2O. An aliquot (50 µl) of the assay mixture was derivatized in an autosampler (model 712 WISP, Waters, Milford, MA) with 50 µl of 30 mM o-phthaldialdehyde (in 3.1% Brij-35, 50 mM 2-mercaptoethanol, and 40 mM sodium borate, pH 9.5), and 50 µl of the derivatized mixture was injected in a Supelco 3-mm reversed phase C18 column (150x4.6 mm 1D). Polyamines were separated using a solvent gradient consisting of solution A (0.1 M sodium acetate, 2 mM SDS, 0.5% tetrahydrofuran, 9% methanol, pH 7.2) and solution B (methanol and 2 mM SDS). For ornithine analysis, 50 µl of neutralized cell extracts were mixed with 10 µl of 1.2% benzoic acid and 100 µl HPLC H2O. An aliquot (50 µl) of the assay mixture was derivatized with 50 ml of 30 µM o-phthalaldialdehyde. Fifty microliters of the derivatized mixture was used for HPLC separation of ornithine as described for polyamine separation, except that 2 mM SDS was omitted form solvents A and B. Putrescine, spermidine, spermine and ornithine in samples were quantified on the basis of authentic standards, using Millenium-32 Software (Waters)

Caspase activity assay
Cells were lysed in caspase assay buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.1% CHAPS, 10% sucrose and 5 mM DTT. Aliquots of 6 mg of crude cell lysate were incubated with caspase-3 substrate Ac-DEVD-AMC (Pharmingen, San Diego, CA) at 37°C for 30 min. The caspase-3 activity was quantified in a versofluofluorometer with excitation at 380 nm and emission at 440 nm.

Western blot analysis
Cells were lysed in lysis buffer containing 50 mM HEPES (pH 7.5), 1 mM DTT, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM orthovanadate, 10 mg/ml leupeptin, 10 mg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride and kept at 4°C for 30 min. Lysates (20 mg) were resolved electrophoretically on 10% SDS–PAGE and transferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) using a tank blot procedure (Bio-Rad Mini Protean 11). The filters were incubated with 1:1000 dilution of anti p21 (Santa Cruz Biotechnology, SC) or 1:3000 dilution of anticaspase-3 (65906E) for 2 h. The washed filters were incubated with 1:1000 dilution of respective horseradish peroxidase linked secondary antibodies for 1 h. Immunoreactive bands were visualized with the help of ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). The relative intensities of the bands were quantified by densitometric analysis (Personal Densitometer SI, Molecular Dynamics, Sunnyvale, CA).

TUNEL assay
The TUNEL assay was performed using ApoAlert DNA Fragmentation Assay Kit from Clontech (Palo Alto, CA). Cells (3x106) were collected by centrifugation, washed twice with PBS, and fixed in 1% formaldehyde–PBS at 4°C for 20 min. The cells were collected, washed with PBS, resuspended in 500 ml of PBS, and stored overnight in 70% ethanol. The cells were then collected, washed, and gently resuspended in equilibration buffer. Nucleotide mixture and terminal deoxynucleotidyl (TdT) transferase enzyme were added, and cells were incubated at 37°C for 1 h. Cells were washed and analyzed in a Becton Dickinson Flow Cytometer.

Statistical analysis
All data are presented as means ± SEM. The differences between means were analyzed by two-way ANOVA. The mean values of ornithine and polyamine levels were obtained from three separate experiments in each group and P values <=0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
SAM-486A and DFMO inhibited cell proliferation of MDA-MB-468 cells
Figure 1Go shows the results of SAM-486A (3 mM) and DFMO (5 mM), either alone or in combination, on the cell proliferation of human breast cancer cell line MDA-MB-468. The concentrations of these drugs were chosen based on our initial dose–response studies (data not shown). We observed that both the drugs had antiproliferative effects, either alone or in combination. SAM-486A and DFMO led to 85 and 63% growth inhibition, respectively, compared with control cells after day 6. However, when used in combination, these drugs induced cytostasis. We did not observe any significant cell death (<5%) even after day 6 of treatment with either drug when used alone or in combination.



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Fig. 1. Effect of SAM-486A and DFMO on the proliferation of MDA-MB-468 cells. Cells (1x106) were plated in 100 mm plates and allowed to grow. SAM-486A (3 mM) and DFMO (5 mM) were added to the cells. The medium was changed and fresh drugs were added after every 48 h. The cells were harvested after different days and the number of viable cells was counted. Results are expressed as mean cell numbers from three independent experiments; ± SEM.

 
SAM-486A and DFMO arrested MDA-MB-468 cells in the G0–G1 phase of the cell cycle
Figure 2Go shows the percentage of cells in different phases of the cell cycle after days 3 and 6 following these drug treatments, either alone or in combination. The distribution of cells in the different phases did not change significantly at day 3 but at day 6, a significant increase was observed in the G0–G1 phase with SAM-486A or DFMO treatment, either alone or in combination, when compared with untreated control cells. DFMO was more potent than SAM-486A in arresting the cells at the G0–G1 phase. Again, we observed that there was a significant decrease of cells in the S-phase following these drug treatments, either alone or in combination, and only a marginal increase in the G2–M phase after day 6.



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Fig. 2. Effect of SAM-486A and DFMO on the cell-cycle in MDA-MB-468 cells. Cells (1x106) were plated in 100 mm plates and allowed to grow. SAM-486A (3 mM) and DFMO (5 mM) were added to the cells. The medium was changed and fresh drugs were added after every 48 h. The cells were harvested after days 3 and 6 and 1x106 cells were stained with propidium iodide, and fluorescence activated cell-sorting analysis was done for each treatment group. Results are expressed as mean of three different experiments; ± SEM (*indicates that values are significantly different than the control, P < 0.05).

 
SAM-486A and DFMO-induced p21 expression in MDA-MB-468 cells
The effect of these inhibitors, SAMDC and ODC on the expression of p21 was assessed by western blot analysis of cells treated with these drugs for up to day 6. Figure 3Go shows that the levels of p21 were significantly induced following the treatment with SAM-486A and DFMO, either alone or in combination, as early as day 2 of treatment. DFMO appeared to be a more potent inducer of p21 than SAM-486A. This early induction of p21 remained elevated during later time points.



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Fig. 3. Western blot analysis of the effect of SAM-486A and DFMO on the expression of p21 in MDA-MB-468 cells from days 1–6. Cells (2x106) were treated with SAM-486A and DFMO as described in Figure 1Go, lysed in lysis buffer and 20 mg of cell lysate were run on 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes. The membranes were immunoblotted using anti-p21 antibody. Results are from a single experiment that is representative of three separate experiments.

 
Effect of SAM-486A and DFMO treatment on the intracellular ornithine and polyamine content of MDA-MB-486 cells
Table IGo shows the concentrations of ornithine and different polyamines after SAM-486A and DFMO treatment, either alone or in combination, after days 3 and 6. SAM-486A treatment increased the putrescine concentration by 81% (day 3) and 137% (day 6) as compared with the control group. However, treatment with SAM-486A decreased the spermidine concentration by 54% (day 3) and 73% (day 6). On the other hand, there was only a minor change in the intracellular L-ornithine values after days 3 and 6.


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Table I. Effect of SAM-486A and DFMO on the intracellular polyamine levels
 
We observed a decrease in the intracellular concentrations of putrescine and spermidine levels by 96% (day 3) and 98% (day 6) with DFMO treatment. However, the levels of intracellular spermine concentration were decreased only by 34% (day 3) and 49% (day 6) with DFMO treatment. The intracellular levels of L-ornithine were increased by 92% (day 3) and 112% (day 6) compared with control values.

We also studied the effects of a combination of SAM-486A and DFMO on the intracellular levels of ornithine and the different polyamines. Putrescine levels were found to be decreased by 95% (day 3) and 97% (day 6). The levels of spermidine were found to be decreased by 93% (day 3) and 98% (day 6). The spermine levels decreased by 74% (day 3) and 79% (day 6). However the levels of L-ornithine were found to be increased by 76% (day 3) and 96% (day 6).

Effect of SAM-486A and DFMO pre-treatment on NOHA-induced changes in intracellular L-ornithine and polyamines
Table IIGo shows the intracellular concentrations of different polyamines in the various groups. The cells were pre-treated with SAM-486A (3 mM) and DFMO (5 mM), either alone or in combination, for 4 days followed by NOHA (1 mM) treatment for another 48 h. NOHA, on its own, decreased the intracellular concentration of L-ornithine by 67% after 48 h of treatment and the levels of putrescine, spermidine and spermine were decreased by 80%, 54% and 64%, respectively.


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Table II. Effect of SAM-486A and DFMO pre-incubation for 4 days, either alone or in combination, of the NOHA (1 mM)-induced changes in polyamines
 
In cells pre-treated with SAM-486A for 4 days followed by NOHA treatment for another 48 h, the putrescine levels were found to be 230% of the control group whereas the levels of spermidine and spermine were found to be 36% and 22%, respectively, compared with control group. The intracellular L-ornithine levels were found to be 48% of the control group.

DFMO pre-treatment followed by NOHA treatment changed the putrescine, spermidine, and spermine levels to 6%, 5% and 69% compared with the control group. However the intracellular L-ornithine levels were only found to be 113% compared with the control group. Pre-treatment with the combination of SAM-486A and DFMO for 4 days followed by NOHA treatment changed the putrescine, spermidine and spermine levels to 6%, 12% and 30%, respectively. The intracellular L-ornithine levels were found to be 67% of the control group.

SAM-486A and DFMO treatment did not induce caspase-3 activation and apoptosis
We measured caspase-3 activation as an indicator of apoptosis induction since different upstream pathways leading to apoptosis depend on caspase-3 induction for final apoptotic execution. Figure 4Go shows the effect of SAM-486A and DFMO, at the level of caspase-3 induction. There was no significant increase in caspase-3 activity until day 6 with these drugs, either alone or in combination, as compared with the NOHA-induced caspase-3 activity after 48 h. We further performed western blot analysis with caspase-3 antibody using 20 mg of cell lysates obtained after similar drug treatment to analyze the proteolytic cleavage of the caspase-3 protein (Figure 5Go). No caspase-3 cleavage was observed under these conditions confirming that these drugs when used alone or in combination were unable to activate caspase-3. Also, these drugs did not induce apoptosis as measured by TUNEL assay either alone or in combination until day 6 (data not shown).



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Fig. 4. Effect of SAM-486A and DFMO on the caspase-3 activity in MDA-MB-468 cells. Cells (1x106) were treated with SAM-486A and DFMO as described in Figure 1Go, lysed in insect lysis buffer, and 6 mg of cell lysates from each treatment group were used for caspase-3 assay using Ac-DEVD-AMC substrate. In a parallel experiment, cells were treated with 1 mM NOHA for 48 h as a positive control for caspase-3 activity. Results are expressed as mean of three different experiments; ± SEM (*indicates that values are significantly different than the control, P < 0.05).

 


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Fig. 5. Western blot analysis of the effect of SAM-486A, DFMO and NOHA on the activation and proteolytic cleavage of caspase-3 in MDA-MB-468 cells. Cells (2x106) were treated with SAM-486A and DFMO, either alone or in combination, for days 3 and 6 and with NOHA (1 mM) until day 2 (48 h), lysed in lysis buffer, and 20 mg of cell lysate were run on 10% SDS–PAGE. The proteins were transferred to a polyvinylidene difluoride membrane and immunoblotted using anti-caspase-3 antibody. Results are from a single experiment that is representative of three separate experiments.

 
SAM-486A and DFMO pre-treatment inhibited NOHA-induced caspase-3 induction and apoptosis
SAM-486A and DFMO pre-treatment altered the polyamine profile of NOHA-treated MDA-MB-486 cells as shown in Table IIGo. In order to assess whether these altered polyamine levels could modify the caspase-3 induction by NOHA under similar conditions, we performed enzymatic assay for caspase-3 with the cell lysates obtained after various treatment regimens. Pre-treatment of the cells for 4 days with just the vehicle without any drug followed by NOHA treatment for 48 h led to an increase of caspase-3 activity by 10-fold compared with the control group. When the cells were pre-incubated with SAM-486A, DFMO, or a combination of both drugs or vehicle, alone for 4 days followed by NOHA treatment for another 48 h, we observed a 55%, 81% and 69% inhibition of the caspase-3 activity in the drug treated groups, respectively, when compared to the vehicle group treated with NOHA alone (Figure 6Go). We performed TUNEL assay to test whether similar pre-treatment with these drugs either alone or in combination were able to inhibit the apoptosis induction by NOHA treatment. We observed a significant inhibition in DNA fragmentation after pre-incubation with these drugs compared with NOHA treatment alone (data not shown). In addition, we also performed western blot analysis with caspase-3 antibody using 20 mg of the same cell lysates obtained after various drug treatments as enumerated above to assess the effects of NOHA-induced proteolytic cleavage of caspase-3. Figure 7Go show that SAM-468A, DFMO or combined pre-incubation inhibited the caspase-3 proteolytic cleavage induced by NOHA to a comparable degree as observed with enzymatic assay shown in Figure 6Go. However, when SAM-486A and DFMO either alone or in combination were added simultaneously with NOHA, they were unable to inhibit NOHA-induced apoptosis or caspase-3 induction (data not shown) indicating that these drugs did not have any intrinsic antiapoptotic actions of their own.



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Fig. 6. Effect of SAM-486A and DFMO pre-treatment for 4 days, either alone or in combination, on the NOHA induce caspase-3 activity. Cells (1x106) were treated with SAM-486A and DFMO, either alone or in combination, for 4 days. Media were changed and fresh drugs were added along with 1 mM NOHA for another 48 h. In a parallel experiment NOHA was added to a control plate pre-incubated without any treatment for 4 days and kept for another 48 h. Cells were lysed in insect lysis buffer and 6 mg of cell lysates from each treatment group were used for caspase-3 assay using Ac-DEVD-AMC as substrate. Results are expressed as mean of four different experiments; ± SEM (*indicates that values are significantly different than the control, P < 0.05;**indicates that values are significantly different than *value, P < 0.05).

 


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Fig. 7. Western blot analysis of the effect of SAM-486A and DFMO pre-treatment for 4 days, either alone or in combination, on the NOHA-induced proteolytic cleavage of caspase-3. Cells (1x106) were treated with SAM-486A, DFMO, and NOHA exactly as described in Figure 6Go, lysed in lysis buffer and 20 mg of cell lysate were run on 10% SDS–PAGE. The proteins were transferred to a PVDF membrane and immunoblotted using anti-caspase-3 antibody. Results are from a single experiment that is representative of three separate experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We (13), and others (2), have demonstrated previously that polyamines are essential for proliferation of some breast tumor cells and that inhibition of polyamine biosynthesis leads to inhibition of cell proliferation followed by apoptosis (13,1517). The polyamine biosynthesis occurs from arginine, which is converted by arginase to ornithine (18). Ornithine is converted to putrescine by ODC, which catalyzes the first and rate-limiting step in polyamine biosynthesis (18). An increase in ODC activity is one of the early biochemical events associated with the induction of cellular proliferation and numerous investigators have utilized DFMO, which is a specific and irreversible inhibitor of ODC to assess the role of polyamines in cell proliferation. SAMDC which converts SAM to decarboxylated SAM is required for the conversion of putrescine to spermidine. SAM-486A, is a highly specific inhibitor of SAMDC and both DMFO or SAM-486A used, either alone or in combination, have been shown to reduce proliferation of various types of malignant cells (6,10) accompanied by reduction in intracellular polyamine levels.

The primary objective of the present study was to assess whether the inhibition of cell proliferation and apoptotic actions of NOHA on MDA-MB-468 cells which we had reported previously (13), were solely a result of a reduction of intracellular polyamine content or whether other actions of NOHA were involved in this process. In this study we, therefore, used two other key inhibitors of the polyamine biosynthetic pathways, i.e. SAM-486A and DFMO to decrease intracellular polyamine content of MDA-MB-468 cells and assess whether the effects of these two compounds were similar or were different from the effects brought about by NOHA.

In this study some similarities as well as significant differences in the effects of DFMO and SAM-486A when compared with NOHA were observed. DFMO and SAM-486A, when used alone, inhibited cell proliferation, whereas, when used in combination led to cytostasis and no significant activation of caspase-3 was observed. On the other hand, NOHA led to activation of caspase-3 and induction of apoptosis (13). Similarly, cells treated with either DFMO or SAM-486A were arrested in the G0–G1 phase accompanied by a decrease in the S-phase of the cell cycle whereas the cells treated with NOHA were arrested in the S-phase with concomitant decrease of cells in the G0–G1 and G2–M phases. However, there were some similarities as well. The inhibition of cell proliferation induced by SAM-486A, DFMO and NOHA were in all cases accompanied by a significant increase in the expression of p21. This increase in p21 occurred by a p53-independent mechanism as p53 is mutant in this cell line (19). A significant decrease in the intracellular levels of both spermidine and spermine also occurred with the use of all of the three inhibitors. It therefore appears that the effects of NOHA in inhibiting cell proliferation, immediately followed by activation of caspase-3 and apoptosis cannot be explained just on the basis of a reduction in the intracellular levels of spermidine and spermine alone and that other actions of NOHA need to be considered.

Also, MDA-MB-468 cells have very little nitric oxide synthase (NOS) activity and, therefore, the apoptotic action of NOHA, an intermediate formed during the synthesis of NO by NOS and which is also a substrate of NOS, cannot be explained on the basis of NO formation from NOHA by these cells. Moreover, the apoptotic actions of NOHA were abolished in the presence of L-ornithine (13), further confirming that the apoptotic actions of NOHA cannot be ascribed to NO formation and, therefore, were most probably because of a decrease in the synthesis of ornithine.

Other investigators (20) have utilized epidermal growth factor (EGF) to reduce intracellular polyamine levels of MDA-MB-468 cells and also observed apoptosis, which could be reversed by the addition of putrescine or spermidine but not by spermine. However, the inability of DFMO and SAM-486A to induce caspase-3 activation in this study, in spite of it being able to significantly reduce intracellular concentrations of putrescine, spermidine and spermine, indicates that other potential mechanisms need to be considered in order to explain as to why all compounds that are able to reduce the intracellular concentrations of some polyamines inhibit proliferation of cells, whereas only one of these compounds, like NOHA, activated caspase-3 and induced apoptosis (13), even when the same cell line was utilized.

Arginase II (a mitochondrial enzyme) is the major isoform of arginase in MDA-MB-468 cells, but these cells also express arginase I (a cytosolic enzyme) (1). Thus, ornithine is produced from arginine primarily in the mitochondria of MDA-MB-468 cells. In mammalian cells, ornithine catabolism is initiated by mitochondrial ornithine aminotransferase to form pyrroline-5-carboxylate (P5C) (18). The latter enters the cytosol for conversion into proline by NADPH-dependent P5C reductase, thereby regulating the cellular NADPH/NADP+ ratio or redox state. Interestingly, previous studies have demonstrated that an increase in the conversion of P5C into proline is coupled with increase in pentose cycle activity for provision of both ribose-5-phosphate and NADPH in tumor cells (23). The enhanced generation of ribose-5-phosphate results in increased purine synthesis and cell proliferation (23). Further, as NADPH is required for synthesis of many important molecules, including fatty acids, superoxide, and nitric oxide, and for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH), changes in ornithine metabolism may play an important role in the cellular redox state and function.

We initially ruled out the possibility that SAM-486A and DFMO had some intrinsic antiapoptotic action of its own as when we added NOHA simultaneously with SAM-486A or DFMO, NOHA was still able to induce apoptosis and caspase-3 activation. We, therefore, considered the possibility that the intracellular levels of ornithine may be critical in determining whether a cell undergoes apoptosis. This is because NOHA, by inhibiting arginase reduced intracellular ornithine levels and activated caspase-3 and the cells underwent apoptosis, whereas, following the use of the other two inhibitors, either an increase or no change in ornithine levels was observed and caspase-3 was not activated. The mitochondria is now known to play an important role in cell respiration and generation of ATP and factors that adversely affect mitochondrial function can initiate activation of downstream caspases and apoptosis (21). Ornithine transport across the mitochondria involves the exchange of ornithine+ for citrulline and one proton (22). Although not known, it is possible that in situations of low intracellular ornithine levels, this exchange is impaired leading to mitochondrial dysfunction followed by activation of caspases and apoptosis.

This could also be a potential explanation as to why once cytostasis was induced by DFMO, which actually increased intracellular ornithine levels, NOHA, which was only able to lower ornithine levels to that observed in control cells, failed to induce apoptosis. DFMO inhibits ornithine decarboxylase, the first and rate-limiting step in polyamine biosynthesis and thus there was almost doubling of the intracellular ornithine level. This increase in intracellular ornithine level may, therefore, have been able to significantly attenuate the apoptotic action of NOHA. However, this explanation would not be valid for cells initially treated with SAM-486A as NOHA was able to significantly lower ornithine levels and yet in these cells, NOHA was not able to activate caspase-3 to the same extent when compared with that observed when the cells were not pre-treated with either SAM-486A or DFMO.

It is also possible that for NOHA to significantly activate caspase and induce apoptosis, the cells need to be in the S-phase of the cell cycle as observed during NOHA-induced apoptosis. Following treatment of cells with either DFMO or SAM-486A, the cells were arrested in the G0–G1 phase of the cell cycle which may have rendered ineffective the apoptotic action of NOHA. It is also possible that the apoptotic action of NOHA is independent of its action in inhibiting arginase. Further studies are in progress to elucidate this aspect by utilizing other specific inhibitors of arginase (24) and comparing their actions with that of NOHA in inducing apoptosis and the mechanisms involved.


    Notes
 
4 To whom correspondence should be addressed Email: gchaudhuri{at}mednet.ucla.edu Back


    Acknowledgments
 
We thank Janis Cuevas and Svetlana Arutyunova for their technical assistance. This work was supported in part by Palomba Weingarten, the Allegra Charach Cancer Research Fund, and US Public Health Service Grant CA-78357 (to G.C.). Dr G. Wu is an established investigator of the American Heart Association.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 30, 2001; revised June 27, 2001; accepted July 3, 2001.