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Carcinogenesis Advance Access originally published online on September 16, 2005
Carcinogenesis 2006 27(3):533-540; doi:10.1093/carcin/bgi228
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

Diallyl trisulfide, a constituent of processed garlic, inactivates Akt to trigger mitochondrial translocation of BAD and caspase-mediated apoptosis in human prostate cancer cells

Dong Xiao and Shivendra V. Singh *

Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

* To whom correspondence should be addressed. Tel: +1 412 623 3263; Fax: +1 412 623 7828; Email: singhs{at}upmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown previously that apoptosis induction by diallyl trisulfide (DATS), a constituent of processed garlic, in PC-3 and DU145 human prostate cancer cells is associated with c-Jun N-terminal kinase and extracellular signal-regulated kinase-mediated phosphorylation of Bcl-2. However, pharmacological inhibition of these kinases offers only partial protection against the cell death caused by DATS. Here, we demonstrate that DATS inactivates Akt to trigger apoptosis in prostate cancer cells. Treatment of PC-3/DU145 cells with apoptosis inducing concentration of DATS (40 µM) resulted in a rapid decrease in Ser473 and Thr308 phosphorylation of Akt leading to inhibition of its kinase activity. The DATS-mediated inactivation of Akt was associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation. DATS treatment (40 µM) also caused a decrease in Ser155 and Ser136 phosphorylation of BAD (a proapoptotic protein), which is a downstream target of Akt. Phosphorylation sequesters BAD in the cytoplasm owing to increased binding with 14-3-3 proteins. The interaction between BAD and 14-3-3ß was reduced markedly upon a 4 h treatment with 40 µM DATS in both cell lines. Consistent with these results, DATS treatment (40 µM, 4 h) promoted mitochondrial translocation of BAD as revealed by immunocytochemistry. Ectopic expression of constitutively active Akt conferred statistically significant protection against DATS-induced apoptosis. The DATS-induced apoptosis in both cell lines was significantly attenuated in the presence of pan caspase inhibitor zVAD-fmk and caspase 9 specific inhibitor zLEHD-fmk. In conclusion, the present study demonstrates that DATS-induced apoptosis in human prostate cancer cells is mediated, at least in part, by inactivation of Akt signaling axis.

Abbreviations: BSA, bovine serum albumin; Cdk1, cyclin-dependent kinase 1; JNK, c-Jun N-terminal kinase; CA-Akt, constitutively active Akt; DAPI, 4',6-diamidino-2-phenylindole; DADS, diallyl disulfide; DATS, diallyl trisulfide; ERK, extracellular signal-regulated kinase; IGF-1R, insulin-like growth factor receptor 1; MAPK, mitogen-activated protein kinase; OSCs, organosulfur compounds; PARP, poly(ADP-ribose)polymerase; PBS, phosphate buffered saline; PrEC, prostate epithelial cell line; ROS, reactive oxygen species; SDS–PAGE, sodium-dodecyl sulfate–polyacrylamide gel electrophoresis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies continue to support the premise that dietary intake of Allium vegetables, especially garlic, may offer protection against the risk of different types of malignancies including cancer of the prostate (1–4). For example, Hsing et al. (4) examined the association between Allium vegetable intake and prostate cancer risk in a population-based study involving 238 histologically confirmed cases and 471 controls. The risk of prostate cancer was found to be significantly lower in men consuming >10 g/day of total Allium vegetables than in men with total Allium vegetable intake of <2.2 g/day (4). Laboratory studies indicate that anticarcinogenic effect of Allium vegetables is because of organosulfur compounds (OSCs) that are generated upon processing (cutting or chewing) of these vegetables (5,6).

Garlic-derived OSCs including diallyl sulfide, diallyl disulfide (DADS) and diallyl trisulfide (DATS) have been shown to offer significant protection against cancer in animal models induced by a variety of chemical carcinogens (7–13). For example, cancer prevention by naturally occurring OSC analogs has been observed against dimethylhydrazine-induced colon cancer in rats, benzo[a]pyrene-induced forestomach and pulmonary carcinogenesis in mice, azoxymethane-induced colon cancer in rats, and N-methyl-N-nitrosourea and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary cancer in rats (7–13). Prevention of chemically induced cancers in animal models by OSCs is believed to be owing to their ability to increase detoxification of activated carcinogenic metabolites through induction of Phase II enzymes such as glutathione transferases and/or inhibit carcinogen activation through inhibition of Phase I enzymes (14–17). In addition, oral administration of DADS significantly suppresses growth of H-ras oncogene transformed NIH 3T3 tumor xenografts in athymic mice by inhibiting membrane association of p21H-ras (18,19).

Evidence is accumulating to indicate that certain naturally occurring OSC analogs can inhibit proliferation of cultured cancer cells by causing cell-cycle arrest and apoptosis induction (20–32). For example, DADS inhibited proliferation of HCT-15 human colon cancer cells by causing G2–M phase cell-cycle arrest in association with accumulation of cyclinB1, reduced complex formation between cyclin-dependent kinase 1 (Cdk1) and cyclinB1, and hyperphosphorylation (inactivation) of Cdk1 (21,22). We have shown recently that the DATS-induced cell-cycle arrest in human prostate cancer cells is mediated by reactive oxygen species (ROS) and is associated with activation of checkpoint kinase 1 (30–32). The DADS-induced apoptosis in HCT-15 cells was shown to correlate positively with an increase in the level of intracellular free calcium (20). The DADS-induced cell death in MDA-MB-231 human breast cancer cell line was attributed to induction of Bax, downregulation of Bcl-xL and activation of caspase 3 (23). In spite of these advances, the mechanism(s) by which garlic-derived OSCs cause cell death is not fully defined. This knowledge is essential for further development of OSCs as clinically useful anticancer agents.

We have shown recently that DATS-induced cell death in PC-3 and DU145 cells is associated with c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Bcl-2 (29). However, pharmacological inhibition of these kinases offers only partial protection against the cell death caused by DATS (29). Moreover, ectopic expression of Bcl-2, through stable transfection in PC-3 cells, confers partial resistance to DATS-induced cell death (29). These results suggested involvement of additional mechanism(s) in DATS-induced apoptosis. Here, we provide experimental evidence to indicate that DATS-induced apoptosis in PC-3 and DU145 cells is mediated by inactivation of Akt leading to mitochondrial translocation of BAD (a proapoptotic protein) and activation of caspases 3 and 9.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
DATS was purchased from LKT Laboratories (St Paul, MN). Tissue culture media and fetal bovine serum were from Gibco (Grand Island, NY). The antibodies against Akt (cat. no. sc-8312), BAD (cat. no. sc-8044), phospho-(Ser155)-BAD (cat. no. sc-12970-R), phospho-(Ser136)-BAD (cat. no. sc-12969-R) and 14-3-3ß (cat. no. sc-629) were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against insulin-like growth factor receptor 1 (IGF-1R; cat. no. GR31L), phospho-tyrosine (cat. no. PT04B) and actin (cat. no. CP01) were from Oncogene Research Products (Boston, MA) and the antibodies specific for cleaved caspase 3 (cat. no. 9661), cleaved poly(ADP-ribose)polymerase (PARP; cat. no. 9546), phospho-(Ser473)-Akt (cat. no. 9271) and phospho-(Thr308)-Akt (cat. no. 9275) were from Cell Signaling Technology (Beverly, MA). The caspase inhibitors were purchased from Enzyme Systems (Dublin, CA).

Cell culture and cell survival assay
Monolayer cultures of PC-3 and DU145 cell lines were maintained as described by us previously (29,30). Effect of DATS on cell survival was determined by sulforhodamine B assay as described previously (29). Stock solution of DATS was prepared in dimethyl sulfoxide (DMSO) and an equal volume of DMSO (final concentration 0.2%) was added to the controls.

Immunoblotting
Desired cell line was treated with DATS for specified time period and lysed as described previously (29,30). Cell lysate was cleared by centrifugation at 21 000 g for 15 min. Supernatant proteins were resolved by sodium-dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto PVDF membrane. After blocking with 5% (w/v) non-fat dry milk solution in Tris buffered saline containing 0.05% Tween-20, the membrane was incubated with the desired primary antibody for 1 h at room temperature. The membrane was then treated with appropriate secondary antibody, and the immunoreactive bands were visualized using enhanced chemiluminescence method. Membrane was stripped and reprobed with anti-actin antibody to ensure equal protein loading. Change in protein level was determined by densitometric scanning of the immunoreactive bands followed by correction for actin loading control.

Akt kinase assay
Akt kinase assay was performed using reagents supplied by the manufacturer (Cell Signaling Technology). Briefly, 2 x 106 cells were plated and allowed to attach by overnight incubation. The cells were then treated with 40 µM DATS for specified time period and lysed with a solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of lysate proteins (200 µg) from control and DATS-treated PC-3 or DU145 cells were incubated overnight at 4°C with agarose-coupled anti-Akt antibody. Immunoprecipitated complexes were washed four times with the lysis buffer and twice with kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4 and 10 mM MgCl2]. The immunoprecipitates were resuspended in 40 µl of kinase buffer supplemented with 200 µM of ATP and 1 µg of GSK-3{alpha}/ß fusion protein. After incubation at 30°C for 30 min, the kinase reaction was terminated with the addition of 40 µl of 2x SDS sample buffer. An aliquot (30 µl) was subjected to immunoblotting and probed with anti-phospho-(Ser21/9)-GSK-3{alpha}/ß antibody.

Immunoprecipitation
PC-3 or DU145 cells were treated with DATS and lysed as described above. Equal amounts of lysate proteins (200 µg) from control and DATS treated PC-3 or DU145 cells were incubated with 4 µg anti-14-3-3ß antibody overnight at 4°C with gentle shaking. Protein A–agarose (50 µl, Santa Cruz Biotechnology) was then added to each sample, and the incubation was continued for an additional 3 h at 4°C. The immunoprecipitates were washed five times with lysis buffer and subjected to SDS–PAGE followed by immunoblotting using anti-BAD or anti-14-3-3ß antibody. In another experiment, immunoprecipitation was performed using anti-IGF-1R antibody followed by immunoblotting using anti-phospho-tyrosine antibody.

Immunofluorescence analysis for mitochondrial localization of BAD
PC-3 cells (2 x 105) were plated on coverslips, and treated with 40 µM DATS or DMSO (control) for 4 h. The cells were then treated for 1 h with 100 nM mitochondria-specific dye MitoTracker Red (Molecular Probes, Eugene, OR) at 37°C. After washing with phosphate buffered saline (PBS), the cells were fixed with 3% paraformaldehyde and permeabilized using 0.1% Triton X-100. After blocking with normal goat serum (1:20 dilution) diluted with 0.5% bovine serum albumin (BSA) and 0.15% glycine in PBS (BSA buffer) for 45 min, the cells were treated with anti-BAD antibody (1:400 dilution) for 2 h. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:1000 dilution) for 1 h. After washing with BSA buffer, the cells were treated with 10 ng/ml 4',6-diamidino-2-phenylindole (DAPI) for 1 min to stain DNA. Cells were washed with PBS, mounted and photomicrographs were obtained using a Leica DC300F fluorescence microscope.

Transient transfection
DU145 cells were transiently transfected with pCMV6 vector containing constitutively active Akt1 (Myr-Akt1-HA; kindly provided by Dr Daniel Altschuler, University of Pittsburgh, PA) or empty vector using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA). Briefly, DU145 cells were plated at a density of 2 x 105 cells/ml and allowed to attach overnight. Cells were transfected with expression constructs encoding the constitutively active Akt or empty vector. After 6 h, the medium was replaced with fresh complete medium, and the cells were treated with 40 or 80 µM DATS or DMSO (control) for 8 h. The cells were collected and processed for cell survival assay or apoptosis assay.

Apoptosis assay
Apoptosis induction in DATS treated PC-3 or DU145 cell line was assessed by analysis of cytoplasmic histone associated DNA fragmentation. Briefly, 104 cells were plated in 96-well plates and allowed to attach by overnight incubation. The cells were then exposed to desired concentrations of DATS for specified time period. Cytoplasmic histone-associated DNA fragmentation was determined using a kit from Roche Diagnostics GmbH (Mannheim, Germany) according to the manufacturer's instructions.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
DATS inhibited Akt kinase activity
Our previous study indicated that JNK and ERKmediated phosphorylation of Bcl-2 alone cannot fully account for DATS-induced apoptosis in PC-3 or DU145 human prostate cancer cells (29). In the present study, we used the same cell lines to test whether DATS-induced apoptosis was mediated by inactivation of Akt, a kinase known to promote cell survival and block apoptosis (33,34). Initially, we determined the effect of DATS treatment on activating phosphorylations of Akt (Ser473 and Thr308) by immunoblotting, and the results are shown in Figure 1A. The levels of Ser473 and Thr308 phosphorylated Akt were reduced rapidly on treatment of PC-3 cells with 40 µM DATS, a concentration that reduces PC-3 cell viability by >80% (29) but minimally affects proliferation of a normal prostate epithelial cell line (PrEC) (30). However, DATS treatment did not alter the level of total Akt protein (data not shown). Treatment of PC-3 cells with 40 µM DATS for 1 and 4 h resulted in an ~39 and 68% reduction in Akt kinase activity, respectively, compared with control (Figure 1B). One mechanism by which activated Akt promotes cell survival involves phosphorylation of pro-apoptotic Bcl-2 family member BAD (35). The phosphorylation of BAD at Ser155 (Figure 1C) and Ser136 (data not shown) was reduced markedly on treatment of PC-3 cells with 40 µM DATS (Figure 1C).


Figure 1
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  		Fig. 1. (A) Immunoblotting for phospho-(Ser473)-Akt (top panel), phospho-(Thr308)-Akt (bottom panel) using lysates from PC-3 cells treated with 40 µM DATS for the indicated time periods. (B) Akt kinase activity in lysates from PC-3 cells treated with 40 µM DATS for the indicated time periods. Activity of Akt was determined by immunoprecipitation kinase assay by monitoring Ser21/9 phosphorylation of GSK-3{alpha}/ß. (C) Immunoblotting for phospho-(Ser155)-BAD using lysates from PC-3 cells treated with 40 µM DATS for the indicated time periods. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The immunoblotting for each protein was performed at least twice, and the results were comparable.

 
To test whether DATS-mediated inactivation of Akt was unique to the PC-3 cell line, we determined the effect of DATS treatment on Ser473 phosphorylation and kinase activity of Akt using DU145 prostate adenocarcinoma cells. Similar to PC-3 cells, the Ser473 phosphorylation of Akt was reduced by ~30–50% on treatment of DU145 cells with DATS when compared with control (Figure 2A). The DATS-mediated suppression of Akt phosphorylation in DU145 cells was coupled to a marked inhibition in the kinase activity of Akt (Figure 2B). The Ser155 phosphorylation of BAD was also reduced in DU145 cells on treatment with 40 µM DATS (Figure 2C). These results indicated that DATS-mediated inactivation of Akt as well as dephosphorylation of BAD was not restricted to the PC-3 cell line.


Figure 2
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  		Fig. 2. (A) Immunoblotting for phospho-(Ser473)-Akt using lysates from DU145 cells treated with 40 µM DATS for the indicated time periods. (B) Akt kinase activity in lysates from DU145 cells treated with 40 µM DATS for the indicated time periods. (C) Immunoblotting for phospho-(Ser155)-BAD using lysates from DU145 cells treated with 40 µM DATS for the indicated time periods. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Similar results were observed in at least two independent experiments.

 
DATS downregulated IGF-1R protein expression
Activation of Akt is mediated by receptor tyrosine kinases (e.g. IGF-1R) which, upon ligand binding, are autophosphorylated and cause activation of phosphatidylinositide-3'-kinase (36,37). Activated phosphatidylinositide-3'-kinase generates lipid second messengers, which facilitate recruitment of Akt to the plasma membrane for its activation (37). To gain insights into the mechanism of suppression of Akt kinase in our model, we determined the effect of DATS treatment on IGF-1R protein level by immunoblotting. As can be seen in Figure 3A, DATS treatment (40 µM) caused a rapid and marked decrease in the level of IGF-1R protein in PC-3 cells, which was coupled to a reduction in autophosphorylation of IGF-1R (Figure 3B). The protein level of 85 kDa regulatory subunit of phosphatidylinositide-3'-kinase was also reduced in DATS-treated PC-3 cells (Figure 3C). These results suggested that the DATS-mediated inactivation of Akt may be owing to downregulation of IGF-1R protein level and inhibition of its autophosphorylation.


Figure 3
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  		Fig. 3. (A) Immunoblotting for IGF-1R using lysates from PC-3 cells treated with 40 µM DATS for the indicated time periods. (B) Immunoblotting for phospho-tyrosine using IGF-1R immunoprecipitates from DATS-treated (40 µM) PC-3 cell lysates. (C) Immunoblotting for PI3K using lysates from PC-3 cells treated with 40 µM DATS for the indicated time periods. In panels (A) and (C), the blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The immunoblotting for each protein was performed at least twice, and the results were comparable.

 
DATS treatment reduced interaction between BAD and 14-3-3ß
The proapoptotic activity of BAD is regulated by phosphorylation (35). In its dephosphorylated state, BAD is localized to the outer mitochondrial membrane where it binds to and antagonizes pro-survival Bcl-2 family proteins such as Bcl-xL (38,39). Growth factor-mediated phosphorylation of BAD causes its cytoplasmic sequestration owing to increased binding with 14-3-3 proteins, which prevents interaction of BAD with anti-apoptotic Bcl-2 family members (39–43). To test whether DATS treatment affected interaction between BAD and 14-3-3 proteins, we immunoprecipitated 14-3-3ß using lysates from DMSO-treated control and DATS-treated (40 µM, 4 h) cells, and the immunoprecipitated complexes were then subjected to immunoblotting using anti-BAD antibody. As shown in Figure 4A, the complex formation between BAD and 14-3-3ß was reduced markedly in DATS-treated PC-3 and DU145 cells. The same blot was reprobed with anti-14-3-3ß antibody to ensure equal immunoprecipitation from control and DATS treated lysates (Figure 4A).


Figure 4
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  		Fig. 4. (A) Immunoblotting for BAD or 14-3-3ß using immunoprecipitates from PC-3 and DU145 cell lysates treated for 4 h with DMSO (control) or 40 µM DATS. Equal amounts of lysate proteins (200 µg) from control and DATS-treated cells were used for immunoprecipitation. (B) Microscopic analysis of BAD localization in PC-3 cells following a 4 h treatment with DMSO (control) or 40 µM DATS. The staining for BAD, mitochondria and nuclear DNA is indicated by green, red and blue fluorescence, respectively. The images were merged to detect mitochondrial localization of BAD. Experiment was performed twice, and the results were comparable.

 
DATS promoted mitochondrial translocation of BAD
Because DATS treatment reduced the interaction between 14-3-3ß and BAD (Figure 4), we tested the possibility whether DATS promoted translocation of BAD to the mitochondria. Figure 4B depicts merged images for BAD (green fluorescence), mitochondrial (red fluorescence) and nuclear DNA staining (blue fluorescence) in representative cells from DMSO treated control and DATS treated PC-3 cultures. In DMSO treated controls, a large fraction of mitochondria were stained red and the BAD immunostaining was predominant in the cytoplasm (Figure 4B). The mitochondria of a large fraction of DATS treated PC-3 cells were stained yellow-orange due to merge of red and green fluorescence indicating mitochondrial translocation of BAD (Figure 4B). Higher magnification was necessary to observe these effects, and thus representative data from a single cell are shown in Figure 4B. Collectively, these results indicated that DATS-mediated inactivation of Akt was associated with dephosphorylation and mitochondrial translocation of BAD.

Ectopic expression of constitutively active Akt conferred significant protection against DATS-induced apoptosis
Next, we determined the effect of overexpression of constitutively active Akt on DATS-induced cell death using DU145 cells. Cells transfected with the empty vector were used as controls. Overexpression of constitutively active Akt (CA-Akt) in transfected cells was confirmed by immunoblotting using antibodies against total Akt and phospho-(Ser473)-Akt (Figure 5A). In vector-transfected DU145 cells, the DATS treatment (40 and 80 µM for 8 h) caused a statistically significant increase in cytoplasmic histone-associated DNA fragmentation (a measure of apoptotic cell death) compared with vehicle treated control. However, the DATS-induced cytoplasmic histone-associated DNA fragmentation was not observed in DU145 cells overexpressing constitutively active Akt (Figure 5B). Consistent with these results, sulforhodamine B assay revealed that the cells transfected with CA-Akt were resistant to cell killing by DATS, whereas DATS treatment caused a dose-dependent and statistically significant reduction in survival of vector-transfected DU145 cells (Figure 5C).


Figure 5
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  		Fig. 5. (A) Immunoblotting for phospho-(Ser473)-Akt and total Akt using lysates from DU145 cells transiently transfected with empty vector or vector containing constitutively active Akt (CA-Akt). The blot was stripped and re-probed with anti-actin antibody to ensure equal protein loading. (B) Analysis of cytoplasmic histone associated DNA fragmentation in DU145 cells transiently transfected with empty vector or CA-Akt following an 8 h treatment with DMSO (white bar), 40 µM DATS (shaded bar) and 80 µM DATS (black bar). (C) Survival of DU145 cells transiently transfected with empty vector or CA-Akt following an 8 h treatment with DMSO (white bar), 40 µM DATS (shaded bar) and 80 µM DATS (black bar) as determined by sulforhodamine B assay. Similar results were observed in two independent experiments. Data are mean ± SE (n = 3). *, P < 0.05, significantly different compared with DMSO treated control by one-way ANOVA followed by Dunnett's test.

 
Caspase inhibitors attenuated DATS-induced apoptosis in PC-3 and DU145 cells
We have shown previously that DATS treatment causes cleavage of caspase 3 in PC-3 cells (29). To further examine caspase dependence in our model, we determined the effects of zVAD-fmk (a pan caspase inhibitor) and zLEHD-fmk (a caspase 9 specific inhibitor) on DATS-induced cleavage of caspase 3. As can be seen in Figure 6A, a 24 h treatment of PC-3 cells with 40 µM DATS resulted in cleavage of procaspase 3 as evidenced by appearance of 19 kDa cleaved intermediate. The DATS-induced cleavage of procaspase 3 was blocked in the presence of 40 µM pan caspase inhibitor zVAD-fmk and caspase 9 specific inhibitor zLEHD-fmk. The DATS-induced cleavage of PARP, a substrate of activated caspase 3, was significantly blocked in the presence of zVAD-fmk and zLEHD-fmk. Similar inhibitory effects of zVAD-fmk and zLEHD-fmk on DATS-induced cleavage of caspase 3 and PARP were also observed in DU145 cells (results not shown). Consistent with these results, the DATS-induced apoptosis, judged by analysis of cytoplasmic histone-associated DNA fragmentation, was significantly attenuated in the presence of zVAD-fmk and zLEHD-fmk in both cell lines (Figure 6B). These results indicated that DATS-induced apoptosis in PC-3 and DU145 cells was mediated by activation of caspases 9 and 3, and that DATS-induced apoptosis was inhibited in the presence of caspase inhibitors.


Figure 6
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  		Fig. 6. (A) Immunoblotting for cleaved caspase 3 and cleaved PARP using lysates from PC-3 cells treated with DATS and/or caspase inhibitors (zVAD-fmk or zLEHD-fmk). PC-3 cells were pretreated for 2 h with 40 µM zVAD-fmk or zLEHD-fmk and then exposed to 40 µM DATS for 24 h in the presence of the inhibitor. Cell lysates were prepared, and used for immunoblotting using antibodies specific for detection of cleaved caspase 3 and cleaved PARP. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. (B) Cytoplasmic histone associated DNA fragmentation in PC-3 (white bars) and DU145 (shaded bars) cells treated with 40 µM DATS (24 h) and/or 40 µM zVAD-fmk or zLEHD-fmk. Data are mean ± SE (n = 3). a, P < 0.05, significantly different compared with DMSO treated control; and b, P < 0.05, significantly different compared with DATS alone treatment group by one-way ANOVA followed by Bonferroni's multiple comparison test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study was undertaken to investigate possible involvement of Akt in apoptosis induction by DATS. The rationale for examining the effect of DATS on Akt signaling axis was based on the following considerations: (i) the DATS-induced apoptosis in PC-3/DU145 cells was not fully rescued either by pharmacological inhibition of JNK or ERK or by forced expression of Bcl-2 (29), (ii) Akt modulates the function of numerous proteins involved in the regulation of cell survival, cell-cycle progression and cell death (reviewed in Ref. 37), (iii) Akt activation has been observed in spontaneously developing tumors of a transgenic mouse model of prostate cancer (TRAMP mouse) (44) and (iv) Akt inactivation is implicated in regulation of apoptosis by agents including a cruciferous vegetable-derived cancer chemopreventive agent indole-3-carbinol (45,46). The present study reveals that DATS treatment causes a rapid decrease in activating phosphorylations of Akt (Ser473 and Thr308) in both PC-3 and DU145 cells. The Ser473 and Thr308 phosphorylation of Akt occurs in response to growth factor stimulus and is necessary for maximal activation of Akt (36,37,47). The DATS-mediated suppression of Akt phosphorylation in PC-3 and DU145 cells is coupled to inhibition of the kinase activity of Akt as judged by immunoprecipitation kinase assay. Relative resistance of DU145 cells transfected with constitutively active Akt towards DATS-induced apoptosis compared with vector transfected controls (Figure 5) further supports that Akt inactivation contributes to the cell death caused by DATS. However, further studies are needed to probe into the question whether the association between DATS-mediated Akt inactivation and cell death is unique to the prostate cancer cells.

Activated Akt can phosphorylate several apoptosis regulating proteins including pro-apoptotic Bcl-2 family member BAD (37,48–50). BAD promotes cell death by interacting with anti-apoptotic Bcl-2 members such as Bcl-xL, which allows the multidomain pro-apoptotic Bcl-2 family members Bax and Bak to aggregate and cause release of apoptogenic molecules (e.g. cytochrome c) from mitochondria to the cytosol culminating into caspase activation and cell death (51,52). Growth factor-stimulated phosphorylation of BAD at Ser112, Ser136 and/or Ser155 induces a conformational change in BAD that reduces its ability to interact with Bcl-xL (39–43). However, phosphorylation of BAD promotes its interaction with 14-3-3 proteins, which sequesters BAD in the cytoplasm (41–43). The present study indicates that DATS-mediated inactivation of Akt in our model is associated with reduced Ser136 and Ser155 phosphorylation of BAD. We also found that DATS treatment reduces interaction between BAD and 14-3-3ß as revealed by immunoprecipitation–immunoblotting experiment (Figure 4A) leading to mitochondrial localization of BAD.

Activated Akt can also phosphorylate apoptosis signal-regulating kinase 1 (Ask-1) at Ser83 resulting in inhibition of Ask-1 activity (53). Ask-1 acts upstream of JNK and p38 mitogen-activated protein kinase (MAPK) (54). DATS-mediated inactivation of Akt in our model is likely to relieve Ask-1 phosphorylation resulting in activation of JNK and/or P38 MAPK. It is interesting to note that DATS treatment causes rapid and sustained activation of JNK, but not P38 MAPK, in both PC-3 and DU145 cells (29). Activation of JNK in response to treatment with other naturally occurring OSC analogs has also been reported (26,27). Thus, it is possible that the DATS-mediated JNK activation in PC-3 and DU145 cells (29) may be linked to reduced Ser83 phosphorylation of Ask-1. However, further studies are needed to experimentally verify this possibility.

Activation of caspases leads to cleavage and inactivation of key cellular proteins such as PARP. We showed previously that DATS treatment caused cleavage of caspases 9 and 3 in PC-3 cells (29). In the present study, we extended these observations and determined the role of caspases 9 and 3 in DATS-induced apoptosis using PC-3 and DU145 cells. The DATS-induced cleavage of caspase 3 and PARP is significantly blocked on pretreatment with both pan caspase inhibitor and caspase 9 specific inhibitor (Figure 6A). Because DATS-induced apoptosis is also significantly attenuated in the presence of zVAD-fmk and zLEHD-fmk (Figure 6B), we postulate involvement of intrinsic caspase cascade in cell death caused by DATS.

Recent studies from our laboratory have indicated that the initial signal for cellular effects of DATS (apoptosis and cell-cycle arrest) is derived from generation of reactive oxygen species (29,30). This conclusion is based on the following observations: (i) DATS treatment causes generation of ROS in both PC-3 and DU145 cells (30), (ii) the DATS-induced activation of JNK, but not ERK, is significantly blocked by adenovirus-mediated overexpression of H2O2 scavenger catalase (29), (iii) catalase overexpression offers significant protection against DATS-induced cell death (29) and (iv) the DATS-induced destruction of cell-cycle regulatory protein Cdc25C, hyperphosphorylation of Cdc25C at Ser216 and G2/M phase cell-cycle arrest is significantly attenuated in the presence of N-acetylcysteine, a well-known antioxidant (30). Involvement of ROS in cellular effects of DADS, an analog of DATS, has also been postulated in other cellular systems (26,27). However, the precise mechanism by which DATS or other OSC analogs cause formation of ROS remains to be elucidated.

The DATS-induced apoptosis and cell-cycle arrest in prostate cancer cells have been observed at 20–40 µM concentrations (29–32). It is unclear if such concentrations of DATS are achievable in humans owing to lack of pharmacokinetic data. The pharmacokinetic parameters for S-allylcysteine, a water soluble OSC analog, have been determined in rodents and dogs (55). The plasma concentrations following oral administration of 12.5, 25 and 50 mg S-allylcysteine/Kg body wt were ~50, 112 and 227 µM, respectively (55). Detailed pharmacokinetic evaluation of DATS in rodent models and humans is necessary for its further development as a potential chemopreventive or therapeutic agent.

In conclusion, the present study demonstrates that DATS treatment inactivates Akt to relieve BAD phosphorylation, which leads to reduced interaction between BAD and 14-3-3ß and mitochondrial translocation of BAD. To the best of our knowledge, the present study is the first published report to link modulation of Akt signaling axis in cell death caused by a naturally occurring OSC analog.


   Acknowledgments
 
This work was supported by USPHS grant CA113363 awarded by the National Cancer Institute.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. You,W.C., Blot,W.J., Chang,Y.S., Ershow,A., Yang,Z.T., An,Q., Henderson,B.E., Fraumeni,J.F. and Wang,T.G. (1989) Allium vegetables and reduced risk of stomach cancer. J. Natl Cancer Inst., 18, 162–164.
  2. Challier,B., Perarnau,J.M. and Viel,J.F. (1998) Garlic, onion and cereal fibre as protective factors for breast cancer: a French case–control study. Eur. J. Epidemiol., 14, 737–747.[CrossRef][Web of Science][Medline]
  3. Gao,C.M., Takezaki,T., Ding,J.H., Li,M.S. and Tajima,K. (1999) Protective effect of allium vegetables against both esophageal and stomach cancer: a simultaneous case-referent study of a high-epidemic area in Jiangsu Province, China. Jpn. J. Cancer Res., 90, 614–621.[CrossRef][Web of Science]
  4. Hsing,A,W, Chokkalingam,A.P., Gao,Y.T., Madigan,M.P., Deng,J., Gridley,G. and Fraumeni,J.F. (2002) Allium vegetables and risk of prostate cancer: a population-based study. J. Natl Cancer Inst., 94, 1648–1651.[Abstract/Free Full Text]
  5. Block,E. (1992) The organosulfur chemistry of the genus Allium- implications for the organic chemistry of sulfur. Angew. Chem. Int. Ed. Engl., 31, 1135–1178.[CrossRef]
  6. Milner,J.A. (2001) A historical perspective on garlic and cancer. J. Nutr., 131, 1027s–1031s.[Medline]
  7. Wargovich,M.J. (1987) Diallyl sulfide, a flavor component of garlic (Allium sativum) inhibits dimethylhydrazine-induced colon cancer. Carcinogenesis, 8, 487–489.[Abstract/Free Full Text]
  8. Wargovich,M.J., Woods,C., Eng,V.W.S., Stephens,L.C. and Gray,K. (1988) Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide. Cancer Res., 48, 6872–6875.[Abstract/Free Full Text]
  9. Sparnins,V.L., Barany,G. and Wattenberg,L.W. (1988) Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse. Carcinogenesis, 9, 131–134.[Abstract/Free Full Text]
  10. Takahashi,S., Hakoi,K., Yada,H., Hirose,M., Ito,N. and Fukushima,S. (1992) Enhancing effects of diallyl sulfide on hepatocarcinogenesis and inhibitory actions of the related diallyl disulfide on colon and renal carcinogenesis in rats. Carcinogenesis, 13, 1513–1518.[Abstract/Free Full Text]
  11. Reddy,B.S., Rao,C.V., Rivenson,A. and Kelloff,G. (1993) Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res., 53, 3493–3498.[Abstract/Free Full Text]
  12. Schaffer,E.M., Liu,J.Z., Green,J., Dangler,C.A. and Milner,J.A. (1996) Garlic and associated allyl sulfur components inhibit N-methyl-N-nitrosourea induced rat mammary carcinogenesis. Cancer Lett., 102, 199–204.[CrossRef][Web of Science][Medline]
  13. Suzui,N., Sugie,S., Rahman,K.M., Ohnishi,M., Yoshimi,N., Wakabayashi,K. and Mori,H. (1997) Inhibitory effects of diallyl disulfide or aspirin on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary carcinogenesis in rats. Jpn. J. Cancer Res., 88, 705–711.[CrossRef][Web of Science]
  14. Hu,X., Benson,P.J., Srivastava,S.K., Mack,L.M., Xia,H., Gupta,V., Zaren,H.A. and Singh,S.V. (1996) Glutathione S-transferases of female A/J mouse liver and forestomach and their differential induction by anti-carcinogenic organosulfides from garlic. Arch. Biochem. Biophys., 336, 199–214.[CrossRef][Web of Science][Medline]
  15. Hu,X. and Singh,S.V. (1997) Glutathione S-transferases of female A/J mouse lung and their induction by anticarcinogenic organosulfides from garlic. Arch. Biochem. Biophys., 340, 279–286.[CrossRef][Web of Science][Medline]
  16. Andorfer,J.H., Tchaikovskaya,T. and Listowsky,I. (2004) Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds. Carcinogenesis, 25, 359–367.[Abstract/Free Full Text]
  17. Brady,J.F., Ishizaki,H., Fukuto,J.M., Lin,M.C., Fadel,A., Gapac,J.M. and Yang,C.S. (1991) Inhibition of cytochrome P-450 2E1 by diallyl sulfide and its metabolites. Chem. Res. Toxicol., 4, 642–647.[CrossRef][Web of Science][Medline]
  18. Singh,S.V., Mohan,R.R., Agarwal,R., Benson,P.J., Hu,X., Rudy,M.A., Xia,H., Katoh,A., Srivastava,S.K., Mukhtar,H., Gupta,V. and Zaren,H.A. (1996) Novel anti-carcinogenic activity of an organosulfide from garlic: inhibition of H-RAS oncogene transformed tumor growth in vivo by diallyl disulfide is associated with inhibition of p21H-ras processing. Biochem. Biophys. Res. Commun., 225, 660–665.[CrossRef][Web of Science][Medline]
  19. Singh,S.V. (2001) Impact of garlic organosulfides on p21(H-ras) processing. J. Nutr., 131, 1046s–1048s.[Medline]
  20. Sundaram,S.G. and Milner,J.A. (1996) Diallyl disulfide induces apoptosis of human colon tumor cells. Carcinogenesis, 17, 669–673.[Abstract/Free Full Text]
  21. Knowles,L.M. and Milner,J.A. (1998) Depressed p34cdc2 kinase activity and G2/M phase arrest induced by diallyl disulfide in HCT-15 cells. Nutr. Cancer, 30, 169–174.[Web of Science][Medline]
  22. Knowles,L.M. and Milner,J.A. (2000) Diallyl disulfide inhibits p34(cdc2) kinase activity through changes in complex formation and phosphorylation. Carcinogenesis, 21, 1129–1134.[Abstract/Free Full Text]
  23. Nakagawa,H., Tsuta,K., Kiuchi,K., Senzaki,H., Tanaka,K., Hioki,K. and Tsubura,A. (2001) Growth inhibitory effects of diallyl disulfide on human breast cancer cell lines. Carcinogenesis, 22, 891–897.[Abstract/Free Full Text]
  24. Robert,V., Mouille,B., Mayeur,C., Michaud,M. and Blachier,F. (2001) Effects of the garlic compound diallyl disulfide on the metabolism, adherence and cell cycle of HT-29 colon carcinoma cells: evidence of sensitive and resistant sub-populations. Carcinogenesis, 22, 1155–1161.[Abstract/Free Full Text]
  25. Shirin,H., Pinto,J.T., Kawabata,Y., Soh,J.W., Delohery,T., Moss,S.F., Murty,V., Rivlin,R.S., Holt,P.R. and Weinstein,I.B. (2001) Antiproliferative effects of S-allylmercaptocysteine on colon cancer cells when tested alone or in combination with sulindac sulfide. Cancer Res., 61, 725–731.[Abstract/Free Full Text]
  26. Filomeni,G., Aquilano,K., Rotilio,G. and Ciriolo,M.R. (2003) Reactive oxygen species-dependent c-Jun NH2-terminal kinase/c-Jun signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide. Cancer Res., 63, 5940–5949.[Abstract/Free Full Text]
  27. Xiao,D., Pinto,J.T., Soh,J.W., Deguchi,A., Gundersen,G.G., Palazzo,A.F., Yoon,J.T., Shirin,H. and Weinstein,I.B. (2003) Induction of apoptosis by the garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH2-terminal kinase 1 activation. Cancer Res., 63, 6825–6837.[Abstract/Free Full Text]
  28. Lan,H. and Lu,Y. (2004) Allitridi induces apoptosis by affecting Bcl-2 expression and caspase-3 activity in human gastric cancer cells. Acta Pharmacol. Sin., 25, 219–225.[Web of Science][Medline]
  29. Xiao,D., Choi,S., Johnson,D.E., Vogel,V.G., Johnson,C.S., Trump,D.L., Lee,Y.J. and Singh,S.V. (2004) DATS-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2. Oncogene, 23, 5594–5606.[CrossRef][Web of Science][Medline]
  30. Xiao,D., Herman-Antosiewicz,A., Antosiewicz,A., Xiao,H., Brisson,M., Lazo,J.S. and Singh,S.V. (2005) DATS-induced cell cycle arrest in human prostate cancer cells is caused by reactive oxygen species-dependent destruction and hyperphosphorylation of Cdc25C. Oncogene, 24, 6256–6268.[CrossRef][Web of Science][Medline]
  31. Herman-Antosiewicz,A. and Singh,S.V. (2004) Signal transduction pathways leading to cell cycle arrest and apoptosis induction in cancer cells by Allium vegetable-derived organosulfur compounds: a review. Mutation Res., 555, 121–131.[Medline]
  32. Herman-Antosiewicz,A. and Singh,S.V. (2005) Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells. J. Biol. Chem., 280, 28519–28528.[Abstract/Free Full Text]
  33. Hemmings,B.A. (1997) Akt signaling: linking membrane events to life and death decisions. Science, 275, 628–630.[Free Full Text]
  34. Kennedy,S.G., Kandel,E.S., Cross,T.K. and Hay,N. (1999) Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol. Cell. Biol., 19, 5800–5810.[Abstract/Free Full Text]
  35. Datta,S.R., Dudek,H., Tao,X., Masters,S., Fu,H., Gotoh,Y. and Greenberg,M.E. (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 91, 231–241.[CrossRef][Web of Science][Medline]
  36. Scheid,M.P. and Woodgett,J.R. (2001) PKB/AKT: functional insights from genetic models. Nat. Rev. Mol. Cell. Biol., 2, 760–768.[CrossRef][Web of Science][Medline]
  37. Fresno Vara,J.A., Casado,E., de Castro,J., Cejas,P., Belda-Iniesta,C. and Gonzalez-Baron,M. (2004) PI3K/Akt signalling pathway and cancer. Cancer Treat. Rev., 30, 193–204.[CrossRef][Web of Science][Medline]
  38. Yang,E., Zha,J., Jockel,J., Boise,L.H., Thompson,C.B. and Korsmeyer,S.J. (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell, 80, 285–291.[CrossRef][Web of Science][Medline]
  39. Zha,J., Harada,H., Yang,E., Jockel,J. and Korsmeyer,S.J. (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell, 87, 619–628.[CrossRef][Web of Science][Medline]
  40. Downward,J. (1999) How BAD phosphorylation is good for survival. Nat. Cell Biol., 1, E33–E35.[CrossRef][Web of Science][Medline]
  41. Datta,S.R., Katsov,A., Hu,L., Petros,A., Fesik,S.W., Yaffe,M.B. and Greenberg,M.E. (2000) 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol. Cell, 6, 41–51.[CrossRef][Web of Science][Medline]
  42. Virdee,K., Parone,P.A. and Tolkovsky,A.M. (2000) Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr. Biol., 10, 1151–1154.[CrossRef][Web of Science][Medline]
  43. Tan,Y., Demeter,M.R., Ruan,H. and Comb,M.J. (2000) BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J. Biol. Chem., 275, 25865–25869.[Abstract/Free Full Text]
  44. Shukla,S., MacLennan,G.T., Marengo,S.R., Resnick,M.I. and Gupta,S. (2005) Constitutive activation of PI3K-Akt and NF-{kappa}B during prostate cancer progression in autochthonous transgenic mouse model. Prostate, 64, 224–239.[CrossRef][Web of Science][Medline]
  45. Nakashio,A., Fujita,N., Rokudai,S., Sato,S. and Tsuruo,T. (2000) Prevention of phosphatidylinositol 3'-kinase-Akt survival signaling pathway during topotecan-induced apoptosis. Cancer Res., 60, 5303–5309.[Abstract/Free Full Text]
  46. Chinni,S.R. and Sarkar,F.H. (2002) Akt inactivation is a key event in indole-3-carbinol-induced apoptosis in PC-3 cells. Clin. Cancer Res., 8, 1228–1236.[Abstract/Free Full Text]
  47. Alessi,D.R., Andjelkovic,M., Caudwell,B., Cron,P., Morrice,N., Cohen,P. and Hemmings,B.A. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J., 15, 6541–6551.[Web of Science][Medline]
  48. del Peso,L., Gonzalez-Garcia,M., Page,C., Herrera,R. and Nunez,G. (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science, 278, 687–689.[Abstract/Free Full Text]
  49. Cardone,M.H., Roy,N., Stennicke,H.R., Salvesen,G.S., Franke,T.F., Stanbridge,E., Frisch,S. and Reed,J.C. (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science, 282, 1318–1321.[Abstract/Free Full Text]
  50. Shin,I., Bakin,A.V., Rodeck,U., Brunet,A. and Arteaga,C.L. (2001) Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1. Mol. Biol. Cell, 12, 3328–3339.[Abstract/Free Full Text]
  51. Cheng,E.H., Wei,M.C., Weiler,S., Flavell,R.A., Mak,T.W., Lindsten,T. and Korsmeyer,S.J. (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell, 8, 705–711.[CrossRef][Web of Science][Medline]
  52. Wei,M.C., Zong,W.X., Cheng,E.H., Lindsten,T., Panoutsakopoulou,V., Ross,A.J., Roth,K.A., MacGregor,G.R., Thompson,C.B. and Korsmeyer,S.J. (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science, 292, 727–730.[Abstract/Free Full Text]
  53. Kim,A.H., Khursigara,G., Sun,X., Franke,T.F. and Chao,M.V. (2001) Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol. Cell. Biol., 21, 893–901.[Abstract/Free Full Text]
  54. Ichijo,H., Nishida,E., Irie,K., ten Dijke,P., Saitoh,M., Moriguchi,T., Takagi,M., Matsumoto,K., Miyazono,K. and Gotoh,Y. (1997) Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science, 275, 90–94.[Abstract/Free Full Text]
  55. Nagae,S., Ushijima,M., Hatono,S., Imai,J., Kasuga,S., Matsuura,H., Itakura,Y. and Higashi,Y. (1994) Pharmacokinetics of the garlic compound S-allylcysteine. Planta Med., 60, 214–217.[Web of Science][Medline]
Received June 30, 2005; revised August 30, 2005; accepted September 11, 2005.


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