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Carcinogenesis Advance Access originally published online on August 18, 2006
Carcinogenesis 2007 28(1):151-162; doi:10.1093/carcin/bgl144
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1

Sunga Choi, Karen L. Lew, Hui Xiao, Anna Herman-Antosiewicz, Dong Xiao, Charles K. Brown1 and Shivendra V. Singh*

Department of Pharmacology, University of Pittsburgh Cancer Institute University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
1 Surgery, 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
 
D,L-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived isomer L-SFN, suppresses proliferation of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. We used LNCaP (wild-type p53) and PC-3 (p53 deficient) human prostate cancer cells to gain further insights into the mechanism of SFN-induced apoptosis. The LNCaP cell line was relatively more sensitive to SFN-induced apoptosis compared with PC-3. The SFN treatment caused stabilization of p53 protein in LNCaP cells, but SFN-mediated apoptosis was not attenuated by knockdown of p53 protein. Instead, the differential sensitivity of these cells to SFN-induced apoptosis correlated with difference in kinetics of Bax conformational change. Ectopic expression of Bcl-2 failed to confer protection against SFN-induced cell death in LNCaP cells. Treatment of PC-3 cells with SFN resulted in a marked decrease in the levels of inhibitor of apoptosis (IAP) family proteins (cIAP1, cIAP2 and XIAP), which was accompanied by inhibition of nuclear translocation of p65-nuclear factor {kappa}B (NF{kappa}B). The effect of SFN on levels of IAP family proteins as well as transcriptional activity of NF{kappa}B was biphasic in LNCaP cells. The SFN-treated LNCaP and PC-3 cells exhibited a marked increase in protein level of Apaf-1, which was accompanied by an increase in transcriptional activity of E2F1. The SFN-induced apoptosis in both cell lines was significantly attenuated by Apaf-1 protein knockdown. In conclusion, the present study reveals a complex signaling mechanism involving Bax activation, downregulation of IAP family proteins and Apaf-1 induction in regulation of SFN-induced cell death.

Abbreviations: Chk2, checkpoint kinase 2; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; IAP, inhibitor of apoptosis; ITCs, isothiocyanates; L-SFN, L-sulforaphane; PARP, poly-(ADP-ribose) polymerase; PBS, phosphate-buffered saline; SFN, D,L-sulforaphane


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies continue to support the assertion that dietary intake of cruciferous vegetables may be protective against the risk of different types of cancers including prostate cancer (1–4). Anticarcinogenic effect of cruciferous vegetables is attributed to organic isothiocyanates (ITCs), which are generated due to myrosinase-mediated hydrolysis of corresponding glucosinolates (5–7). Broccoli is a rather rich source of the ITC compound (–)-1-isothiocyanato-(4R)-(methylsulfinyl)-butane [L-sulforaphane (L-SFN)]. The L-SFN and its synthetic analogue D,L-sulforaphane (SFN) have generated a great deal of research interest due to their interesting biological effects (8-14). The L-SFN and SFN are equipotent as inducers of quinone reductase activity in Hepa 1c1c7 murine hepatoma cells (8), whereas L-SFN treatment causes transcriptional induction of NQO1, {gamma}-glutamylcysteine synthetase light subunit and microsomal and {alpha}-class glutathione transferases in prostate cancer cells (9). In addition, SFN is an inhibitor of CYP2E1, which is involved in the activation of carcinogenic chemicals (10). Cancer chemoprevention by either natural isomer L-SFN or synthetic racemic SFN has been observed against 9-10-dimethyl-1,2-benzanthracene-induced mammary cancer in rats, azoxymethane-induced colonic aberrant crypt foci in rats and benzo[a]pyrene-induced forestomach cancer in mice (11,13,14).

More recent studies including those from our laboratory have revealed that SFN or natural isomer L-SFN can inhibit proliferation of cultured cancer cells by causing G2–M phase cell cycle arrest and/or apoptosis induction (15–23). The SFN was recently shown to inhibit histone deacetylase activity (24). Our own work has revealed that oral gavage of SFN significantly retards growth of PC-3 human prostate cancer xenografts in athymic mice without causing weight loss or any other side effects (20).

An understanding of the mechanism by which SFN causes cell cycle arrest and apoptosis induction is critical for its further development as a clinically useful cancer preventive/therapeutic agent since this knowledge could lead to identification of mechanism-based biomarkers potentially useful in future clinical trials. Recent studies have offered novel insights into the mechanism by which SFN blocks G2–M progression (15,16,19,21). For instance, the SFN-mediated G2–M phase cell cycle arrest in prostate cancer cells is associated with checkpoint kinase 2 (Chk2)-mediated phosphorylation of cell division cycle 25C (Cdc25C) at Ser216 leading to its sequestration in the cytosol and accumulation of inactive cyclin-dependent kinase 1 (21).

Using PC-3 and DU145 human prostate cancer cells as a model, which are androgen-independent and lack functional p53 protein, we have demonstrated that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (23). Even though these studies suggest that p53 is probably not required for the cell death caused by SFN, it was of interest to determine whether the presence of wild-type p53 affects cellular sensitivity to SFN-induced apoptosis. In the present study, we addressed this question using a wild-type p53 expressing human prostate cancer cell line (LNCaP). Even though SFN treatment caused stabilization of p53 protein in LNCaP cells, the p53 protein knockdown failed to confer significant protection against SFN-induced cell death. We also demonstrate that SFN treatment differentially modulates the levels of inhibitor of apoptosis (IAP) family proteins in LNCaP and PC-3 cells which correlates with its effect on transcriptional activity of nuclear factor {kappa}B (NF{kappa}B). Moreover, SFN treatment causes induction of Apaf-1 protein in both cell lines, and Apaf-1 knockdown confers significant protection against SFN-induced apoptosis. In conclusion, the present study reveals a complex signaling mechanism in regulation of SFN-induced cell death.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
SFN (purity 98.3%) and L-SFN (purity 99%) were purchased from LKT Laboratories (St Paul, MN, USA). Reagents for cell culture including F-12K Nutrient Mixture, DMEM, RPMI1640 medium, McCoy’s 5A medium, penicillin/streptomycin antibiotic mixture, and fetal bovine serum were procured from Gibco (Grand Island, NY, USA) or Mediatech (Herndon, VA, USA). Propidium iodide, RNaseA, 4',6-diamidino-2-phenylindole (DAPI) were from Sigma (St Louis, MO, USA). The antibodies against Bax (cat. # sc493), Bak (cat. # sc832), Bcl-xL (cat. # sc8392), cIAP2 (cat. #sc7944) and p65-NFkB (for immunocytochemistry; cat. # sc8008) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Bcl-2 antibody (cat. # M0887) was from Dako (Carpinteria, CA, USA), the antibodies against Apaf-1 (cat. # 559683), cIAP1 (cat. # 556533) and XIAP (cat. # 610717) were from BD Pharmingen (Palo Alto, CA, USA), the antibody against p53 (cat. # OP43) was from Calbiochem (La Jolla, CA, USA), anti-poly-(ADP-ribose)polymerase (PARP) antibody (cat. # SA250) was from Biomol (Butler pike, PA, USA), the monoclonal antibody specific for detection of conformational change of Bax (clone 6A7) was from Pharmingen (Cat. # 556467), antibody against p65-NF{kappa}B (for immunoblotting; cat. # PC-137) and anti-actin antibody (cat. #CP-01) were from Oncogene Research Products (San Diego, CA, USA).

Cell culture and cell survival assay
Monolayer cultures of PC-3 cells were maintained in F-12K Nutrient Mixture (Kaighn’s Modification) supplemented with 10% (v/v) non-heat inactivated fetal bovine serum and antibiotics. The LNCaP cell line was maintained in RPMI1640 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 0.2% glucose, and 10% (v/v) fetal bovine serum and antibiotics. The wild-type HCT116 cells and XIAP–/– and Chk2–/– variants of HCT116 cells were generously provided by Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA) and maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum and antibiotics.

The effect of SFN or L-SFN on survival of LNCaP cells was determined by trypan blue dye exclusion assay. Briefly, 1 x 104 cells in 1 ml of complete medium were plated in 12-well plates, and allowed to attach overnight. The medium was replaced with fresh complete medium containing desired concentrations of SFN or L-SFN, and the plates were incubated for 24 h at 37°C. Stock solutions of SFN and L-SFN were prepared in dimethyl sulfoxide (DMSO), and an equal volume of DMSO (final concentration 0.1%) was added to the controls. At the end of the incubation, both floating and adherent cells were collected and suspended in 25 µl of phosphate-buffered saline (PBS). The cells were then mixed with 5 µl of 0.4% trypan blue solution, and live (unstained) and dead (stained) cells were counted under an inverted microscope.

Apoptosis assays
Apoptosis induction by SFN was assessed by (i) fluorescence microscopic analysis of cells with condensed and fragmented DNA following staining with DAPI, (ii) quantification of cytoplasmic histone-associated DNA fragmentation and/or (iii) flow cytometric analysis of cells with sub-G0/G1 DNA. For DAPI assay, cells (2 x 104) were plated on coverslips, allowed to attach overnight and exposed to DMSO or SFN for desired time period at 37°C. Cells were washed with PBS, and fixed with 3% paraformaldehyde for 1 h at room temperature. The cells were washed three times with PBS, permeabilized with 0.1% Triton X-100 for 15 min, washed again with PBS and stained by incubation with 10 ng/ml DAPI for 20 min. Cells with condensed and fragmented DNA (apoptotic cells) were counted under a fluorescence microscope. The effect of SFN on cytoplasmic histone-associated DNA fragmentation was determined using a kit from Roche Diagnostics (Mannheim, Germany) according to the manufacturer’s recommendations. The sub-G0/G1 fraction in control and SFN-treated cells was analyzed by flow cytometry as described by us previously (21).

Immunoblotting
Cells were treated with SFN as described above. Both floating and attached cells were collected, washed with ice-cold PBS, and lysed as described by us previously (21,22). The cell lysate was cleared by centrifugation at 14 000 r.p.m. for 30 min. Lysate proteins were resolved by sodium-dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membrane. After blocking with Tris-buffered saline containing 0.05% Tween-20 and 5% (w/v) non-fat dry milk, the membrane was treated with the desired primary antibody for 2 h at room temperature or overnight at 4°C. Following incubation with appropriate secondary antibody, the immunoreactive bands were visualized using enhanced chemiluminescence method. The blots were stripped and re-probed with anti-actin antibody to correct for differences in protein loading. Change in protein level was determined by densitometric scanning of the immunoreactive bands and corrected for actin loading control.

Quantitative RT–PCR for p53
Total RNA from LNCaP cells treated for 24 h with DMSO (control) or SFN (20 or 40 µM) was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized by reverse transcription and quantitative RT–PCR for p53 was performed using Human P53 Oncogene and GADPH Gene Dual PCR kit from Maxim Biotech (San Francisco, CA, USA).

RNA interference of p53
RNA interference of p53 was performed using p53-specific siRNA duplexes purchased from Santa Cruz (cat # sc-29435; Santa Cruz Biotechnology). For transfection, LNCaP cells were seeded in 6-well plates and transfected at 30% confluency with 100 nM siRNA using OligofectAMINE (Invitrogen) according to the manufacturer’s recommendations. Cells transfected with a non-specific siRNA (Qiagen, cat.#1022076) were used as controls. After 24 h of transfection, cells were treated with DMSO or desired concentration of SFN for 24 h. Both floating and adherent cells were collected, washed with PBS, and processed for analysis of cytoplasmic histone-associated DNA fragmentation and p53 immunoblotting.

Analysis of Bax conformation change
PC-3 and LNCaP cells were treated with the desired concentrations of SFN for specified time periods, and lysed using a solution containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail. Aliquots containing 500 µg lysate protein in 0.5 ml of lysis buffer were incubated overnight at 4°C with 2 µg of anti-Bax 6A7 monoclonal antibody. Protein G-agarose beads (40 µl; Santa Cruz Biotechnology) were then added to each sample and the incubation was continued for 2 h at 4°C. The immunoprecipitated complexes were washed with lysis buffer, and subjected to immunoblotting using polyclonal anti-Bax antibody.

Bcl-2 transfection
LNCaP cells were transiently transfected with pSFFV-Bcl-2 and pSFFV-neo plasmids using Fugene6 transfection reagent (cat. #11815091001, Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s recommendations. Briefly, LNCaP cells were seeded, allowed to attach and transfected with pSFFV-Bcl-2 or pSFFV-neo plasmid. Twenty-four hours after transfection, the cells were treated with DMSO or SFN for 24 h, collected and processed for immunoblotting or analysis of cytoplasmic histone-associated DNA fragmentation.

Preparation of nuclear and cytosolic fractions
Nuclear and cytosolic fractions from control (DMSO-treated) and SFN-treated PC-3 cells were prepared using a nuclear extraction kit from Panomics (Redwood, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were lysed and centrifuged at 15 000 g for 3 min. The supernatant (cytosolic fraction) and nuclear pellet were collected. Nuclear pellet was re-suspended in nuclear buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT and protease inhibitors), vortexed, passed (15–20 times) through a 27 gauge needle, and centrifuged at 15 000 g for 5 min. The supernatant fraction was collected and used for immunoblotting of p65-NF{kappa}B.

Immunohistochemical localization of p65-NFkB
PC-3 cells were cultured on coverslips, and treated with DMSO (control) or 40 µM SFN for 16 h. After washing with PBS, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with normal goat serum (1:20 dilution) in PBS for 45 min. Subsequently, the cells were treated with anti-p65-NFkB antibody (1:500 dilution) for 2 h. The cells were washed with PBS, and incubated with Alexa Fluor 488-conjugated secondary antibody (1:1000 dilution, Molecular probes) for 1 h. After washing, cells were counterstained with DAPI (10 ng/ml) for 10 min. The cells were visualized under a fluorescence microscope.

Luciferase reporter assay
Cells were plated at a density of 2 x 105 cells/well in 12-well plates, allowed to attach overnight and transfected with 0.8 µg of total plasmid containing 0.78 µg/well reporter vector and 20 ng/well of pCMV-pRL internal control vector (Promega) using lipofectamine (Invitrogen). The NF{kappa}B-luciferase reporter plasmid was generously provided by Dr Anning Lin (University of Chicago, Chicago, IL, USA). The E2F1-luciferase reporter construct containing –728/+77 region of E2F1 gene promoter was a generous gift from Dr Stephen Safe (Texas A & M University, College Station, TX, USA). After transfection, the cells were treated with SFN for desired time period, washed with ice-cold PBS and harvested in reporter lysis buffer. After centrifugation, 20 µl supernatant fraction was used for measurement of dual luciferase activity (Promega) using a luminometer. The luciferase activity was normalized against protein concentration and expressed as relative luciferase activity (a ratio of firefly luciferase to renilla luciferase units).

RNA interference of Apaf-1 and Chk2
RNA interference of Apaf-1 was performed using Apaf-1-specific siRNA duplexes from Qiagen (cat. #1024594). The Chk2-specific siRNA duplexes were purchased from Dharmacon (Lafayette, CO, USA), which has been described by us previously (21). For transfection, desired cell line was seeded in 6-well plates and transfected at 30% confluency with siRNA duplexes (200 nM) using OligofectAMINE (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Cells transfected with non-specific siRNA (Qiagen, cat. #1022076 for Apaf-1 and Dharmacon, cat. #D-001206-08-20 for Chk2) were used as controls for direct comparison. After 24 h of transfection, cells were treated with DMSO or SFN (20 µM) for desired time period. Both floating and adherent cells were collected, and processed for analysis of cytoplasmic histone-associated DNA fragmentation or immunoblotting.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of SFN and L-SFN on survival of LNCaP cells
The L-SFN and SFN have been shown to be equipotent as inducers of quinone reductase activity in Hepa 1c1c7 cells indicating that the chirality of the sulfoxide does not affect inducer potency (8). Even though previous studies including those from our laboratory have indicated that synthetic SFN is a potent suppressor of cancer cell proliferation (18–24), it was of interest to determine whether cancer cell growth suppressing potency of SFN is comparable with that of L-SFN. In the present study, we addressed this question by determining the effects of SFN and L-SFN on survival of LNCaP cells by trypan blue dye exclusion assay, and the results are summarized in Table I. Survival of LNCaP cells was reduced significantly upon a 24 h exposure to SFN as well as L-SFN in a concentration-dependent manner (Table I). However, the synthetic compound was relatively more effective against LNCaP cells compared with L-SFN. For example, the survival of LNCaP cells was reduced by ~87% upon a 24 h exposure to 20 µM SFN relative to DMSO-treated control. On the other hand, LNCaP cell survival was reduced by only ~68% by a similar treatment with 20 µM L-SFN (P < 0.05, SFN versus L-SFN at 20 µM concentration by paired t-test). Because SFN was relatively more effective than L-SFN (Table I) and our previous cellular and animal studies were conducted using SFN (20–23), we focused on the synthetic compound to gain further insights into the mechanism of its anticancer effect.


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  		Table I Trypan blue dye exclusion assay for effect of synthetic SFN and naturally occurring L-SFN on survival of LNCaP cells

 
Differential sensitivity of LNCaP and PC-3 cells to SFN-induced apoptosis
We have shown previously that SFN treatment causes dose-dependent apoptotic cell death in PC-3 human prostate cancer cell line, which lacks functional p53 protein (20). Even though these results suggested that p53 might not be required for SFN-induced apoptosis, it was of interest to determine whether the presence of wild-type p53 affects cellular sensitivity to the cell death caused by SFN because p53 is known to regulate apoptosis in response to different stimuli (25,26). We addressed this question by determining sensitivity of LNCaP cells (wild-type p53) toward SFN-induced apoptosis by microscopic analysis of apoptotic cells after staining with DAPI. The PC-3 cell line was included for direct comparison. Figure 1A depicts representative DAPI staining in cultures of LNCaP and PC-3 cells following a 24 h treatment with either DMSO (control) or 20 µM SFN. Apoptotic cells with condensed DNA were clearly visible in SFN-treated LNCaP/PC-3 cultures but rarely seen in DMSO-treated controls (Figure 1A). As can be seen in Figure 1B, SFN treatment caused a concentration-dependent increase in apoptotic cells with condensed DNA in both LNCaP and PC-3 cell lines. However, the LNCaP cells were relatively more sensitive to apoptosis induction by SFN compared with PC-3 cells. For instance, a 24 h treatment of LNCaP cells with 10 µM SFN resulted in ~13-fold increase in apoptotic cells compared with DMSO-treated control (Figure 1B). A similar treatment of PC-3 cells with SFN (10 µM, 24 h) resulted in ~3-fold increase in percentage of apoptotic cells compared with DMSO-treated control (Figure 1B). Moreover, the SFN-induced apoptosis was statistically significantly higher in LNCaP cells than in PC-3 at both 10 and 20 µM concentrations (P < 0.05 by paired t-test). Differential sensitivity of LNCaP and PC-3 cells toward SFN-induced apoptosis was also evident in cytoplasmic histone-associated DNA fragmentation assay, which has emerged as a sensitive method for quantification of apoptotic cells. As can be seen in Figure 1C, SFN-induced cytoplasmic histone-associated DNA fragmentation was relatively more pronounced in LNCaP cells than in PC-3 cells, although the difference did not reach statistical significance at the 10 µM dose. Time course experiments using 20 µM SFN confirmed that LNCaP cells were relatively more sensitive to SFN-induced cytoplasmic histone-associated DNA fragmentation compared with PC-3 cells (Figure 1D). Similar to PC-3 cells (20), however, the apoptosis induction by SFN in LNCaP cells was associated with cleavage of PARP (Figure 1E).


Figure 1
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  		Fig. 1 (A) Representative microscopic images depicting DAPI fluorescence in LNCaP and PC-3 cultures following a 24 h exposure to solvent control (DMSO) or 20 µM SFN. Apoptotic cells with condensed DNA were rarely seen in control LNCaP and PC-3 cultures but clearly visible in both cell lines after treatment with SFN. (B) Quantification of apoptotic cells (DAPI assay) in LNCaP and PC-3 cultures following a 24 h treatment with DMSO (control) or SFN (10 or 20 µM). Data are mean ± SE (n = 9). (C) Cytoplasmic histone-associated DNA fragmentation in LNCaP and PC-3 cells following a 24 h treatment with DMSO or SFN (10 or 20 µM). (D) Cytoplasmic histone-associated DNA fragmentation in LNCaP and PC-3 cells following 8 or 16 h treatment with DMSO or 20 µM SFN. Data in panel C and D are mean of two determinations from a representative experiment that was repeated with similar results. Error bars are shown to indicate data scatter. In Panels B–D, *significantly different (P < 0.05) compared with DMSO-treated control in respective cell line by one-way ANOVA followed by Dunnett’s test, and !=significantly different (P < 0.05) between LNCaP and PC-3 cells by paired t-test. (E) Immunoblotting for PARP cleavage using lysates from LNCaP cells treated with 20 µM SFN for the indicated time periods. The blot was stripped and re-probed with anti-actin antibody to ensure equal protein loading.

 
p53 protein knockdown does not affect SFN-induced apoptosis
The p53 tumor suppressor protein, which is transcriptionally inert in the absence of stress, is involved in regulation of apoptosis by different stimuli (25,26). The p53 protein is stabilized under stress and transcriptionally regulates the expression of certain genes involved in apoptosis signaling such as Bax (25–27). We designed experiments to determine whether SFN-induced apoptosis in LNCaP cells was regulated by p53. As can be seen in Figure 2A, SFN treatment caused an increase in protein level of p53 in control siRNA transfected as well as in untransfected LNCaP cells (data not shown), which was not due to an increase in p53 transcript as revealed by quantitative RT–PCR (Figure 2B). The SFN-mediated stabilization of p53 protein was markedly reduced in LNCaP cells transiently transfected with a p53-specific siRNA (Figure 2A). Next, we determined the effect of p53 protein knockdown on SFN-induced cytoplasmic histone-associated DNA fragmentation, and the results are summarized in Figure 2C. The DNA fragmentation resulting from a 24 h exposure to 10 or 20 µM SFN did not differ significantly between LNCaP cells transfected with control siRNA and p53-specific siRNA as judged by paired t-test (Figure 2C). Collectively, these results indicated that p53 was not involved in regulation of SFN-induced apoptosis.


Figure 2
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  		Fig. 2 (A) Immunoblotting for p53 protein using lysates from control siRNA transfected and p53-specific siRNA transfected LNCaP cells following 24 h treatment with DMSO (control) or different concentrations of SFN. The blot was stripped and re-probed with anti-actin antibody to ensure equal protein loading. (B) Effect of SFN treatment on p53 transcript level determined by quantitative RT–PCR. Lane 1, positive control for p53; lane 2, positive control for GAPDH; lane 3, LNCaP cells treated with DMSO for 24 h; lane 4, LNCaP cells treated with 20 µM SFN for 24 h; lane 5, LNCaP cells treated with 40 µM SFN for 24 h. (C) Cytoplasmic histone-associated DNA fragmentation in LNCaP cells transfected with control siRNA and p53-specific siRNA following 24 h treatment with DMSO or the indicated concentrations of SFN. Data are mean ± SE (n = 3). *Significantly different (P < 0.05) compared with corresponding DMSO-treated control by one-way ANOVA followed by Dunnett’s test. The difference in DNA fragmentation between SFN-treated control siRNA and p53 siRNA transfected cells was not statistically significant at either drug concentration.

 
Effect of SFN treatment on levels of Bcl-2 family proteins in LNCaP cells
The Bcl-2 family proteins have emerged as critical regulators of apoptosis by functioning as either promoters (e.g. Bax and Bak) or inhibitors (Bcl-2 and Bcl-xL) of the cell death process (28–30). We have shown previously that the SFN-induced cell death in PC-3 cells is associated with induction of Bax and downregulation of Bcl-2 leading to a change in Bax:Bcl-2 ratio favoring apoptosis (20). To address the question of whether the SFN-mediated change in Bax and Bcl-2 protein level was restricted to PC-3 cells due to its unique characteristics, we determined the effect of SFN treatment on levels of Bcl-2 family proteins in LNCaP cells by immunoblotting, and the results are shown in Figure 3A. Similar to PC-3 cells (20), the level of Bax protein was increased markedly on treatment of LNCaP cells with SFN (Figure 3A). The SFN-mediated induction of Bax protein was evident as early as 4 h after treatment (~2.8-fold increase over control as determined by densitometric scanning of the immunoreactive band and corrected for actin loading control) and increased gradually with increasing exposure time. On the other hand, SFN treatment resulted in a decrease in Bak protein level especially at 24 h time point. After an initial induction at 2–4 h time points (between 1.3- and 1.9-fold increase relative to control) the level of Bcl-2 protein was reduced by >80% upon treatment of LNCaP cells with 20 µM SFN for 16 and 24 h. Interestingly, SFN treatment caused an increase in the protein level of Bcl-xL in LNCaP cells (Figure 3A). Collectively, these results suggested that the SFN-induced cell death in LNCaP cells might be regulated by Bax and Bcl-2.


Figure 3
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  		Fig. 3 (A) Immunoblotting for Bcl-2 family proteins using lysates from LNCaP cells treated with 20 µM SFN for the indicated time periods. The blots were stripped and re-probed with anti-actin antibody to ensure equal protein loading. (B) Analysis of conformational change of Bax using lysates from LNCaP and PC-3 cells treated with 20 µM SFN for the indicated time periods. (C) Analysis of conformational change of Bax using lysates from LNCaP and PC-3 cells treated for 24 h with DMSO (control) or the indicated concentrations of SFN. Bax protein was immunoprecipitated from equal amounts of lysate protein using anti-Bax monoclonal antibody 6A7, which recognizes activated Bax. Immunoprecipitated complexes were subjected to immunoblotting using polyclonal anti-Bax antibody. (D) Immunoblotting for Bcl-2 protein using lysates from LNCaP/neo and LNCaP/Bcl-2 cells following 24 h treatment with DMSO (control) or the indicated concentrations of SFN. The blots were stripped and re-probed with anti-actin antibody to ensure equal protein loading. (E) Cytoplasmic histone-associated DNA fragmentation in LNCaP/neo and LNCaP/Bcl-2 cells following 24 h treatment with DMSO (control) or the indicated concentrations of SFN. Data are mean ± SE (n = 3). *Significantly different (P < 0.05) compared with DMSO-treated control in respective cell line by one-way ANOVA followed by Dunnett’s test. The difference in DNA fragmentation between SFN-treated LNCaP/neo and LNCaP/Bcl-2 was statistically insignificant at both drug concentrations.

 
SFN treatment causes Bax activation in PC-3 and LNCaP cells
The Bax protein exists in an inactive form in the cytosol but changes conformation and translocates to the mitochondria upon apoptotic stimuli to trigger release of apoptogenic molecules including cytochrome c (31,32). We raised the question of whether differential sensitivity of LNCaP and PC-3 cells toward SFN-induced cell death was due to differences in Bax activation profile. We addressed this question by immunoprecipitation of Bax from lysates of control and SFN-treated PC-3 and LNCaP cells using a monoclonal antibody (6A7) that recognizes an epitope at the NH2 terminus of activated Bax followed by immunoblotting using polyclonal anti-Bax antibody. In time course experiments using 20 µM SFN, Bax activation compared with control was observed in both LNCaP and PC-3 cells at 16–24 h time points (Figure 3B). However, SFN-mediated Bax activation was relatively more pronounced in LNCaP cells than in PC-3 (Figure 3B). In dose-response experiments using LNCaP cells, Bax activation was evident following a 24 h treatment with 10, 20 and 40 µM SFN (Figure 3C). The SFN-mediated activation of Bax in PC-3 cells was observed only at 40 µM SFN concentration (Figure 3C). These results suggested that the differential sensitivity of LNCaP and PC-3 cells to apoptosis induction by SFN might, at least in part, be due to difference in Bax activation profile between these two cell lines.

Bcl-2 overexpression fails to protect against SFN-induced apoptosis in LNCaP cells
Next, we raised the question of whether downregulation of Bcl-2 contributed to SFN-induced apoptosis in LNCaP cells. We addressed this possibility by determining the effect of ectopic expression of Bcl-2, by transient transfection, on cell death caused by SFN. Overexpression of Bcl-2 in LNCaP cells transiently transfected with a Bcl-2 plasmid (LNCaP/Bcl-2) was confirmed by immunoblotting and the results are shown in Figure 3D. Similar to untransfected LNCaP cells (Figure 3A), SFN treatment (20 and 40 µM for 24 h) caused a marked reduction in the protein level of Bcl-2 in both LNCaP/Bcl-2 and vector transfected control LNCaP/neo cells (Figure 3D). Data on effect of Bcl-2 overexpression on SFN-induced cytoplasmic histone-associated DNA fragmentation are summarized in Figure 3E. Similar to untransfected LNCaP cells (Figure 1C) SFN treatment caused a dose-dependent and statistically significant increase (P < 0.05 by one-way ANOVA followed by Dunnett’s test) in cytoplasmic histone-associated DNA fragmentation in both LNCaP/neo and LNCaP/Bcl-2 cells compared with corresponding DMSO-treated control (Figure 3E). However, the DNA fragmentation resulting from a 24 h treatment with 20 or 40 µM SFN did not differ significantly between LNCaP/neo and LNCaP/Bcl-2 cells (P > 0.05 by paired t-test; Figure 3E). Collectively, these results indicated that SFN-induced apoptosis in LNCaP cells was not regulated by Bcl-2.

SFN downregulates IAP family proteins differentially between PC-3 and LNCaP cells
The IAP family proteins, including X-linked IAP (XIAP), cIAP1 and cIAP2, play an important role in regulation of apoptosis by binding to and inhibiting effector caspases (33–35). Because SFN treatment has been shown to cause activation of both mitochondria-mediated intrinsic caspase pathway and death receptor-mediated extrinsic caspase cascade (20,23), we raised the question of whether SFN-induced apoptosis in our model was regulated by IAP family proteins. We addressed this question by determining the effect of SFN treatment on levels of IAP family proteins by immunoblotting, and the results are summarized in Figure 4. As can be seen in Figure 4A, the levels of cIAP1, cIAP2 and XIAP proteins were reduced markedly on treatment of PC-3 cells with SFN, especially at 16 and 24 h time points. The SFN-mediated reduction in the protein levels of IAP family proteins, especially XIAP, in PC-3 cells was also observed at lower drug concentrations (Figure 4B). Interestingly, SFN treatment exhibited a biphasic response on levels of IAP family proteins in LNCaP cells with initial induction followed by a decline in their protein levels at 16 and 24 h time points (Figure 4C). Because the effect of SFN on XIAP protein was relatively more pronounced than cIAP1 or cIAP2 (e.g. dose-response data in Figure 4B), we explored the role of XIAP in regulation of SFN-induced apoptosis using HCT116 human colon cancer cells with genetic disruption of XIAP. The HCT116 derived XIAP–/– cells were relatively more sensitive to apoptosis induction by SFN compared with wild-type (XIAP+/+) cells as judged by DAPI assay following a 24 h (Figure 5A) or 36 h drug exposure (Figure 5B). Relative resistance of wild-type cells in comparison with XIAP–/– variant of HCT116 cells toward SFN-induced apoptosis was also evident in analysis of sub-diploid fraction (Figure 5C) and cytoplasmic histone-associated DNA fragmentation (Figure 5D). Collectively, these results indicated that the SFN-induced apoptosis was partially inhibited by XIAP protein.


Figure 4
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  		Fig. 4 Immunoblotting for cIAP1, cIAP2 and XIAP proteins using lysates from (A) PC-3 cells treated with 40 µM SFN for the indicated time periods, (B) PC-3 cells treated for 24 h with the indicated concentrations of SFN and (C) LNCaP cells treated with 40 µM SFN for the indicated time periods. The blots were stripped and re-probed with anti-actin antibody to ensure equal protein loading.

 


Figure 5
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  		Fig. 5 Quantification of apoptotic cells by DAPI assay in wild-type HCT116 cells and HCT116-derived XIAP-/- cells following (A) 24 h treatment or (B) 36 h treatment with DMSO (control) or the indicated concentrations of SFN. (C) Percentage of subdiploid fraction (apoptotic cells) in wild-type HCT116 and HCT116-derived XIAP–/– cultures following 24 h treatment with DMSO (control) or the indicated concentrations of SFN. (D) Quantification of cytoplasmic histone-associated DNA fragmentation in wild-type HCT116 cells and HCT116-derived XIAP–/– cells following 36 h treatment with DMSO (control) or the indicated concentrations of SFN. Data are mean ± SE (n = 3–6). *Significantly different (P < 0.05) compared with corresponding DMSO-treated control by one-way ANOVA followed by Dunnett’s test. !=Significantly different (P < 0.05) between wild-type HCT116 cells and XIAP–/– variant of HCT116 cells by paired t-test.

 
Effect of SFN treatment on NF{kappa}B localization and transcriptional activity
The expression of IAP family genes is regulated by the transcription factor NF{kappa}B (36–38), which is constitutively activated in a variety of cancer cell lines including prostate cancer cells (39–41). It was shown recently that SFN treatment inhibited transcriptional activity of NF{kappa}B in PC-3 cells (42), which explains our results on SFN-mediated downregulation of the IAP family proteins in this cell line (Figure 4A). To confirm inhibition of NF{kappa}B in PC-3 cells in our experimental conditions, we determined the effect of SFN treatment on nuclear translocation of p65-NF{kappa}B, which is necessary for its activation. Initially, we performed immunoblotting for p65-NF{kappa}B using nuclear fractions prepared from PC-3 cells treated with 40 µM SFN for different time periods. As can be seen in Figure 6A, SFN treatment caused a marked decrease in the nuclear level of p65-NF{kappa}B that was evident as early as 1 h after treatment. The SFN-mediated inhibition of nuclear translocation of p65-NF{kappa}B was evident even at 20 µM concentration (Figure 6B). The membranes were stripped and re-probed with anti-PARP and/or {alpha}-tubulin antibodies to ensure equal protein loading as well as to rule out cross-contamination of nuclear and cytoplasmic fractions (Figure 6B). Next, we performed immunocytochemistry to further examine the effect of SFN treatment on nuclear/cytoplasmic localization of p65-NF{kappa}B, and the results are shown in Figure 6C. In DMSO-treated control PC-3 cells, the p65-NF{kappa}B immunostaining was evident in both nucleus and cytoplasm. The p65-NF{kappa}B staining in a large fraction of SFN-treated PC-3 was restricted to the cytoplasm indicating inhibition of its nuclear translocation (Figure 6C). These results indicated that SFN inhibited nuclear translocation of NF{kappa}B in PC-3 cells.


Figure 6
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  		Fig. 6 (A) Immunoblotting for p65-NF{kappa}B using nuclear fraction prepared from PC-3 cells following treatment with 40 µM SFN for the indicated time periods. The blot was stripped and re-probed with anti-PARP antibody to ensure equal protein loading. (B) Immunoblotting for p65-NF{kappa}B using nuclear and cytoplasmic fractions prepared from PC-3 cells following treatment with DMSO (control) or 20 µM SFN for 16 h. The blot was stripped and re-probed with anti-PARP and anti-{alpha}-tubulin antibody to ensure equal protein loading as well as to rule out cross-contamination of nuclear and cytoplasmic fractions. (C) Immunocytochemical analysis for p65-NF{kappa}B localization in PC-3 cells following 16 h treatment with DMSO (control) or 40 µM SFN. Staining for p65-NF{kappa}B and DNA is indicated by green and blue fluorescence, respectively. (D) Transcriptional activity of NF{kappa}B in LNCaP cells following treatment with 20 or 40 µM SFN for the indicated time periods as determined by luciferase reporter gene assay. Data are mean ± SE (n = 3–4 except for the solvent controls and the 16 h time point at 40 µM SFN where n = 2). *Significantly different (P < 0.05) compared with control by one-way ANOVA followed by Dunnett’s test.

 
We were intrigued by our observations that SFN treatment initially caused induction of IAP family proteins in LNCaP cells (Figure 4C), which raised a possibility that SFN treatment might increase transcriptional activity of NF{kappa}B in this cell line. We explored this possibility by determining the effect of SFN treatment on transcriptional activity of NF{kappa}B using a luciferase reporter gene assay, and the results are shown in Figure 6D. Indeed, an increase in transcriptional activity of NF{kappa}B was observed in SFN-treated LNCaP cells. Similar to expression of IAP family proteins (Figure 4C), SFN exhibited a biphasic response on transcriptional activity of NF{kappa}B with initial activation followed by an inhibition at 24 h time point (Figure 6D). Thus, the PC-3 and LNCaP cells behave differently with respect to effect of SFN on transcriptional activity of NF{kappa}B.

SFN treatment causes E2F1-dependent induction of Apaf-1
Next, we determined the effect of SFN treatment on the level of Apaf-1 protein, which plays an important role in regulation of apoptosis (43–45). As can be seen in Figure 7A, SFN treatment caused an increase in Apaf-1 protein level in PC-3 cells in a time- and concentration-dependent manner. Studies have shown that Apaf-1 is a direct transcriptional target of p53 as well as E2F1 transcription factors (46). Because PC-3 cell line lacks functional p53 protein, we reasoned that SFN-mediated induction of Apaf-1 protein might be due to transcriptional activation of E2F1. We directly tested this possibility by determining the effect of SFN treatment on transcriptional activity of E2F1 by luciferase assay (47). As can be seen in Figure 7B, SFN treatment indeed caused an increase in E2F1-dependent luciferase activity that was statistically significant at 24 h time point compared with control. Similar to PC-3 cells, SFN-mediated induction of Apaf-1 protein expression was also observed in LNCaP cells (Figure 7C). Next, we tested the role of Apaf-1 in regulation of SFN-induced apoptosis using siRNA technology. As can be seen in Figure 8A, the level of Apaf-1 protein was reduced by >90% by transfection of PC-3 cells with Apaf-1 targeting siRNA. In addition, Apaf-1 knockdown significantly inhibited SFN-induced apoptosis in PC-3 (Figure 8B) as well as in LNCaP cells (results not shown). These results indicated that Apaf-1 protein plays an important role in regulation of SFN-induced apoptosis.


Figure 7
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  		Fig. 7 (A) Immunoblotting for Apaf-1 protein using lysates from PC-3 cells treated with the indicated concentrations of SFN for specified time periods. (B) Transcriptional activity of E2F1 in PC-3 cells treated with 20 or 40 µM SFN for the indicated time periods as determined by luciferase reporter gene assay. Data are mean ± SE (n = 4 except for the controls where n = 2). *Significantly different (P < 0.05) compared with control by one-way ANOVA followed by Dunnett’s test. (C) Immunoblotting for Apaf-1 using lysates from LNCaP cells treated with the indicated concentrations of SFN for specified time periods.

 


Figure 8
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  		Fig. 8 (A) Immunoblotting for Apaf-1 protein using lysates from PC-3 cells transiently transfected with control siRNA (lane 1) and Apaf-1 targeted siRNA (lane 2). (B) Cytoplasmic histone-associated DNA fragmentation in PC-3 cells transiently transfected with control siRNA and Apaf-1 targeted siRNA following 16 h treatment with DMSO (control) or 20 µM SFN. Data are mean ± SE (n = 3). aSignificantly different (P < 0.05) compared with corresponding DMSO-treated control, and bsignificantly different (P < 0.05) between SFN-treated control siRNA and Apaf-1 siRNA transfected PC-3 cells by one-way ANOVA followed by Bonferroni’s multiple comparison test.

 
The E2F1 transcription factor is a downstream target of Chk2 kinase (48), which is an intermediary of DNA damage checkpoint (49). Because our previous work indicated activation of Chk2 in SFN-treated PC-3 cells (21), we raised the question of whether SFN-mediated transcriptional activation of E2F1 was linked to Chk2 activation. We addressed this question using wild-type HCT116 cells and HCT116-derived Chk2–/– cells. Similar to PC-3 and LNCaP cells (Figure 7), SFN treatment resulted in induction of Apaf-1 protein in wild-type HCT116 cells (data not shown). Interestingly, SFN-mediated induction of Apaf-1 was also observed in HCT116 derived Chk2–/– cells (data not shown). Moreover, Chk2 deficiency did not confer protection against SFN-induced apoptosis as judged by DAPI assay or analysis of subdiploid fraction (data not shown). Consistent with these results, siRNA-based knockdown of Chk2 protein in PC-3 cells failed to confer protection against SFN-mediated induction of Apaf-1 protein (results not shown). These results indicated that SFN-mediated induction of Apaf-1 in our model was not regulated by Chk2.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study reveals that the synthetic SFN is modestly but statistically significantly more potent than L-SFN in reducing survival of LNCaP cells (Table I). Interestingly, previous studies have shown that SFN and L-SFN are equipotent as inducers of the quinone reductase activity in Hepa 1c1c7 cells (8). Even though the reasons for differential potency of SFN versus L-SFN against LNCaP cell proliferation remain to be elucidated, we focused on the synthetic compound to extend our previous observations (20–23) concerning the mechanism of SFN-induced cell death. We found that the LNCaP cell line, which expresses wild-type p53, is relatively more sensitive to apoptosis induction by SFN compared with p53 deficient PC-3 cells. Because p53 tumor suppressor protein plays an important role in regulation of apoptosis by different stimuli (25–27), we reasoned that the differential sensitivity of LNCaP and PC-3 cells toward SFN-induced cell death might be due to difference in p53 status between these two cell lines. Even though SFN treatment causes stabilization of p53 protein in LNCaP cells, p53 knockdown fails to confer protection against SFN-induced cell death. Thus, we conclude that the difference in p53 status cannot explain differential sensitivity of LNCaP and PC-3 cells toward SFN-induced cell death. The LNCaP cell line is androgen responsive whereas PC-3 cells are androgen-independent. It is possible that difference in androgen responsiveness between LNCaP and PC-3 cells may contribute to their differential sensitivity toward SFN-induced cell death. Further studies are needed to systematically explore this possibility.

The multidomain pro-apoptotic Bcl-2 family members Bax and Bak play an important role in regulation of apoptosis in different cellular systems (15,16,20,22,23,30,50,51). The Bax protein in normal cells exists in an inactive form mainly in the cytosol, but can be induced to change conformation and translocate to the mitochondria in response to certain apoptotic stimuli (31,32). Activated Bax oligomerizes on the outer mitochondrial membrane and induces release of apoptogenic molecules to the cytoplasm (31,32). Recent studies have indicated that microtubule damaging agents cause Bax activation to trigger the cell death (52). Because SFN was recently shown to disrupt tubulin polymerization (19), we hypothesized that apoptosis induction by this phytochemical may be linked to Bax activation. Indeed SFN treatment causes conformational change of Bax in both PC-3 and LNCaP cell lines (Figure 3). Interestingly, the SFN-mediated Bax activation is relatively more pronounced in LNCaP cells than in PC-3, which may partly explain the differential sensitivity of these cells to SFN.

Studies have indicated that SFN-induced apoptosis in different cellular systems is associated with downregulation of Bcl-2 and/or Bcl-xL proteins (15,16,20,42), which are inhibitors of the cell death process (28,29). Despite downregulation of Bcl-2 in SFN-treated LNCaP cells the SFN-induced apoptosis is not affected by overexpression of Bcl-2 (Figure 3E). Collectively, these results indicate that SFN-mediated downregulation of Bcl-2 is an effect rather than cause of cell death, at least in human prostate cancer cells.

The IAP family proteins have emerged as critical regulators of apoptosis and serve to inhibit activation of initiator as well as effectors caspases (33,34). We found that SFN treatment differentially modulates the levels of IAP family proteins between LNCaP and PC-3 cells. The SFN-treated PC-3 cells exhibit a decrease in the protein levels of cIAP1, cIAP2 and XIAP, whereas the effect of SFN on levels of IAP family proteins is biphasic in LNCaP cells with an initial induction followed by a decline in their protein levels (Figure 4). The expression of IAP family proteins is regulated by the transcription factor NF{kappa}B (36–38). The present study suggests that the difference in response of PC-3 and LNCaP cells to SFN-mediated modulation of IAP family proteins is probably attributable to the differential effect of SFN on transcriptional activity of NF{kappa}B. Previous studies have shown that the transcriptional activity of NF{kappa}B is inhibited in PC-3 cells on treatment with SFN (42). The present study supports this conclusion since nuclear translocation of p65-NF{kappa}B is inhibited on treatment of PC-3 cells with SFN (Figure 6). On the other hand, similar to the effect on IAP family protein levels, SFN exhibits a biphasic response with respect to effect on transcriptional activity of NF{kappa}B in LNCaP cells (Figure 6D). The luciferase reporter gene assay reveals activation followed by an inhibition in transcriptional activity of NF{kappa}B in SFN-treated LNCaP cells (Figure 6D). Even though further studies are needed to elucidate the mechanism for biphasic response of SFN on transcriptional activity of NF{kappa}B in LNCaP cells, it is possible that the differential response between PC-3 and LNCaP cells is due to differences in basal activity of NF{kappa}B. This transcription factor is constitutively active in PC-3 cells but its basal activity is barely detectable in LNCaP cells (Figure 6).

The Apaf-1 is essential for activation of caspase-9 in stress- and oncogene-induced apoptosis (44,45). Apaf-1 binds to cytochrome c, which is released by apoptotic stimuli and facilitates recruitment and activation of procaspase-9 in the apoptosome. We have shown previously that the SFN-induced apoptosis in PC-3 cells is associated with release of cytochrome c to the cytosol and activation of caspase-9 (23). Moreover, the cell death caused by SFN is inhibited significantly in the presence of pan caspase inhibitor zVAD-fmk as well as zLEHD-fmk, a specific inhibitor of caspase-9 (20). The present study provides experimental evidence to indicate that the SFN-induced cell death in both PC-3 and LNCaP cells is regulated by Apaf-1 induction that is accompanied by an increase in transcriptional activity of E2F1 as revealed by luciferase reporter assay. A role for Apaf-1 in regulation of SFN-induced apoptosis is further substantiated by our observations that Apaf-1 protein knockdown confers statistically significant protection against SFN-induced cell death in PC-3 (Figure 8) as well as in LNCaP cells (results not shown).

In conclusion, the present study demonstrates that (i) SFN is relatively more potent than L-SFN against proliferation of LNCaP cells, (ii) the p53 protein status has no effect on the cell death caused by SFN, at least in prostate cancer cells, (iii) the differential sensitivity of LNCaP and PC-3 cells towards cell killing by SFN may, at least in part, be due to differences in Bax activation profile between these cell lines, (iv) SFN treatment differentially modulates IAP family protein level in PC-3 and LNCaP cells which correlates with NF{kappa}B activity, (v) SFN treatment causes E2F1-dependent induction of Apaf-1 and (vi) Apaf-1 knockdown confers significant protection against SFN-induced apoptosis.


   Acknowledgments
 
This study was supported by NCI grants CA 115498 and CA101753. We thank Dr Fred Bunz and Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA) for generous gift of wild-type and Chk2–/– and XIAP–/– variants of HCT116 cells, Dr Anning Lin (University of Chicago, Chicago, IL, USA) for generous gift of NF{kappa}B luciferase reporter plasmid, Dr Stephen Safe (Texas A & M University, College Station, TX, USA) for generous gift of E2F1 luciferase reporter plasmid, and Dr Stanley Korsmeyer for pSFFV-neo and pSFFV-Bcl-2 plasmids.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Verhoeven D.T., Goldbohm R.A., van Poppel G., Verhagen H., van den Brandt P.A. (1996) Epidemiological studies on Brassica vegetables and cancer risk. Cancer Epidemiol. Biomarkers Prev. 5:733–748.[Abstract]
  2. Cohen J.H., Kristal A.R., Stanford J.L. (2000) Fruit and vegetable intakes and prostate cancer risk. J. Natl Cancer. Inst. 92:61–68.[Abstract/Free Full Text]
  3. Zhang S.M., Hunter D.J., Rosner B.A., Giovannucci E.L., Colditz G.A., Speizer F.E., Willett W.C. (2000) Intakes of fruits, vegetables, and related nutrients and the risk of non-Hodgkin’s lymphoma among women. Cancer Epidemiol. Biomarkers Prev. 9:477–485.[Abstract/Free Full Text]
  4. Ambrosone C.B., McCann S.E., Freudenheim J.L., Marshall J.R., Zhang Y., Shields P.G. (2004) Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. J. Nutr. 134:1134–1138.[Abstract/Free Full Text]
  5. Hecht S.S. (2000) Inhibition of carcinogenesis by isothiocyanates. Drug Metab. Rev. 32:395–411.[CrossRef][ISI][Medline]
  6. Talalay P. and Fahey J.W. (2001) Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J. Nutr. 131:(suppl.), 3027s–3033s.
  7. Fahey J.W., Zalcmann A.T., Talalay P. (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5–51.[CrossRef][ISI][Medline]
  8. Zhang Y., Talalay P., Cho C.G., Posner G.H. (1992) A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. Natl Acad. Sci. USA 89:2399–2403.[Abstract/Free Full Text]
  9. Brooks J.D., Paton V.G., Vidanes G. (2001) Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol. Biomarkers Prev. 10:949–954.[Abstract/Free Full Text]
  10. Barcelo S., Gardiner J.M., Gescher A., Chipman J.K. (1996) CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane. Carcinogenesis 17:277–282.[Abstract/Free Full Text]
  11. Zhang Y., Kensler T.W., Cho C.G., Posner G.H., Talalay P. (1994) Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc. Natl Acad. Sci. USA 91:3147–3150.[Abstract/Free Full Text]
  12. Fahey J.W., Zhang Y., Talalay P. (1997) Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl Acad. Sci. USA 94:10367–10372.[Abstract/Free Full Text]
  13. Chung F.L., Conaway C.C., Rao C.V., Reddy B.S. (2000) Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate. Carcinogenesis 21:2287–2291.[Abstract/Free Full Text]
  14. Fahey J.W., Haristoy X., Dolan P.M., Kensler T.W., Scholtus I., Stephenson K.K., Talalay P., Lozniewski A. (2002) Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proc. Natl Acad. Sci. USA 99:7610–7615.[Abstract/Free Full Text]
  15. Gamet-Payrastre L., Li P., Lumeau S., Cassar G., Dupont M.A., Chevolleau S., Gasc N., Tulliez J., Terce F. (2000) Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 60:1426–1433.[Abstract/Free Full Text]
  16. Fimognari C., Nüsse M., Cesari R., Iori R., Cantelli-Forti G., Hrelia P. (2002) Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane. Carcinogenesis 23:581–586.[Abstract/Free Full Text]
  17. Misiewicz I., Skupinska K., Kasprzycka-Guttman T. (2003) Sulforaphane and 2-oxohexyl isothiocyanate induce cell growth arrest and apoptosis in L-1210 leukemia and ME-18 melanoma cells. Oncol. Rep. 10:2045–2050.[ISI][Medline]
  18. Wang L., Liu D., Ahmed T., Chung F.L., Conaway C.C., Chiao J.W. (2004) Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention. Int. J. Oncol. 24:187–192.[ISI][Medline]
  19. Jackson S.J.T. and Singletary K.W. (2004) Sulforaphane: a naturally occurring mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization. Carcinogenesis 25:219–227.[Abstract/Free Full Text]
  20. Singh A.V., Xiao D., Lew K.L., Dhir R., Singh S.V. (2004) Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo. Carcinogenesis 25:83–90.[Abstract/Free Full Text]
  21. Singh S.V., Herman-Antosiewicz A., Singh A.V., Lew K.L., Srivastava S.K., Kamath R., Brown K.D., Zhang L., Baskaran R. (2004) Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2 mediated phosphorylation of Cdc25C. J. Biol. Chem. 279:25813–25822.[Abstract/Free Full Text]
  22. Choi S. and Singh S.V. (2005) Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable derived cancer chemopreventive agent. Cancer Res. 65:2035–2043.[Abstract/Free Full Text]
  23. Singh S.V., Srivastava S.K., Choi S., et al. (2005) Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species. J. Biol. Chem. 280:19911–19924.[Abstract/Free Full Text]
  24. Myzak M.C., Hardin K., Wang R., Dashwood R.H., Ho E. (2006) Sulforaphane inhibits histone deacetylase activity in BPH-1, LnCaP and PC-3 prostate epithelial cells. Carcinogenesis 27:811–819.[Abstract/Free Full Text]
  25. Agarwal M.L., Taylor W.R., Chernov M.V., Chernova O.B., Stark G.R. (1998) The p53 network. J. Biol. Chem. 273:1–4.[Free Full Text]
  26. Sheikh M.S. and Fornace A.J. (2000) Role of p53 family members in apoptosis. J. Cell. Physiol. 182:171–181.[CrossRef][ISI][Medline]
  27. Miyashita T. and Reed J.C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299.[CrossRef][ISI][Medline]
  28. Reed J.C. (1997) Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Semin. Hematol. 34:9–19.[ISI][Medline]
  29. Chao D.T. and Korsmeyer S.J. (1998) Bcl-2 family: regulators of cell death. Annu. Rev. Immunol. 16:395–419.[CrossRef][ISI][Medline]
  30. 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., Korsmeyer S.J. (2001) Proapoptotic Bax and Bak: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730.[Abstract/Free Full Text]
  31. Wolter K.G., Hsu Y.T., Smith C.L., Nechushtan A., Xi X.G., Youle R.J. (1997) Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139:1281–1292.[Abstract/Free Full Text]
  32. Yamaguchi H. and Wang H.G. (2001) The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene 20:7779–7786.[CrossRef][ISI][Medline]
  33. LaCasse E.C., Baird S., Korneluk R.G., MacKenzie A.E. (1998) The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17:3247–3259.[CrossRef][ISI][Medline]
  34. Deveraux Q.L. and Reed J.C. (1999) IAP family proteins—suppressors of apoptosis. Genes Dev. 13:239–252.[Free Full Text]
  35. Cummins J.M., Kohli M., Rago C., Kinzler K.W., Vogelstein B., Bunz F. (2004) X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res. 64:3006–3008.[Abstract/Free Full Text]
  36. Chu Z.L., McKinsey T.A., Liu L., Gentry J.J., Malim M.H., Ballard D.W. (1997) Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control. Proc. Natl Acad. Sci. USA 94:10057–10062.[Abstract/Free Full Text]
  37. You M., Ku P.T., Hrdlickova R., Bose H.R. (1997) ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the anti-apoptotic activity of the v-Rel oncoprotein. Mol. Cell. Biol. 17:7328–7341.[Abstract]
  38. Stehlik C., de Martin R., Kumabashiri I., Schmid J.A., Binder B.R., Lipp J. (1998) Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J. Exp. Med. 188:211–216.[Abstract/Free Full Text]
  39. Bargou R.C., Emmerich F., Krappmann D., et al. (1997) Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J. Clin. Invest. 100:2961–2969.[ISI][Medline]
  40. Shattuck-Brandt R.L. and Richmond A. (1997) Enhanced degradation of I-kappaB alpha contributes to endogenous activation of NF-kappaB in Hs294T melanoma cells. Cancer Res. 57:3032–3039.[Abstract/Free Full Text]
  41. Palayoor S.T., Youmell M.Y., Calderwood S.K., Coleman C.N., Price B.D. (1999) Constitutive activation of IkappaB kinase alpha and NF-kappaB in prostate cancer cells is inhibited by ibuprofen. Oncogene 18:7389–7394.[CrossRef][ISI][Medline]
  42. Xu C., Shen G., Chen C., Gelinas C., Kong A.N. (2005) Suppression of NF-kappaB and NF-kappaB-regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene 24:4486–4495.[CrossRef][ISI][Medline]
  43. Cecconi F., Alvarez-Bolado G., Meyer B.I., Roth K.A., Gruss P. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94:727–737.[CrossRef][ISI][Medline]
  44. Yoshida H., Kong Y., Yoshida R., Elia A.J., Hakem A., Hakem R., Penninger J.M., Mak T.W. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94:739–750.[CrossRef][ISI][Medline]
  45. Soengas M.S., Alarcón R.M., Yoshida H., Giaccia A.J., Hakem R., Mak T.W., Lowe S.W. (1999) Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 284:156–159.[Abstract/Free Full Text]
  46. Moroni M.C., Hickman E.S., Lazzerini-Denchi E., Caprara G., Colli E., Cecconi F., Müller H., Helin K. (2001) Apaf-1 is a transcriptional target for E2F and p53. Nature Cell Biol. 3:552–558.[CrossRef][ISI][Medline]
  47. Wang W., Dong L., Saville B., Safe S. (1999) Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Mol. Endocrinol. 13:1373–1387.