Abstract
Purpose
D,L-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo efficacy against prostate cancer in experimental rodents. This study was undertaken to determine the role of vimentin and plasminogen activator inhibitor-1 (PAI-1) in anti-cancer effects of SFN.
Methods
Effect of SFN on levels of different proteins was determined by western blotting or immunofluorescence microscopy. RNA interference of vimentin and PAI-1 was achieved by transient transfection. Apoptosis was quantified by flow cytometry. Transwell chambers were used to determine cell migration.
Results
Exposure of PC-3 and DU145 human prostate cancer cells to SFN resulted in induction of vimentin protein, which was accompanied by down-regulation of E-cadherin protein expression. The SFN-mediated induction of vimentin was also observed in a normal human prostate epithelial cell line. RNA interference of vimentin did not have any appreciable effect on early or late apoptosis resulting from SFN exposure. On the other hand, SFN-mediated inhibition of PC-3 and DU145 cell migration was significantly augmented by knockdown of the vimentin protein. Knockdown of vimentin itself was inhibitory against cell migration. The SFN-treated cells also exhibited induction of PAI-1, which is an endogenous inhibitor of urokinase-type plasminogen activator system. Similar to vimentin, PAI-1 knockdown resulted in a modest augmentation of PC-3 cell migration inhibition by SFN. Tumors from SFN-treated Transgenic Adenocarcinoma of Mouse Prostate mice showed a 1.7-fold increase in vimentin protein level compared with control tumors.
Conclusion
The present study indicates that vimentin and PAI-1 inductions confer modest protection against SFN-mediated inhibition of prostate cancer cell migration.
Keywords: Sulforaphane, Vimentin, Prostate Cancer, Chemoprevention
Introduction
D,L-Sulforaphane (SFN) is a promising chemopreventive agent with in vivo activity in rodent cancer models [1, 2]. SFN occurs naturally as L-isomer in edible cruciferous vegetables such as broccoli [3]. Research interest in anti-cancer effects of SFN and other structurally-related small molecules (eg, phenethyl isothiocyanate) was initially sparked by data from population-based case-control studies suggesting an inverse association between dietary intake of cruciferous vegetables and the risk of different cancers including cancer of the prostate [1, 4, 5]. Chemopreventive effect of SFN was first documented against 9,10-dimethyl-1,2- benzanthracene-induced breast cancer in rats [6]. Cancer chemopreventive efficacy of SFN was subsequently extended to other chemical carcinogens [7, 8]. For example, SFN administration (both pre- and post-initiation) resulted in suppression of azoxymethane-induced colonic aberrant crypt foci in rats [7]. Previous studies from our laboratory have shown that oral administration of 6 μmol SFN (three times per week) inhibited incidence and burden of prostatic intraepithelial neoplasia and/or well-differentiated prostate cancer as well as pulmonary metastasis multiplicity in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without causing any side effects [9]. Consistent with these data, TRAMP mice fed with 240 mg of broccoli sprouts/day exhibited a significant decrease in prostate tumor growth in another study [10]. We have also shown previously that growth of PC-3 human prostate cancer cells subcutaneously implanted in male athymic mice is retarded significantly after oral treatment with SFN [11].
Cellular systems, including prostate cancer cells, have been utilized to elucidate the mechanisms underlying anti-cancer effects of SFN. Mechanisms potentially contributing to SFN-mediated inhibition of pre-initiation and post-initiation cancer development include inhibition of CYP2E1, cell cycle arrest, apoptosis induction, suppression of angiogenesis, inhibition of histone deacetylase, and epigenetic repression of human telomerase reverse transcriptase [12–19]. SFN is capable of inhibiting various oncogenic pathways, including nuclear factor-κB (NF-κB), androgen receptor, and signal transducer and activator of transcription 3 [20–23]. Interestingly, SFN treatment causes activation of Notch signaling in human prostate cancer cells, but Notch activation is largely dispensable for cellular effects of SFN [24].
Previous studies from our laboratory have indicated that benzyl isothiocyanate, which is a structural analogue of SFN, is a potent inhibitor of epithelial-mesenchymal transition (EMT) in human breast cancer cells [25]. Because EMT is associated with aggressiveness of cancers [26], the present study was undertaken to determine whether anti-cancer effect of SFN in prostate cancer cells involves inhibition of the EMT phenotype.
Materials and methods
Ethics statement
Tumor tissues from our published study [9] were used to determine the effect of SFN on expression of vimentin and PAI-1 proteins in vivo. Use of mice and their care was in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee guidelines.
Reagents
Stock solution of SFN was prepared in dimethyl sulfoxide (DMSO), stored at −20°C, and diluted with fresh complete media immediately before use. Same volume of DMSO (final concentration 0.1%) was added to the controls. Cell culture reagents including fetal bovine serum, antibiotics mixture, phosphate-buffered saline (PBS), and trypsin were purchased from Invitrogen-Life Technologies (Grand Island, NY). Antibodies against vimentin and actin were purchased from Sigma-Aldrich (St Louis, MO). An antibody against E-cadherin was from BD Biosciences (San Diego, CA), whereas antibodies against snail, slug, Zeb1, and plasminogen activator inhibitor-1 (PAI-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Vimentin and PAI-1 targeted small-interfering RNA (siRNA), and anti-vimentin antibody used for immunofluorescence microscopy were from Santa Cruz Biotechnology. A kit for flow cytometric analysis of apoptosis was from BD Biosciences. Transwell Permeable chambers used for cell migration assay were purchased from Corning (Corning, NY).
Cell lines
PC-3 and DU145 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to the supplier’s instructions. A normal human prostate stromal cell line (PrSC) and a normal human prostate epithelial cell line (PrEC) were purchased from Lonza (Walkersville, MD) and cultured according to provider’s recommendations.
Western blotting
After treatment, cells were collected and lysed as described by us previously [27]. The western blotting experiments for each protein were performed using independently prepared lysates from 3 (PC-3) or 2 (DU145, PrEC, PrSC) different experiments. Actin normalization was done for each individual experiment. Immunoreactive bands were visualized using enhanced chemiluminescence method [27]. Actin bands are the same for some western blots due to multiplexing (stripping and reprobing). Densitometric quantitation was done using UN-SCAN-IT software version 5.1 (Silk Scientific Corporation, Orem, UT).
Immunofluorescence microscopy for E-cadherin and vimentin
Cells (0.5 × 104 cells/ml) were cultured on coverslips in 12-well plates, allowed to attach by overnight incubation, and then exposed to DMSO (control), or SFN (10 or 20 μM) for 8 or 24 h. The cells were fixed with 2% paraformaldehyde for 1 h, permeabilized with PBS containing 0.05% Triton X-100 for 10 min, and blocked with 0.5% bovine serum albumin in PBS for 1 h at room temperature. Cells were incubated overnight at 4°C or at room temperature for 1 hour with anti-E-cadherin or anti-vimentin antibody, washed with PBS, and incubated with Alexa Fluor 568-conjugated secondary antibody for 1 h at room temperature. Nuclei were stained with 4′,6 diamidino-2-phenylindole (200 μg/ml; 10 min at room temperature). Cells were washed twice with PBS, mounted, and examined under a Leica DC300F fluorescence microscope. Images were captured at 100 × magnification.
RNA interference
PC-3 or DU145 cells (1 × 105 cells/well) were seeded in 6-well plates 1 day before transfection. The cells were washed thrice with serum/growth factor-free OptiMEM (Invitrogen-Life Technologies) and then transfected with 200 nM vimentin-targeted siRNA or control siRNA using OligofectAMINE. For RNA interference of PAI-1, cells were transfected using 100 nM siRNA. Forty-eight hours after transfection, cells were treated with DMSO or SFN for an additional 24 h. Cells were collected and processed for immunoblotting, apoptosis assay or migration assay.
Detection of apoptosis
Cells were treated with DMSO or SFN for 24 h, harvested by trypsinization, and washed with PBS. Cells (1 × 105) were then suspended in 100 μl of binding buffer, and stained with 4 μl Annexin V and 2 μl propidium iodide (PI) solution for 15 min at room temperature in dark. Samples were then diluted with 200 μl of binding buffer. Stained cells were analyzed using a CyAn ADP Analyzer (Beckman Coulter, Fullerton, CA).
Cell migration assay
Cell migration was determined as described previously [24]. The migrated cells on the bottom face of the membrane were fixed with methanol followed by staining with hematoxylin and eosin. Four randomly selected fields were examined under a microscope at 10 × magnification.
Statistical analysis
Each numeric value is presented as mean ± SD. Statistical significance of difference was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Results
SFN treatment caused vimentin induction in prostate cancer cells
Initial objective of this study was to determine if SFN treatment inhibits EMT in human prostate cancer cells. Induction of vimentin concomitant with suppression of E-cadherin expression is a biochemical hallmark of EMT [26]. Effect of SFN treatment on levels of EMT-related proteins was determined by western blotting, and the results are shown in Figure 1a. Exposure of PC-3 and DU145 human prostate cancer cells to 10 and 20 μM SFN resulted in induction of vimentin protein (Fig. 1b). Immunoblotting for vimentin revealed a doublet in both PC-3 and DU145 cells possibly due to post-translational modification of the protein. The SFN-mediated induction of vimentin was accompanied by suppression of E-cadherin protein level especially at the 24 h time point in PC-3 cells and 16 and 24 h time points in DU145 cells (Fig. 1a,b). Moreover, an increase in protein levels of E-cadherin transcriptional repressors snail, slug, and Zeb1 was also observed in SFN-treated PC-3 and DU145 cells (Fig. 2a).
Next we raised the question of whether SFN-mediated induction of vimentin was unique to cancer cells. We addressed this question using a normal human prostate epithelial cell line (PrEC) as well as a normal human prostate stromal cell line (PrSC). As can be seen in Figure 2b and c, basal expression of E-cadherin was detectable in PrEC cells, whereas vimentin protein was expressed in both cells. E-cadherin protein expression was not detectable in the PrSC cell line. Similar to prostate cancer cells, however, SFN treatment resulted in induction of vimentin protein level in PrEC cells (Fig. 2b,c). On the other hand, SFN-mediated induction of vimentin was either modest or not observed in the PrSC cell line (Fig. 2b,c). These results indicated that normal and cancerous epithelial cells were sensitive to SFN-mediated induction of vimentin.
Immunofluorescence microscopy was performed using PC-3 and DU145 cells to confirm SFN-mediated changes in E-cadherin and vimentin proteins. In agreement with the results of immunoblotting, SFN-treated PC-3 (Fig. 3a) and DU145 cells (Fig. 3b) exhibited down-regulation of E-cadherin protein but induction of vimentin. Collectively, these results indicated induction of vimentin after treatment with SFN in prostate epithelial cells.
Effect of vimentin knockdown on apoptosis induction by SFN
We next performed RNA interference studies to determine the functional significance of vimentin induction in anti-cancer effects of SFN. Initially, we performed a dose-response study using 100, 200, and 300 nM vimentin-targeted siRNA to optimize the conditions for knockdown. Knockdown was partial but comparable with 200 and 300 nM siRNA (results not shown). The cells were transfected with 200 nM siRNA in follow-up experiments. Protein level of vimentin was decreased by about 60% upon transient transfection of PC-3 cells with a vimentin-targeted siRNA in comparison with cells transfected with a control (non-specific) siRNA (Fig. 4a). In control siRNA transfected PC-3 cells, SFN treatment resulted in a dose-dependent increase in early (Fig. 4b) as well as late apoptosis (Fig. 4c). The SFN-induced apoptosis was maintained even after RNA interference of vimentin. The possibility that lack of an effect of vimentin RNA interference on SFN-induced apoptosis is due to incomplete knockdown can’t be excluded. Because vimentin induction did not have any meaningful impact on early or late apoptosis resulting from SFN exposure in PC-3 cells, these experiments were not performed in the DU145 cell line.
Vimentin knockdown modestly augmented SFN-mediated inhibition of cancer cell migration
Because vimentin protein is implicated in cancer cell motility [28], we questioned whether SFN-mediated inhibition of prostate cancer cell migration was affected by vimentin protein. Figure 5a shows microscopic images for PC-3 and DU145 cell migration after knockdown of vimentin protein and/or treatment with SFN. The PC-3 and DU145 cell migration was decreased significantly after 24 h treatment with 10 μM SFN (Fig. 5b). Vimentin protein knockdown itself was inhibitory against PC-3 and DU145 cell migration. Furthermore, the SFN-mediated inhibition of prostate cancer cell migration was modestly but significantly augmented by RNA interference of vimentin in both cell lines (Fig. 5b). These results indicated that vimentin induction by SFN conferred modest protection against in vitro inhibition of prostate cancer cell migration by this agent.
SFN treatment caused induction of PAI-1 in human prostate cancer cells
PAI-1 is an endogenous inhibitor of urokinase-type plasminogen activator system, which constitutes an integral component of extracellular matrix proteolysis and cell-extracellular matrix interaction as well as cell signaling involving receptor tyrosine kinases [29, 30]. Effect of SFN treatment on protein level of urokinase-type plasminogen activator or its receptor was not consistent (results not shown). On the other hand, SFN treatment resulted in an increase in PAI-1 protein levels in both PC-3 and DU145 cells (Fig. 6a,b). The SFN-mediated induction of PAI-1 was, however, variable between PC-3 and DU145 cells. For example, induction of PAI-1 protein level resulting from SFN exposure was observed at 16, and 24 h time points in PC-3 cells (Fig. 6a,b). On the other hand, SFN-mediated induction of PAI-1 protein was robust only at the 24 h time point in DU145 cells. The reasons for the cell line-specific differences in induction of PAI-1 by SFN treatment are not yet clear. We next proceeded with RNA interference studies to determine functional significance of SFN-mediated induction of PAI-1 protein. The level of PAI-1 protein was decreased between 70–90% upon its RNA interference (Fig. 6c). Similar to vimentin, knockdown of PAI-1 protein significantly inhibited PC-3 and DU145 cell migration (Fig. 6d). In addition, the SFN-mediated inhibition of cell migration was modestly but significantly augmented by RNA interference of PAI-1 at least in the PC-3 cell line. These results indicated that PAI-1 induction also conferred modest protection against SFN-mediated inhibition of PC-3 cell migration.
SFN administration caused induction of vimentin protein in TRAMP tumor
We have shown previously that SFN administration inhibits prostate cancer development in TRAMP mice [9]. We used tumor tissues from this study to determine the in vivo effect of SFN treatment on vimentin and PAI-1 protein expression. Mean intensity for vimentin protein was about 70% higher in the tumors from SFN-treated TRAMP mice compared with control mice (Fig. 7a). On the other hand, SFN-mediated induction of PAI-1 in vivo was not observed at least in the TRAMP tumors (Fig. 7b).
Discussion
The present study reveals that SFN is a potent inducer of vimentin in human prostate cancer cells. Vimentin is a major constituent of the intermediate filament system and responsible for maintenance of cellular integrity [28]. The SFN-mediated induction of vimentin is not confined to prostate cancer cells as this effect is also observed in a normal human prostate epithelial cell line (PrEC). Moreover, the SFN-mediated prevention of prostate cancer in TRAMP mice is associated with induction of vimentin (present study). Several transcription factors are implicated in regulation of vimentin, including nuclear NF-κB, AP-1, PEA3, and Sp1, and β-catenin [31–34]. Even though further work is needed to determine the mechanism underlying SFN-mediated induction of vimentin, this effect is probably not mediated by NF-κB as this transcription factor is inhibited by SFN treatment in prostate cancer cells [20].
Vimentin is overexpressed in various epithelial cancers including prostate cancer [28]. In prostate cancer, vimentin is mainly detectable in poorly-differentiated cancers and bone metastases [35, 36]. On the other hand, vimentin expression is largely undetectable in well- or moderately-differentiated tumors [35, 36]. Vimentin expression is associated with motility in prostate cancer cell lines [35]. Consistent with literature data [35, 36], knockdown of vimentin itself is inhibitory against cell migration in both PC-3 and DU145 cells. We also provide evidence to indicate that vimentin induction by SFN modestly impedes its inhibitory effect on cell migration. The precise mechanism by which vimentin protein knockdown augments SFN-mediated inhibition of prostate cancer cell migration is elusive but may involve Scribble. This protein is involved in cell migration, and protected from proteasomal degradation upon interaction with vimentin [37]. It has been suggested that vimentin upregulation during EMT leads to stabilization of Scribble thereby promoting cell migration [37].
Another interesting observation of the present study is that SFN treatment causes induction of PAI-1, which is an endogenous inhibitor of the urokinase-type plasminogen activator system [29, 30]. Similar to vimentin, PAI-1 knockdown itself inhibits PC-3 and DU145 cell migration. The SFN-mediated inhibition of cell migration is modestly augmented by knockdown of PAI-1 protein at least in PC-3 cells. The role of PAI-1 in prostate cancer is somewhat fuzzy based on literature review. For example, overexpression of PAI-1 was shown to be associated with adverse pathologic characteristics as well as higher overall risk of aggressive disease recurrence in men with localized prostate cancer treated with radical prostatectomy [38]. On the other hand, overexpression of PAI-1 was shown to inhibit PC-3 cell growth in nude mice through endothelial apoptosis in the newly established tumor vasculature [39]. Similarly, exogenously applied PAI-1 inhibited angiogenesis in vitro and retarded LNCaP tumor growth in severe-combined immune deficient mice [40]. Nevertheless, the present study suggests that PAI-1 induction confers a modest protection against cell migration inhibition by SFN.
Acknowledgments
This study was supported by the grant CA115498-07, awarded by the National Cancer Institute. This research project used the Flow Cytometry Facility supported in part by a grant from the National Cancer Institute at the National Institutes of Health (P30 CA047904).
Footnotes
Conflict of interest: None.
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