D, L-Sulforaphane was synthesized at the University of Agriculture (Kraków, Poland), according to the method by Schmidt and Karrer (18). Purification of the compound was performed by high-performance liquid chromatography and the identity of the SFN was verified by nuclear magnetic resonance and ultraviolet spectroscopy. A stock solution of SFN (7.2 M in serum-free medium) was stored at −20°C until further use. SFN was diluted with culture medium immediately prior to use. 3-MA, staurosporine (STS) and antibodies against GAPDH (G8795) were purchased from Sigma-Aldrich (Poznań, Poland). Trypsin (0.25%) was obtained from Gibco Life Technologies (Carlsbad, CA, USA), and (6-³H) thymidine was from GE Healthcare Life Sciences (Little Chalfont, UK). Rabbit antibodies against LC3B (3868) and beclin-1 (3495) were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against Bcl-2 (sc-783) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and an antibody against Bax (556467) was obtained from BD Pharmingen (San Diego, CA, USA). Secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (A0545) and anti-mouse IgG antibodies (A9044) were obtained from Sigma-Aldrich. The secondary anti-rabbit IgG HRP-linked antibodies (7074) were from Cell Signaling Technology. All of the other chemicals and reagents used in the experiments of the present study were of analytical grade.

The BE(2)-C human neuroblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA; ATCC number, CRL-2268). The BE(2)-C cell line is a clone of the SK-N-BE(2) neuroblastoma cell line. The cells were cultured in a 1:1 mixture of Eagle’s minimum essential medium and F12 medium, supplemented with 10% fetal calf serum, 1% non-essential amino acid solution, 1 mM sodium pyruvate and 50 μg/ml gentamicin (all from Sigma Aldrich) at 37°C in an atmosphere containing 5% CO₂.

The MTT assay is based on the ability of living cells to reduce the MTT tetrazolium salt to MTT formazan by the mitochondrial enzyme succinate dehydrogenase (19). The BE(2)-C cells (2.5×10⁴/well) were cultured in 96-well plates for 24 h and treated with increasing concentrations of synthetic SFN for the indicated time periods. MTT (Sigma-Aldrich) was added to each well to a final concentration of 0.5 mg/ml. Following a 4-h incubation at 37°C, the formazan crystals were dissolved in isopropanol containing 5 mM HCl. The absorbance was measured at wavelengths of 570 and 630 nm using the SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The 50% maximal inhibitory concentration (IC₅₀) was defined as the concentration that resulted in a 50% decrease in the absorbance of the SFN-treated cells, as compared with the media-grown cells. In separate experiments, the BE(2)-C cells (2.5×10⁴/well) were cultured in 96-well plates for 24 h. The medium was then removed and the cells were treated with either 40 μM synthetic SFN, 10 mM 3-MA or a combination of 40 μM synthetic SFN and 10 mM 3-MA. Untreated control cells were also included in the experiments. After 48 h, the MTT assay was performed on the cells, as described above. The measurements were run in triplicate and the experiments were repeated three times.

The BE(2)-C cells (2.5×10⁴/well) were cultured in 96-well plates for 24 h. The medium was then removed and the cells were treated with either 40 μM synthetic SFN, 10 mM 3-MA or a combination of 40 μM synthetic SFN and 10 mM 3-MA. Untreated control cells were also included in the experiments. After 48 h, the cell cultures were centrifuged at 250 × g for 10 min at room temperature. The medium was removed and the cells were lysed using 1% Triton-X 100 (Sigma-Aldrich) in serum-free culture medium. LDH activity was measured using the Cytotoxicity Detecion kit (LDH) from Roche Diagnostics (Warsaw, Poland) according to the manufacturer’s instructions. The measurements were run in triplicate and the experiments were repeated three times.

The BE(2)-C cells (2.5×10⁴/well) were cultured in 96-well plates for 24 h. The medium was then removed and the cells were treated with either 40 μM synthetic SFN, 10 mM 3-MA or a combination of 40 μM synthetic SFN and 10 mM 3-MA. Untreated control cells were also included in the experiments. After 48 h, the cells were collected and centrifuged at 250 × g for 10 min at room temperature. The culture medium was then removed, the cells were fixed with 0.1% crystal violet (Sigma-Aldrich) in 20% ethanol (Sigma-Aldrich) for 10 min and washed once with distilled water. The cells were observed using a Nikon Eclipse TS100 microscope (Nikon Corooration, Tokyo, Japan). Images of the cells were captured using a digital camera (Canon Powershot A1200; Canon, Inc., Tokyo, Japan).

A thymidine incorporation assay was performed in order to measure the rate of DNA synthesis. The BE(2)-C cells were seeded in a 24-well plate (1.6×10⁵ cells/well) and allowed to attach. After 24 h the medium was refreshed and the indicated concentrations of SFN (10-100 μM) were added. The cells treated with media alone were used as control cells. Following a 24-h incubation, the media was replaced with 400 μl isotope solution (activity, 1 μCurie/ml). The samples were prepared according to the methods by Riss and Sirbasku (20). After 24 h, the cells were fixed with 800 μl cold (4°C) methanol-acetic acid solution (3:1 proportion; POCH, Gliwice, Poland). The solution was then aspirated and each well was washed with 80% methanol and dried for 30 min at room temperature. A total of 125 μl trypsin was added to each well and incubated for 30 min at 37°C. Following incubation, 125 μl 1% SDS solution (Sigma-Aldrich) was added and plates were incubated for 30 min at 25°C. Finally, 20 μl of each sample was mixed with 3 ml scintillator carrier (Akwascynt, POCH). The measurements were performed using the 1211 Rackbeta Liquid Scintillation Counter (LKB Wallac; LKB Instruments, Victoria, Australia). Four cycles of measurements were performed and the first measurement was discarded. The results are presented as plots of counts per minute for 1 μg of whole-cell protein content. The protein concentration was determined using the bicinchoninic acid assay, according to the manufacturer’s instructions (Sigma-Aldrich). The measurements were run in triplicate and the experiment was repeated three times.

The activation of caspases 2, 3, 6, 7, 8, 9 and 10 was determined using the Homogeneous Caspases Assay (Roche Diagnostics) according to the manufacturer’s instructions. The BE(2)-C cells (4×10⁴) were seeded onto 96-well plates with black sides and a transparent, flat bottom (Corning-Costar, Corning, NY, USA), and allowed to attach. Following a 24-h incubation, the medium was replaced and the cells were treated with either the medium alone (control) or the indicated concentrations of SFN and/or 3-MA for the indicated periods of time. The samples were tested in duplicate, the fluorescent signals were measured using the Infinite^® 200 PRO multimode reader (Tecan Group Ltd, Maennedorf, Switzerland).

The BE(2)-C cells (1×10⁶) were grown on 55-mm diameter plates and treated with SFN and/or 3-MA, as described above. Whole cell extracts were obtained according to the methods of Sadowski et al (21). Briefly, following the indicated period of incubation, the media was removed and the cells were collected in cold phosphate-buffered saline (PBS), then washed with PBS and centrifuged at 140 × g for 5 min at 4°C. The samples were then resuspended in 70-100 μl lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM 3-[(3-cholami-dopropyl) dimethylammonio]-1-propanesulfonate, 2 mM EDTA, 1 mM Na₃VO₄, 5 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethanesulfonylfluoride; 10% glycerol) and incubated on ice for 20 min. All reagents were purchased from BioShop Canada, Inc., (Burlington, ON, Canada). The lysates were aspirated through a syringe (0.6-mm diameter) and centrifuged at 11,400 × g for 5 min at 4°C. The supernatants were collected and stored at −20°C until further use. The protein lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Hybond P; Merck Millipore, Warsaw, Poland). The membranes were treated with a solution containing 10 mM Tris (pH 7.4; BioShop Canada), 150 mM NaCl (POCH), 0.05% Tween^® 20 (Sigma-Aldrich) and 5% nonfat dry milk (Cell Signalling Technology, Inc.) and incubated with the desired primary antibody for 1 h at room temperature. Antibodies, raised against human antigens, included rabbit monoclonal anti-beclin-1 antibody (1:1,000) and rabbit monoclonal anti-LC3B antibody (1:1,000), from Cell Signaling Technology, Inc. Rabbit polyclonal anti-Bcl-2 antibody (DC 21, 1:1,000; sc-783) and mouse monoclonal anti-Bax antibody (B-9, 1:1,000; sc-7480) from Santa Cruz Biotechnology, Inc. and mouse monoclonal anti-GAPDH antibody G8795 (1:40,000) from Sigma-Aldrich. Following incubation with these primary antibodies, the membranes were washed three times for 5 min with the solution containing 10 mM Tris (pH 7.4), 150 mM NaCl with 0.1 % Tween® 20. After the washing steps, the membranes were treated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. The secondary antibodies with indicated dilutions were as follows: rabbit anti-mouse IgG-HRP, A9044, 1:50,000 (Sigma-Aldrich); goat anti-rabbit IgG-HRP, A0545, 1:20,000 (Sigma-Aldrich); goat anti-rabbit IgG-HRP, #7074, 1:2,000 (Cell Signaling Technology). After secondary antibodies incubation, membranes were washed as described above. The immunoreactive bands were visualized using an Enhanced Chemiluminescence substrate (Immobilon Western HRP substrate; Merck Millipore), according to the manufacturer’s instructions. The intensity of the immunoreactive bands was determined by densitometric scanning in order to quantify the changes to the protein expression levels (Quest Spot Cutter, Quantity One, version 4.6.9 software; Bio-Rad Laboratories, Hercules, CA, USA). The experiment was repeated between two and four times and representative results are shown.

To measure the mitochondrial membrane potential (∆ψ_m) in the neuroblastoma cells, flow cytometric analyses were performed using the MitoTracker^® Red CMXRos dye (Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were plated and treated as described above. Cells treated with 60 μM STS, an unspecific protein kinase inhibitor, were used as a positive control. The CMXRos staining protocol was performed as follows. The BE(2)-C cells were collected, centrifuged at 250 × g for 10 min at 4°C and stained with 1 ml 500 nM CMXRos solution per 1 ml cell suspension [in medium with 10% fetal bovine serum (FBS), 15 min, 37°C]. The cells were then washed twice with media supplemented with 2% FBS and resuspended in 0.5 ml media. The cells were analyzed using a FACScan (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated three times.

Values are presented as the means. Error bars of the means in Figs. 1B and C and 2A were calculated according to the law of propagation of uncertainty. Otherwise, the error bars denote the standard error of the mean. Experiments were repeated between two and four times. A series of pairwise tests (t-test) comparing the differences between the means of the groups, were also performed. This was calculated using Microsoft Office Excel 2003; Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference (^*P<0.05; ^**P<0.01; ^***P<0.001).

In order to determine the inhibitory influence of SFN on the BE(2)-C neuroblastoma cell line, the cells were cultured with increasing concentrations of the synthetic isothiocyanate (5-100 μM) for 24 and 48 h. Untreated cells were also included. The IC₅₀-value after 24 h was calculated as 38 μM (data not shown). Therefore, based on earlier assays and previously published results (8,22), 40 μM SFN was subsequently selected for further experiments. Microscopic evaluation (data not shown) and crystal violet staining of the SFN-treated cells suggested that SFN treatment caused cell death and after 48 h, the cells began to detach from the culture dish (Fig. 1A). Combinatorial treatment of the cells with SFN with 3-MA resulted in a marked detachment of cultured cells. Treatment with 3-MA alone did not alter the morphology of the cells. To assess the cytotoxicity of SFN on the cells, the cell viability was analyzed using an MTT assay (Fig. 1B). The inhibitory effects of SFN were clearly visible at 48 h. Treatment of the BE(2)-C cells with 40 μM SFN also resulted in decreased cellular levels of LDH, a cytosolic enzyme that leaks from damaged cells (Fig. 1C). Treatment with 3-MA slightly decreased the viability of the cells, as estimated by MTT assay, crystal violet staining and analysis of LDH activity (Fig. 1). However, the combined treatment with SFN and 3-MA resulted in a statistically significant decrease in the cellular parameters measured as compared with SFN treatment alone (Fig. 1B and C; P<0.05 and P<0.01, respectively).

Purified broccoli sprout extracts containing ~90% SFN, used in our preliminary studies, have also shown potent inhibitory effects on the growth of numerous human cancer cell lines, including BE(2)-C neuroblastoma, HepG2 hepatoma and SK-MEL-28 melanoma (data not shown).

To investigate whether apoptosis may be responsible for the decreased survival of the BE(2)-C cells treated with SFN, total activity of caspases 2, 3, 6, 7, 8, 9 and 10 was assayed. The cells (4×10⁴ per well) were cultured in the presence of 40 μM SFN for 48 h. Treatment with SFN resulted in a significant ~1.5-fold increase in the total activity of caspases 2, 3, 6, 7, 8, 9, and 10 (Fig. 2A; P<0.05) compared with the control cells. Furthermore, treatment with autophagy inhibitor 3-MA (10 mM) increased caspase activity when used alone (Fig. 2A). This effect was expected, as the inhibition of autophagy may be associated with increased apoptotic cell death (23). However, treatment with a combination of 3-MA and 40 μM SFN resulted in a significant decrease in caspase activity, possibly due to prolonged incubation with high doses of SFN and 3-MA (Fig. 2A; P<0.01), compared with the control cells and to single agents treatments. As the exact types of caspases involved in the process cannot be distinguished by the test used in the present study, it is possible that caspases of the receptor and mitochondrial pathways are involved in SFN-induced apoptosis (7).

The higher concentrations of SFN also inhibited DNA synthesis. The effects of SFN on its ability to repress ³H-thymidine incorporation into DNA were studied at four different concentrations (10, 50, 75 and 100 μM). The rate of ³H-thymidine incorporation decreased significantly with increasing SFN concentration (Fig. 2B; P<0.001) compared with the control cells. This result is concordant with previous findings reported by Rajendran et al (10), which showed that SFN may cause a severe decrease in DNA content in HCT116 cells treated with 15 μM SFN for 24 h.

To investigate whether autophagy may be induced by SFN treatment in the BE(2)-C cells, the proteolytic process of MAP LC3 (in short LC3) was assessed. The conversion from the endogenous, cytosolic LC3-I protein to the LC3-II protein, which binds to autophagosomal membranes, was detected by immunoblotting with antibodies against LC3 (13,15). These results may provide evidence of early autophagy induction. The cells were treated with 40 μM SFN, 10 mM 3-MA or the two agents simultaneously for 6, 24 and 48 h, and the isolated whole cells extracts were subjected to western blot analysis (Fig. 3). Treatment with SFN led to increased protein expression levels of LC3-I and II at 6, 24 and 48 h (P<0.05) compared with the SFN-treated cells compared with the control cells. These results suggested the involvement of the autophagic process, since the amount of LC3-II usually correlates well with the number of autophagosomes (16). Simultaneous treatment of SFN with 3-MA also resulted in increased expression levels of the two forms of LC3, compared with the control cells, from as early as 6 h and the tendency remained unchanged up to 48 h (Fig. 3A and C; P<0.05). 3-MA treatment alone was used as a control and caused a moderate change in the LC3 protein expression pattern, suggesting that autophagy induction was almost completely blocked. In addition, microscopic observation of the SFN and 3-MA treated cells suggested that the treatment caused cell death and after 48 h the cells began to detach from the culture dish (Fig. 1A).

In order to further investigate the mechanisms of the observed cell death, the BE(2)-C cells were treated with SFN and 3-MA and the protein expression levels of the antiapoptotic gene Bcl-2 were measured (Fig. 4). After 6, 24 and 48 h of SFN treatment, the protein expression levels of Bcl-2 remained unchanged. Combined treatment of SFN with 3-MA decreased the protein expression levels of Bcl-2 as compared with SFN treatment alone (Fig. 4A). Treatment with 3-MA alone decreased the Bcl-2 protein expression levels, particularly at 48 h; however this change was not statistically significant. Furthermore, the protein expression levels of another autophagic factor, beclin-1, were measured and shown to remain unchanged following treatment with the agents tested (Fig. 4B). In addition, Bax protein expression levels were evaluated by immunoblotting and increased levels were observed at 6, 24 and 48 h of SFN treatment (Fig. 4C; P<0.05). Changes in the Bax protein levels were statistically significant at 24 h, suggesting the induction of apoptosis.

Flow cytometry was used to measure changes to the mitochondrial membrane potential upon treatment with SFN. CMXRos served as a dye, which actively accumulates in the mitochondria of live cells. Control cells with functional mitochondrial membranes had a high accumulation of CMXRos (Fig. 5A; channel P3). The positive control apoptotic cells had a significant loss in mitochondrial membrane potential and were obtained following treatment with 60 μM STS, a known proapoptotic compound, for 24 h (channel P5). As compared with STS treatment, treatment with 40 μM SFN also caused a significant decrease in the mitochondrial membrane potential (Fig. 5A). Treatment with 3-MA alone caused only a minimal decrease in mitochondrial membrane potential, but did not alter the effects of SFN.

In addition, the cells were cultured for 3, 6 and 24 h in the presence of 40 μM SFN, 10 mM 3-MA or 60 μM STS. No effect was observed on the mitochondrial membrane depolarization in response to treatment with any of the compounds at 3 h (Fig. 5B), whereas an increase in depolarization was observed in response to STS treatment for 6 h. A significant increase in the number of cells with depolarized mitochondrial membranes was observed in response to treatment with SFN for 24 h and was comparable with the increase caused by STS and combined treatment of SFN with 3-MA. Treatment with 3-MA alone did not affect mitochondrial membrane depolarization.