Sulforaphane was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and dissolved at a concentration of 100 mM in dimethyl sulfoxide (DMSO) as a stock solution, which was stored −20°C. The stock solution was diluted with cell culture medium to the desired concentration prior to use. The maximum concentration of DMSO did not exceed 0.1% (v/v), a concentration at which DMSO did not influence cell growth (data not shown). The selective proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide (CHX) were purchased from Sigma-Aldrich.

HCT116 human colon cancer cells and AGS human gastric cancer cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI-1640 medium (Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO₂ at 37°C. The RPMI-1640 medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 2 mM glutamine (Sigma-Aldrich), 100 U/ml penicillin (Hyclone), and 100 μg/ml streptomycin (Hyclone).

Experiments to investigate the effects of hypoxia were carried out in the hypoxia chamber of an anaerobic system (Thormo, Marietta, OH, USA). The hypoxic condition was 1% O₂ and 5% CO₂. The temperature was maintained at 37°C. The normoxic condition was 21% O₂ and 5% CO₂ (in a standard CO₂ incubator). For hypoxia experiments, HCT116 and AGS cells were grown to 50% confluence in a standard CO₂ incubator at 37°C. Twenty-four hours prior to the experiment, cell culture media were placed in normoxic and hypoxic chambers to allow equilibration. Immediately before each experiment, cell culture media were withdrawn from HCT116 and AGS cells and replaced with fresh media that were equilibrated to normoxic and hypoxic conditions for 24 h.

Cell survival was quantified by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasolium bromide; Sigma-Aldrich) assay to measure mitochondrial activity in viable cells. Cells seeded at a density of 1×10⁵ per well were allowed to adhere overnight, after which the culture media were replaced with fresh media. Cells were exposed to sulforaphane at concentrations of 12.5, 25 and 50 μM for 6 h under normoxic and hypoxic conditions. The control groups were treated with DMSO equal to the highest percentage (<0.1%) used in the experimental conditions for the MTT assay. After 6 h, the medium was replaced. MTT was freshly prepared at a concentration of 5 mg/ml in PBS and passed through a filter (pore size, 0.2 μm). An aliquot of 2 ml of MTT stock solution was added to each well and the plate was incubated at 37°C for 4 h in a humidified 5% CO₂ atmosphere. After 2 h, media were removed. To each well, 2 ml of DMSO was added in order to solubilize the formazan crystals, which were measured after 10 min. The optical density of each well was measured with a spectrophotometer equipped with a 540-nm filter.

Cells were harvested and washed twice in PBS at 4°C. Total cell lysates were lysed in lysis buffer [40 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 100 μg/ml phenymethylsulfonyl fluoride (PMSF)]. The supernatants were collected and protein concentrations were measured with protein assay reagents (Pierce, Rockford, IL, USA). Equal amounts of protein were denatured by boiling at 100°C for 5 min in sample buffer (0.5 M Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.1% bromophenol blue, 10% β-mercaptoethanol) at a 1:1 ratio. Equal amount of the total proteins were subjected to 6–15% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 buffer (TBS-T) (20 mM Tris, 100 mM NaCl, pH 7.5, and 0.1% Tween-20) for 1 h at room temperature, after which the membranes were incubated overnight at 4°C with the primary antibodies. The membranes were washed thrice for 10 min with TBS-T buffer and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The membranes were washed 4 times for 10 min with TBS-T buffer. Antigen-antibody complexes were detected using the enhanced chemiluminescence (ECL) detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA).

To analyze VEGF secretion, HCT116 cells were seeded in 12-well plates, cultured to 50% confluence, pretreated with sulforaphane or DMSO (control treatment) for 30 min, and switched to fresh media that were pre-conditioned in normoxic or hypoxic conditions. Cells were incubated with or without sulforaphane at the corresponding conditions for 24 h. The supernatants in the wells were collected, cleared by centrifugation, and stored at −20°C. ELISA was performed using the human VEGF Quantikine kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Recombinant human VEGF was used for calibration. Experiments were carried out at least 3 times in triplicate.

ell migration assays were performed using 24-well modified Boyden chambers (Corning Life Science, Corning, NY, USA). A cell migration kit was used for the cell migration assay according to the manufacturer's protocol. Confluent cells were added to the inner chamber of the insert in 100 μl of serum-free medium. Medium (600 μl) with 10% FBS was added to the lower chamber. To determine the effect of sulforaphane on cell migration, 5 or 10 μM sulforaphane was added to the lower chamber (DMSO was used as a control). Cells were fixed and stained with the Diff-Quick Stain kit (Baxter, McGaw Park, IL, USA) following the procedure described by the manufacturer. The number of migrating cells was counted under a microscope (×200 magnification) and the results were expressed as the percentage of invaded cells per field for each condition.

Results are expressed as the mean ± SD of 3 separate experiments. Data were analyzed by Student's t-test. Means were considered significantly different at p<0.05 or p<0.01.

To investigate the effects of hypoxia-induced HIF-1α, HCT116 and AGS cells were grown to 70% confluence in a standard CO₂ incubator at 37°C (normoxic conditions of 21% O₂ and 5% CO₂) and transferred to a hypoxia chamber (1% O₂ and 5% CO₂). Cell cultures were exposed to hypoxia and harvested at various time-points. Hypoxic conditions dramatically induced HIF-1α protein expression in HCT116 and AGS cells (Fig. 1A). HIF-1α protein induction was observed in cells 2 h after they were transferred to the hypoxic environment and become pronounced from the 2 h time-point through the 8 h time-point, reaching a maximum level at the 4 h and 6 h time-points; therefore, the 6 h time-point was selected for further experiments in HCT116 cells, while the 8 h time-point was selected for AGS cells.

The next study was performed to assess whether sulforaphane suppressed the observed responses to hypoxic conditions in HCT116 and AGS cells. Cells were pretreated with medium containing 12.5–100 μM sulforaphane for 1 h in normoxic conditions and transferred to a hypoxia chamber. Sulforaphane significantly inhibited HIF-1α expression in HCT116 and AGS cells (Fig. 1B). Interestingly, low concentration of sulforaphane slightly induced hypoxia-induced HIF-1α in HCT116 cells. Therefore, to investigate the effects of sulforaphane-induced cytotoxicity under normoxic and hypoxic conditions and determine whether cytotoxicity is responsible for suppression of HIF-1α accumulation, cell viability was determined by MTT assay. No significant concentration-dependent reduction of viability was observed when HCT116 and AGS cells were treated with various concentrations of sulforaphane for 6 and 8 h under normoxic and hypoxic conditions (data not shown). These data indicated that the decrease in HIF-1α abundance under normoxic and hypoxic conditions was not due to cell death.

VEGF is a target gene of HIF-1 that plays a crucial role in tumor angiogenesis. HIF-1 regulates VEGF expression at the transcriptional level (22). To determine whether sulforaphane inhibits VEGF expressions in HCT116 cells, VEGF transcript abundance was measured using an ELISA kit. Cells were incubated under hypoxic conditions with or without 12.5–50 μM sulforaphane. After 24 h of treatment, cell culture media were collected and VEGF transcript abundance was measured. VEGF transcript was increased under hypoxic conditions. However, VEGF induction was decreased in a concentration-dependent manner by sulforaphane (Fig. 2A). Sulforaphane also inhibited hypoxia-related target protein expressions such as VEGF, heme oxygenase (HO)-1 and glucose transporter 1 (GLUT1) (Fig. 2B).

To evaluate the mechanism by which sulforaphane inhibits HIF-1α expression, HCT116 cells were exposed to hypoxic conditions to induce HIF-1α protein expression. The exposure to hypoxic conditions was necessary because very little HIF-1α is detectable under normoxic conditions due to rapid protein degradation. Cyclohexamide (CHX), a protein synthesis inhibitor, is widely used in protein stability studies. HCT116 cells were incubated for 6 h under hypoxic conditions and treated with CHX for 30 min in the presence or absence of 50 μM sulforaphane. Cells were harvested at various time-points and cell lysates were subjected to western blot analysis using anti-HIF-1α antibodies. Under hypoxic conditions, the half-life of HIF-1α was not longer than 1 h when the cells were treated with CHX alone (Fig. 3A). However, the half-life of HIF-1α was ~15 min when the cells were treated with a combination of sulforaphane and CHX (Fig. 3B). These data revealed that sulforaphane reduced the half-life of HIF-1α protein under hypoxic conditions, indicating that sulforaphane treatment regulates HIF-1α expression by decreasing protein stability. These results demonstrate that sulforaphane affected hypoxia-induced HIF-1α protein stability in HCT116 cells.

Next, to examine whether sulforaphane-induced HIF-1α protein degradation is mediated by the proteasome degradation pathway, HCT116 cells were treated with proteasome inhibitor MG132 for 30 min, followed by treatment with medium containing sulforaphane for 30 min, after which the cells were exposed to hypoxic conditions for 6 h, washed with PBS, and lysed inside the hypoxia chamber. Degradation of HIF-1α protein induced by sulforaphane under hypoxic conditions was not completely prevented by MG132 (Fig. 3C and D). These data indicate that sulforaphane did not induce HIF-1α protein degradation through the proteasome degradation pathway.

Accumulating evidence has shown that multiple signaling pathways, particularly phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK pathways, are involved in hypoxia-induced HIF-1α protein accumulation and downstream target gene expression (23). To determine whether sulforaphane inhibits hypoxia-mediated activation of AKT, HCT116 cells were pretreated with various concentrations of sulforaphane for 1 h under normoxic conditions, followed by incubation for 6 h under hypoxic conditions, after which the cells were washed with PBS and lysed inside the hypoxia chamber. Sulforaphane did not inhibit phosphorylation of AKT and ERK under hypoxic conditions (Fig. 4A). Interestingly, hypoxia-induced HIF-1α was slightly increased by low concentration of sulforaphane. To determine whether sulforaphane activates AKT and ERK, cells were exposed to PI3K inhibitor LY294002 and ERK inhibitor PD98059. LY294002 and PD98059 significantly decreased the elevated levels of HIF-1α, pAKT, and pERK protein induced by hypoxic conditions (Fig. 4B). However, combination of the inhibitors with sulforaphane dramatically restored pAKT and pERK expression under hypoxic conditions (Fig. 4B). These results show that the decrease in HIF-1α expression produced by sulforaphane in HCT116 cells under hypoxic conditions is not mediated by regulation of PI3K and ERK pathways.

Hypoxic conditions enhance metastasis of several types of cancer cells, including colon and breast cancer cells. In vitro migration experiments were performed to determine whether sulforaphane inhibits HCT116 cell motility. HCT116 cells were treated with sulforaphane under normoxic and hypoxic conditions. As shown in Fig. 5A, there was little cell migration under normoxic conditions. However, cell migration increased under hypoxic conditions. Treatment with 5 and 10 μM sulforaphane suppressed migration activity. Sulforaphane was not cytotoxic at concentrations of 5 and 10 μM under hypoxic conditions (Fig. 5B). These results indicate that treatment with sulforaphane suppressed hypoxia-induced HCT116 cell migration.