The human ovarian cancer cell line A2780 and derived cell lines resistant to adriamycin A2780/ADR and cisplatin A2780/CP were described earlier (19,20). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Bio-Whittaker, Verviers, Belgium) and 40 mg/ml gentamicin (Lek, Ljubljana, Slovenia) in a humidified atmosphere with 5% CO₂ at 37°C. Exposure to hypoxia was performed in an anaerobic workstation (Ruskin Technologies, Bridgend, UK) in 2% O₂, 5% CO₂, 10% H₂, and 83% N₂ at 37°C. Alternatively, hypoxia was mimicked by 1 mM dimethyloxalylglycine. Sulforaphane was obtained from Sigma-Aldrich (St. Louis, MO, USA) and used at 2.5–10 μM depending on the experimental setting. The cells were first pre-treated with SFN for 4 h and then continuously incubated with SFN in normoxia and/or hypoxia for additional 24 h or for longer periods.

Total RNA was extracted using the Instapure reagent (Eurogentec, Seraing, Belgium) as recommended by the manufacturer. Three micrograms of RNA was transcribed with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) using random heptameric primers. Quantitative real-time PCR was performed on a StepOne Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers (CA9, HIF-1α) and primers for β-actin that served as an internal standard. The primers were as follows: CA9 sense: 5′-CCGAGCGACGCAGCCTTTGA-3′ and CA9 antisense: 5′-GGCTCCAGTCTCGGCTACCT-3′; HIF-1α sense 5′-GCT TGGTGCTGATTTGTGAACC-3′, HIF-1α antisense 5′-GCA TCCTGTACTGTCCTGTGGTG-3′; β-actin sense: 5′-TCCTC CCTGGAGAAGAGCTA-3′ and β-actin antisense: 5′-ACAT CTGCTGGAAGGTGGAC-3′. PCR was performed using DreamTaq™ Green PCR Master Mix (Fermentas, St. Leon-Rot, Germany) and the same primers as shown above.

Human CA9 promoter construct pGL3-CA9 was generated by an insertion of PCR-amplified −174/+37 CA9 genomic fragment upstream of the firefly luciferase gene in pGL3-Basic luciferase reporter vector (Promega, Madison, WI, USA). pRL-TK Renilla vector (Promega) served as a transfection efficiency control. A2780 cells were plated into 35-mm Petri dishes to reach approximately 70% monolayer density on the next day. Transient transfection was performed with 1 μg of pGL3-CA9 plasmid and 100 ng of pRL-TK plasmid using Turbofect reagent (ThermoFisher Scientific) according to the manufacturer’s recommendations. One day later, transfected cells were trypsinized and plated in triplicates into 24-well plates. Transfected cells were allowed to attach overnight, and then transferred to hypoxia for additional 24 h. SFN was added 4 h before the transfer to hypoxia. Reporter gene expression was assessed using the Dual Luciferase Reporter Assay System (Promega), and the luciferase activity was normalized against the Renilla activity.

The cells were washed with PBS and disrupted in lysis buffer containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.5), 0.5% Nonidet P-40, 50 mM NaF, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Total protein extracts (50–100 mg/lane) were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidene fluoride membranes (Immobilon; Millipore, Billerica, MA, USA). Membranes were treated for 1 h in blocking buffer and then incubated either for 1 h with specific antibodies against CA IX (in-house generated M75 in blocking buffer, dilution 1:2), HIF-1α (dilution 1:250; BD Transduction Laboratories, San Jose, CA, USA), GLUT-1 (dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA), and actin (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All membranes were then washed four times for 10 min with the washing buffer (PBS containing 0.2% Nonidet P-40 or 0.1% Tween-20), followed by the incubation with an appropriate secondary antibody conjugated with horseradish peroxidase (Dako, Glostrup, Denmark) for 1 h. After additional washing step, all immunoblots were developed with the ECL detection system.

Cells were harvested in concentration of 1×10⁶ cells/sample and washed in PBS. Labeling was performed in 50 μl of cell suspension with 50 μl of mouse monoclonal antibody M75 for 30 min. Mouse monoclonal antibody against CD45 was used as a negative isotype control. Cells were then washed and labeled with goat anti-mouse F(ab’)2 antibody conjugated with FITC for 30 min at room temperature and analyzed on flow cytometer Coulter Epics Altra. Data were analyzed with FCS Express version 3.0 (De Novo Software, Ontario, Canada).

For assessment of cell viability, the cells were incubated with propidium iodide at a final concentration of 5 μg/ml for 5 min at room temperature. The samples were analyzed using a Guava EasyCyte Plus flow cytometer with Guava Express Pro 2.2.3 software (Millipore).

Cignal^®™ Cell-based Multi-Pathway Activity Assay (SABioscience, Frederick, MD, USA) was performed according to instructions of the manufacturer. Dual-luciferase results were calculated for each transfectant and analyzed by the data analysis software (SABioscience). Changes in the activity of each signaling pathway were determined by comparing the normalized luciferase activities of the reporter in treated vs. untreated transfected cells.

For the proliferation assay, 4×10⁴ A2780 ovarian cancer cells and their chemoresistant variants were seeded into each well of an E-plate 16 (Roche) in DMEM containing 5 μM sulforaphane or DMSO (control samples). For the migration assay, the cells were first pre-treated with sulforaphane or DMSO for 24 h in hypoxia and then seeded into the upper chamber of the CIM-plate 16 (Roche) in the medium containing 0.1% FCS and 5 μM sulforaphane. A chemotactic signal for the cell movement was provided by supplying 10% FCS into the lower chamber. The plates were placed into the real-time cell analyzer (RTCA, xCELLigence, Roche), performing an impedance-based, label-free monitoring of cell proliferation and migration. RTCA analyzer was placed in a hypoxic cabinet with the O₂ controller (2% O₂, COY Laboratory Product, Grass Lake, MI, USA). Data were collected every 15 min during the entire period of measurement and were presented as a dimensionless parameter called the cell index (CI; calculated as a relative change in the measured electrical impedance), graphs show the average of triplicate wells.

Intracellular pH (pHi) was measured using the fluorescent probe 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF; Sigma-Aldrich). SFN-treated cells, untreated controls and cells for calibration curve were plated on 6-well plates and loaded with 8.2 μM BCECF and 5% pluronic acid in PBS buffer, pH 7.48 for 30 min at 37°C in the dark. Afterwards, the cells were washed with PBS buffer and measured. Calibration was performed on untreated cells using PBS/HEPES buffers with different pH values (7.61, 7.48, 7.03, 6.52, 6.32 and 6.01). The fluorescence was excited at 489 nm and measured at 525 nm on the fluorescence scanner BioTek (BioTek, Germany). The pHi signal was calibrated by adding 10 μM of nigericin (Sigma-Aldrich) with 130 mM of KCl. pHi values for samples were calculated from the calibration curve. Extracellular pH was measured in cell culture media as described earlier (21).

Results were analyzed by two-tailed unpaired t-test (Student’s t-test), and P<0.05 was considered significant.

First we investigated how A2780 ovarian carcinoma cells respond to hypoxia and SFN in a broader context of molecular pathways. We exposed A2780 cells for 24 h to 2% of atmospheric oxygen, which corresponds to moderate hypoxia typical for tumor cells present in broader perinecrotic areas less distant from the blood vessels. Western blot analysis of extracts from normoxic vs. hypoxic A2780 cells revealed a hypoxia-related accumulation of HIF-1α protein and its target CA IX suggesting that these cells are sensitive to reduced oxygen and react by induction of a canonical HIF response (Fig. 1A). The A2780 cells grown in a confluent monolayer were then treated with SFN for 28 h, including 4 h pre-treatment in normoxia followed by a 24 h treatment in hypoxia. Cells treated with 2.5, 5 and 10 μM concentrations of SFN were subjected to flow cytometric analysis to assess their viability. As shown on Fig. 1B, hypoxia alone induced 10% cell death of A2780 cells, which was only slightly increased by SFN treatments. Moreover, a real-time monitoring of the proliferation of A2780 cells did not show any pronounced inhibitory effect of 5 μM SFN either in normoxia (not shown), or in hypoxia (Fig. 1C).

Since the concentration of about 5 μM SFN is achievable in vivo (22), we decided to use this SFN concentration in the subsequent experiments. A cell-based reporter assay confirmed the hypoxia-related activation of the HIF pathway (Fig. 1D). In addition, exposure of A2780 cells to hypoxia led to the activation of the MAPK/JNK pathway executed by the AP-1 transcription factor, which was previously linked to hypoxia and VHL deficiency (23,24). SFN treatment considerably reduced the reporter transactivation via HIF and AP-1 pro-oncogenic pathways. On the other hand, five other pathways involved in the negative control of tumor growth were considerably upregulated by SFN during hypoxia, including the pathways resulting in activation of ARE/NRF2, p53, IRF-1, Pax-6 and XRE-driven transcription (Fig. 1D).

We then further followed the HIF-1α response to SFN under hypoxia. First, we analyzed the SFN effect on the HIF-1α mRNA level and found no significant difference between SFN-treated and control A2780 cells exposed to hypoxia, suggesting that SFN did not affect the transcription of the HIF-1α gene (Fig. 2A). In contrast, the western blot analysis showed a considerably reduced level of the HIF-1α protein in the hypoxic A2780 cells subjected to SFN treatment when compared to the control hypoxic cells (Fig. 2B). Since the abundance of the HIF-1α protein can be regulated on the level of translation and/or degradation, we analyzed the stability of the HIF-1α protein in hypoxic conditions following the inhibition of translation by 20 μg/ml cycloheximide (CHX). However, the levels of the HIF-1α protein were similar in the hypoxic A2780 cells incubated with CHX for 10 min whether or not they were pre-treated with SFN (Fig. 2C), indicating that SFN did not induce HIF-1α degradation. This supports the view that SFN acts through the suppression of HIF-1α translation.

In the next step, we analyzed the effects of SFN on the HIF-1α downstream target CA IX. In agreement with the reduced HIF-1α protein level, SFN was able to diminish the hypoxia-induced activation of the CA9 gene promoter almost to its normoxic value as determined by the dual luciferase assay (Fig. 3A). It could also reduce the level of the corresponding transcript as evident from the PCR analysis (Fig. 3B and C) and finally, decrease the level of the CA IX protein as seen in the western blot analysis (Fig. 3D). Moreover, the flow cytometric analysis revealed that SFN treatment led to a reduced proportion of the CA IX-positive tumor cell subpopulation of the hypoxic A2780 cells (Fig. 3E).

Drug resistance is one of the key obstacles in therapy of ovarian cancer, and hypoxia is known to support this phenomenon by activation of molecular mechanisms of multiple drug resistance as well as by selection of cells that are less responsive to drug treatment (5). Therefore, we evaluated the effect of SFN in the chemoresistant variants of A2780 cells under hypoxic conditions using the A2780/CP cell line resistant to cisplatin (expressing the MRP1 gene) and A2780/ADR cell line resistant to adriamycin (expressing the MDR1 gene). Of note, these chemoresistant cell lines showed increased HIF-1α protein levels when exposed to 2% hypoxia similarly to parental chemosensitive A2780 cells (Fig. 4A). This was associated also with the increased CA9 promoter activation (Fig. 4B), increased induction of the CA9 transcript (Fig. 4C) and with the elevated CA IX protein expression (Fig. 4A).

Noteworthy, SFN was able to reduce the molecular response to hypoxia in both cell lines in an extent similar to that in parental A2780 cells, although the inhibitory effect did not go back to the normoxic values either at the level of CA9 mRNA or at the protein levels of both HIF-1α and its CA IX and GLUT-1 downstream targets (see Fig. 4B–D). Moreover, in both chemoresistant cell lines SFN reduced the proportion of the CA IX-positive subpopulation of cells similarly to that observed in the parental A2780 cell line (Fig. 4E and F). Altogether, these data suggest that SFN can exhibit its anticancer effect also in the chemoresistant cell lines.

Since CA IX, cooperating with ion transporters and exchangers, is functionally implicated in acidification of extracellular pH (pHe) and in maintenance of neutral/slightly alkaline intracellular pH (pHi), we evaluated the pH changes in response to 24 h SFN treatment. We found decreased pHi (Fig. 5A) and increased pHe (Fig. 5B) in SFN-treated hypoxic cells compared to non-treated hypoxic controls, suggesting that SFN interferes with the capacity of the pH regulating machinery of the parental A2780 cells as well as of both A2780/ADR and A2780/CP chemoresistant cell lines to maintain the proper acid-base balance in hypoxia.

Finally, we tested whether SFN affects behavior of A2780 cells and of the chemoresistant variants. Since tumor progression to metastasis is associated with hypoxia and induction of migratory phenotype (25), we examined the ability of SFN to modulate migration of A2780 variants. To this end we used an impedance-based approach, which allows for the real-time evaluation of cell migration. We found that SFN reduces migration of hypoxic chemosensitive and chemoresistant A2780 cell lines (Fig. 6A, C, E and G). To exclude the contribution of cell proliferation to the observed effect, we simultaneously monitored this parameter by real-time measurement of the cells plated in parallel, pre-treated in hypoxia and grown on the bottom of impedance plates. This control experiment showed no differences in the proliferation cell index of the analyzed cell lines (Fig. 6B, D and F). Thus, we can conclude that SFN can diminish migration of the ovarian carcinoma cells that were exposed to hypoxia.