Genistein inhibits radiation-induced activation of NF-κB in prostate cancer cells promoting apoptosis and G₂/M cell cycle arrest

The experiments were performed using the PC-3 human prostate carcinoma tumor cell line purchased from American Type Culture Collection (ATCC, Rockville, MD). PC-3 cells were cultured in F-12 K culture medium (CM) (Invitrogen, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum, 2 mmol/L glutamine, 0.1 mmol/L non-essential amino acids, 10 mmol/L HEPES, and 100 U/mL penicillin/streptomycin. Human breast cancer cell line BR231 (MDA-MB-231) was purchased from ATCC. The human renal cell carcinoma (RCC) cell line KCI-18 was established in our laboratory from a primary renal tumor specimen obtained from a patient with papillary RCC (nuclear grade III/IV) [26]. The human RCC RC-2 cell line has been previously described [27]. These cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 4.5 g/L glucose supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 mmol/L HEPES, 100 U/mL penicillin/streptomycin and 50 μg/mL gentamicin. The cultures were incubated at 37°C in a humidified 5% CO₂ incubator.

Genistein was purchased from Toronto Research Chemicals (North York, Ontario, Canada) and dissolved in 0.1 mol/L Na₂CO₃ (Sigma, St. Louis, MO) to make a 10 mmol/L stock solution. Genistein was further diluted in CM to obtain concentrations of 15 μmol/L or 30 μmol/L. Control cells were incubated with equivalent dilutions of Na₂CO₃ in CM. Genistein treatment was administered when cells were 70% to 80% confluent.

Cells in 15 mL tubes, T₂₅ flasks, or T₇₅ flasks were irradiated with photons using a ⁶⁰Co unit (AECL Theratron 780). Tubes were placed at a depth of 2.6 cm in a specially machined lucite block of dimensions 10 cm × 20.3 cm × 12.8 cm [24]. The surface of the block was positioned at 46 cm from the source and tubes were irradiated with a horizontal 25 cm × 25 cm beam at a dose rate was -92 cGy min^-1 [24]. Flasks were irradiated from above with a vertical beam, 2.5 mm of polystyrene build up material was placed on top of the flasks and the surface of the build-up material was at a distance of 76 cm from the source. The dose rate was -32 cGy min^-1.

Cells were plated in T₂₅ flasks at 0.5 × 10⁶ cells/flask in CM. Three days later, 75% confluent cells were washed in CM and treated with genistein at a final concentration of 15 μmol/L in 5 mL CM. After 24 hr exposure to genistein, cells were removed using trypsin-EDTA (Invitrogen, Carlsbad, CA), counted and transferred to 15 mL conical tubes at 2 × 10⁶ cells/5 mL CM for photon irradiation. Following irradiation, cells were plated in triplicate using 6-well plates. For comparison between each treatment group, the number of cells plated after genistein and/or radiation treatment was adjusted relative to untreated cells to predict a measurable survival fraction, as determined in pilot experiments. Based on these data, the number of cells plated in 2 mL CM were as follows: 500 cells/well for control, 1000 cells/well for genistein or radiation alone, and 3000 cells/well for genistein + radiation treatments. After plating, cells in respective treatment groups were supplemented with genistein at a final concentration of 15 μmol/L. Following 10–13 days incubation at 37°C in a 5% CO₂/5% O₂/90% N₂ incubator, colonies were fixed and stained in 2% crystal violet in absolute ethanol, then counted. Clones of at least 50 cells were counted as one colony. The plating efficiency was calculated for each well by dividing the number of colonies by the original number of cells plated. The surviving fraction was normalized to control cell plating efficiency by dividing the plating efficiency of treated cells by that of control cells.

PC-3 cells were plated in T₂₅ flasks at 1 × 10⁶ cells/flask in CM. One day later, when 75% confluent, cells were washed in CM and treated with genistein at a final concentration of 15 μmol/L or 30 μmol/L in 5 mL CM. After 24 hr exposure to genistein, the cell monolayer in T₂₅ flasks was irradiated with 3 Gy photons. On day 4 post-radiation, flasks were washed with Hanks' balanced salt solution (HBSS) and removed using trypsin-EDTA (Invitrogen, Carlsbad, CA). Cells were then washed in phosphate-buffered saline (PBS), counted and 0.5 × 10⁶ cells/100 μl PBS buffer were fixed and permeabilized in 4.5 mL of 70% cold ethanol for 2 hr on ice. Cells were washed again in PBS then stained for 30 min at room temperature with 1 mL DNA fluorochrome solution containing 200 μg propidium iodide (Sigma, St. Louis, MO), 0.1% Triton X-100 (Sigma, St Louis, MO), and 2 mg DNase-free ribonuclease A (Sigma, St Louis, MO). Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Cells were plated in T₇₅ flasks at 1 × 10⁶ cells/flask in CM. Seventy-five percent confluent cells were washed in CM then treated with genistein at a final concentration of 30 μmol/L in 10 mL CM. After 24 hr exposure to genistein, cells were irradiated with 3 Gy photons. After 1 hr, cells were removed using trypsin-EDTA (Invitrogen, Carlsbad, CA) and collected by centrifugation. Nuclear and cytoplasmic proteins were isolated using CelLytic™ NuCLEAR™ Extraction Kit (Sigma, St. Louis, MO) according to the manufacturer's protocol. Extracts were aliqouted, flash frozen in liquid nitrogen, and stored at -80°C for subsequent western blot analyses. Protein concentrations were determined according to Bradford using Protein Assay Kit I (Bio-Rad, Hercules, CA).

Western analysis was performed using 20 μg nuclear extracts as previously described [28]. Briefly, each sample was prepared with an equal volume of 2X loading dye (National Diagnostics, Atlanta, GA), subjected to 10% SDS-PAGE (.75 mm thick; 30% Acrylamide/Bis Solution 29:1) and transferred to a Hybond™ ECL™ nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA). SDS-PAGE progression was monitored using dual color Precision Plus Protein™ Standards (Bio-Rad, Hercules, CA). Upon completion of SDS-PAGE, the region containing the protein(s) of interest was excised and prepared for western analysis while the remaining portion of the gel was stained with GelCode^® Blue Stain Reagent (Pierce, Rockford, IL) to ensure equal quantity of protein was loaded onto the gel. Western blot analysis was accomplished using manufacturer recommended dilutions of rabbit polyclonal anti-PARP (214/215) cleavage site specific antibody (BioSource, Camarillo, CA), mouse monoclonal anti-cyclin B1 antibody (D-11, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonal anti-p21^WAF1/Cip1 antibody (187, Santa Cruz Biotechnology, Santa Cruz, CA). After incubation in recommended dilutions of goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-rabbit IgG-HRP (Cell Signaling Technology, Beverly, MA), membranes were incubated in SuperSignal^® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL), exposed to CL-Xposure Film™ (Pierce, Rockford, IL) and developed using an All-Pro 100 Plus automated X-ray film processor (All-Pro Imaging Corporation, Hicksville, NY). Membranes were stripped using Restore™ buffer (Pierce, Rockford, IL) and reprobed with rabbit polyclonal anti-Rb antibody (C-15, Santa Cruz Biotechnology, Santa Cruz, CA) and developed as described above as an additional control for nuclear protein loading. The resultant bands were quantified using AlphaEaseFC™ imaging software (AlphaInnotech, San Leandro, CA).

PC-3 cells were treated with 30 μmol/L genistein for 24 hr then irradiated with 3 Gy photons. After 30 min, cells were removed using trypsin-EDTA (Invitrogen, Carlsbad, CA) and collected by centrifugation. Nuclear proteins were isolated as previously described [29]. Briefly, the cell pellet was resuspended in 0.5 mL lysis buffer (10 mmol/L Tris-HCl, pH 7.5; 5 mmol/L MgCl₂; 0.05% Triton X-100) and lysed with 20 strokes in a 1 mL Dounce homogenizer. The homogenate was centrifuged at 10,000 g for 15 minutes at 4°C. The pellet was resuspended in equal volume of nuclear extraction buffer A (10 mmol/L Tris-HCl, pH 7.4; 5 mmol/L MgCl₂) and nuclear extraction buffer B (1 mol/L NaCl; 10 mmol/L Tris-HCl, pH 7.4; 4 mmol/L MgCl₂). The resuspended pellet was incubated on ice for 30 min and then centrifuged at 10,000 g for 15 min at 4°C. The supernatant containing the nuclear proteins was removed and 80% glycerol was added to a final glycerol concentration of 20% (vol/vol). Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).

NF-κB DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) as previously described [29]. Briefly, 10 μg nuclear extract was incubated with ³²P-labeled and purified NF-κB consensus double-stranded oligonucleotide and 0.25 mg/mL poly(dI- dC) in 5X binding buffer (20% glycerol, 5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 250 mmol/L NaCl, 50 mmol/L Tris-HCl, pH7.5). After incubation at room temperature for 30 min, samples were loaded on a pre-run 8% polyacrylamide gel and run at 30 mA for 45 min. The gel was dried, exposed to X-ray film overnight at -80°C, then developed using an All-Pro 100 Plus automated X-ray film processor (All-Pro Imaging Corporation, Hicksville, NY). The resultant bands were quantified using AlphaEaseFC imaging software (AlphaInnotech, San Leandro, CA). Anti-Rb immunoblotting with nuclear protein was performed as a loading control as described above.

Comparisons of survival fractions in the clonogenic assays among the various treatment groups were analyzed by two-tailed unpaired Student's t-Test. Comparisons between means in the western blot and EMSA assays among the various treatment groups were analyzed by two-tailed Student's t-Test for independent samples. A p-value less than 0.05 was considered statistically significant.

Doses of 15 μmol/L genistein and 3 Gy radiation were selected based on previous dose titration experiments [24]. Treatment of PC-3 cells with 15 μmol/L genistein or 3 Gy radiation alone promoted approximately 50% inhibition in cell growth, while optimal cell killing was observed after pre-treatment with 15 μmol/L genistein combined with low-dose photon radiation [24]. To confirm this observation, we have repeated the clonogenic assay with an adjusted number of cells plated after genistein and/or radiation treatment in order to predict a measurable survival fraction that permitted enhanced comparisons between single and combined treatments (see Methods section). PC-3 cells were treated with 15 μmol/L genistein for 24 hr then irradiated with 2 Gy or 3 Gy photon radiation. To evaluate the long-term effect of the single or combined treatments, cells were plated in a 10 day clonogenic assay in the presence of 15 μmol/L genistein. Genistein augmented cell growth inhibition induced by 2 Gy or 3 Gy radiation to 72% (p < 0.005) and 80% (p < 0.001), respectively compared to 55% and 65% with 2 Gy or 3 Gy radiation alone; whereas, genistein alone caused only 40% inhibition (Fig. 1A). Based on these data, the conditions of 15 μmol/L genistein and 3 Gy photon radiation were selected for continuation of the studies.

To assess whether the potentiation of radiation-induced cell killing by pre-treatment with genistein is not a phenomenon restricted to PC-3 cells, additional human tumor cell lines were tested (Fig. 1B). The response of PC-3 cells to genistein and radiation was compared to the human breast cancer cell line MDA-MB-231 (BR231) and also two renal cell carcinoma cell lines KCI-18 and RC-2. Cells were pre-treated with 15 μmol/L genistein for 24 hr, then irradiated with 3 Gy photon radiation and plated in a clonogenic assay in the presence of 15 μmol/L genistein. BR231, KCI-18 and RC-2 cell lines showed a comparable inhibition of cell growth when treated with genistein alone, but a lower response to radiation compared to PC-3 cells (Fig. 1B). However, the genistein combined with radiation caused a significant increase in inhibition of colony formation (73–80%, p < 0.05) compared to genistein (33–47%, p < 0.05) or radiation alone (40–50%, p < 0.05) in the three cell lines as observed for PC-3 cells (Fig. 1B).

To address the effect of the sequence and exposure of genistein relative to radiation, we have either pre-treated PC-3 cells with 15 μmol/L genistein for 24 hr followed by 3 Gy photon irradiation or pre-treated the cells with radiation and after 24 hr, treatment with 15 μmol/L genistein for 24 hr. Cells were then plated for the clonogenic assay in the presence or absence of 15 μmol/L genistein for 10 days. The sequence of genistein followed by radiation showed a greater cell growth inhibition with continuous exposure to genistein (88%, p < 0.05) than in the absence of genistein in the clonogenic assay (82%) (Fig. 2A,B). The effect of genistein alone was also more pronounced with continued exposure (55% inhibition vs. 40% without genistein, p < 0.05); whereas, the effect of radiation remained at the level of 68% inhibition (Fig. 2A,B). This sequence of genistein followed by radiation showed a greater effect on cell growth inhibition than the reverse sequence of radiation followed by genistein (p < 0.05) (Fig. 2C,D). Even in this reverse sequence, the effect of genistein alone or combined with radiation was greater with continued exposure of the cells to genistein compared to radiation alone and genistein + radiation (p < 0.05) (Fig. 2C,D).

To investigate the molecular mechanism involved in potentiation of radiation-induced cell killing by genistein, we analyzed cell cycle progression and expression of molecules known to affect cell cycle regulation. PC-3 cells were treated for 24 hr with 15 μmol/L or 30 μmol/L of genistein then irradiated with 3 Gy photons. On Day 3 and 4 after radiation, separate flasks of cells were processed for DNA content and cell cycle analysis as described in the Methods section. From Day 3 after radiation, changes were observed in cell cycle analysis induced by genistein, radiation or both combined, showing an increased trend of cells in G₂/M (Fig. 3A–D). Cell arrest in G₂/M phase was more prominent by Day 4 after radiation (16%, Fig. 3J) and with a higher dose of 30 μmol/L genistein (31%, Fig. 3K) compared to 9% in control cells (Fig. 3I). Cells treated with genistein and radiation showed a further increase to 41% cells in G₂/M cells (Fig. 3L) and a concomitant decrease in cells in G₀/G₁ phase from 84% to 53% (Fig. 3L). An increase in pre-G₀/G₁ peak of apoptotic cells was also noted (arrow, Fig. 3L). The percentage of cells in S phase remained at the level of 6–8%.

Based on these data, we measured the expression of p21^WAF1/Cip1 and cyclin B1, two molecules involved in cell cycle progression. For these short-term molecular studies, and those described below, PC-3 cells were treated with 30 μmol/L genistein for 24 hr, followed by 3 Gy irradiation and processed for extraction of nuclear and cytoplasmic proteins at 1 hr post-irradiation. Following genistein and radiation, the expression of p21^WAF1/Cip1 showed a 1.5-fold increase in nuclear expression associated with 26% decrease in cytoplasmic expression (Fig. 4A). This pattern was comparable to that observed with genistein alone, although at a lower extent (1.1-fold increase in nuclear expression) (Fig. 4A). In contrast, radiation alone showed an significant increase in the expression of p21^WAF1/Cip1 protein both in the nuclear and cytoplasmic fractions (Fig. 4A). Genistein combined with radiation caused a 55% decrease in cyclin B1 expression in the cell nucleus compared to control cells (Fig. 4B). This decrease was greater than that observed with genistein alone (14% at 24 hr post-treatment) (Fig. 4B). No striking effect was observed with radiation alone when cells were tested at 1 hr post-radiation.

We have previously demonstrated that apoptosis induced by genistein alone in PC-3 cells was associated with a decrease in NF-κB DNA binding activity [29], a well-known transcription factor critically involved in the survival of cells. Preliminary studies showed that radiation at 3 Gy or 5 Gy of PC-3 cells caused increased NF-κB DNA binding activity detectable by 30 min post-radiation, persisted up to 3 hr post-radiation, and then a decrease was observed. Based on these data suggesting that activation of NF-κB was an early event in response to radiation, the conditions for testing NF-κB activity in cells treated with genistein and radiation were selected. Cells were treated with 30 μmol/L genistein for 24 hr, followed by 3 Gy irradiation and harvested at 30 min post-radiation and nuclear proteins were extracted for EMSA analysis of NF-κB DNA binding activity. An significant increase (23%, p < 0.0009) in NF-κB was observed at 30 min post-irradiation whereas genistein-treated cells exhibited a significant decrease (21%, p < 0.0015) in NF-κB DNA binding activity (Fig. 5A). Pre-treatment of PC-3 cells with genistein for 24 hr followed by radiation showed that NF-κB DNA binding activity was significantly inhibited (66% decrease, p < 0.00004) (Fig. 5A). This NF-κB DNA binding inhibition induced by genistein combined with radiation persisted for 1 hr and 3 hr post-radiation (data not shown), suggesting its mechanistic role for enhanced apoptosis. Furthermore, the inhibition of NF-κB DNA binding activity by genistein and radiation was consistent with western blot analysis, with these cells showing a 21% increase in NF-κB p65 cytoplasmic protein expression compared to control, untreated cells (data not shown).

To test whether the increased NF-κB DNA binding inhibition observed in PC-3 cells treated with genistein and radiation resulted in increased apoptosis, we analyzed the expression of the 85-kDa cleaved PARP protein, a marker for detecting apoptotic cells. Cells were treated with 30 μmol/L genistein for 24 hr, followed by 3 Gy irradiation and processed for extraction of nuclear proteins at 1 hr post-irradiation. Cleaved PARP expression was significantly and strikingly enhanced by genistein combined with radiation as demonstrated by a 5.6-fold (p < 0.0002) increase in expression compared to 3.6-fold (p < 0.0002) with radiation alone and 1.8-fold (p < 0.004) with genistein alone, relative to control (Fig. 5B).