Genistein inhibits radiation-induced activation of NF-κB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest
© Raffoul et al; licensee BioMed Central Ltd. 2006
Received: 02 February 2006
Accepted: 26 April 2006
Published: 26 April 2006
New cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells.
Tumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-κB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein.
Genistein combined with radiation caused greater inhibition in PC-3 colony formation compared to genistein or radiation alone. Treatment sequence of genistein followed by radiation and continuous exposure to genistein showed optimal effect. Cell cycle analysis demonstrated a significant dose- and time-dependent G2/M arrest induced by genistein and radiation that correlated with increased p21WAF1/Cip1 and decreased cyclin B1 expression. NF-κB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-κB activity was strongly inhibited by genistein pre-treatment. A significant and striking increase in cleaved PARP protein was measured following combined genistein and radiation treatment, indicating increased apoptosis.
A mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-κB, leading to altered expression of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting G2/M arrest and increased radiosensitivity. These findings support the important and novel strategy of combining genistein with radiation for the treatment of prostate cancer.
Prostate cancer (PCa) is an important public health concern in the United States. As our population ages, the number of patients with clinically significant PCa is expected to increase. In the United States, PCa is the most commonly diagnosed cancer in men as well as the second leading cause of male cancer deaths. The American Cancer Society estimates that in 2006 there will be 234,460 new cases of PCa and 27,350 men will die of the disease . Localized PCa is sensitive to conventional radiotherapy using megavoltage photons (X-rays), yet residual disease often causes clinical relapse in a large proportion of patients [2, 3]. While there is continuing debate on the impact of various treatment modalities on the survival of patients with different stages of PCa, the utilization of nutrition as an adjuvant therapy is an attractive idea. The use of dietary supplements, including soy, for cancer therapy has been recently reviewed [4, 5]. To improve the local control and treatment of PCa, we have investigated the combination of genistein with conventional radiation treatment.
Genistein (4',5,7-trihydroxyisoflavone), the most abundant isoflavone found in soybeans, is believed to be a potent anticancer agent [6, 7]. The interest in genistein stems from observations that increased soy consumption in Asian diets, resulting in increased serum isoflavone levels, has been associated with a decreased risk for PCa . Genistein has an heterocyclic diphenolic structure similar to estrogen  and has demonstrated anti-tumor and anti-angiogenic activities [10, 11]. Genistein was found to inhibit tyrosine protein kinases , topoisomerase I and II , and protein histidine kinase . Genistein has also been shown to inhibit cell growth of tumor cell lines from various malignancies including breast, lung, melanoma, prostate, head and neck squamous cell carcinoma, leukemia and lymphoma [15–22].
We have previously shown that genistein inhibited the cell growth of androgen-dependent (LNCaP) and androgen-independent (PC-3) human prostate carcinoma cell lines . Genistein affected the cell cycle and induced apoptosis, establishing it as a cytotoxic agent for PCa. We found that genistein induced G2/M cell cycle arrest leading to cell growth inhibition . Cell growth inhibition was observed with concomitant down-regulation of cyclin B1, up-regulation of the p21WAF1/Cip1 growth inhibitory protein, and induction of apoptosis . We have also demonstrated that genistein augments radiation-induced cell killing of PC-3 prostate cancer cells in vitro . Genistein combined with radiation significantly inhibited DNA synthesis, cell division, and cell growth compared to each modality alone .
We have also demonstrated in vivo that genistein potentiated inhibition of tumor growth by radiation in an orthotopic metastatic PC-3/nude mouse xenograft PCa tumor model . Genistein combined with prostate tumor irradiation led to a greater control of the growth of the primary tumor and metastasis to lymph nodes than genistein or radiation alone, resulting in greater mouse survival . These results suggest the potential for combining genistein with radiation for the treatment of localized PCa in humans.
The goal of our present study was to further elucidate the cellular and molecular interaction between genistein and radiation in vitro. We have investigated the effect of genistein and radiation on cell cycle progression and apoptosis and determined the optimal dose and time kinetics of each. We show that the potentiation of radiation-induced cell killing by genistein was optimal with the sequence of pre-treatment with genistein followed by radiation and continued exposure with genistein. This effect was also observed with other human tumor cell lines from various malignancies, indicating that our treatment strategy is not PCa cell-specific. The G2/M cell cycle arrest observed with genistein was enhanced by combination with radiation. This effect was associated with a greater up-regulation of the p21WAF1/Cip1 growth inhibitory protein and down-regulation of cyclin B1 than that seen with genistein alone, resulting in a significant increase in apoptosis. Moreover, the inhibition of NF-κB DNA binding activity induced by genistein was also enhanced by combining genistein with radiation. We propose a mechanism of increased cell death in PCa cells pre-treated with genistein that may be dependent upon downregulation of radiation-induced NF-κB, thus driving cancer cells toward apoptotic versus survival pathways.
Tumor cell lines
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) . The human RCC RC-2 cell line has been previously described . 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 . 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 . 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.
Analysis of cell survival by clonogenic assay
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.
Analysis of cell cycle progression
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).
Analysis of protein expression by western blot
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 . 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-p21WAF1/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).
Analysis of NF-κB DNA binding activity
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 . 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 . 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.
Enhanced cell growth inhibition of prostate carcinoma cells and other tumor cell lines by genistein and radiation
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).
Sequence and exposure of genistein and radiation treatment
Analysis of cell cycle progression after treatment with genistein and radiation in PC-3 cells
Inhibition of radiation induced NF-κB activation by pre-treatment with genistein and induction of apoptosis in PC-3 cells
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).
The use of soy isoflavones to potentiate conventional cancer treatment is a promising area for investigation. Our laboratory has previously shown that pre-treatment with genistein, the major isoflavone in soy, enhanced radiation-induced cell killing of PC-3 human PCa cells in vitro . In the current study, we investigated the mechanism of interaction between genistein and radiation in vitro at the cellular and molecular levels. Using a highly calibrated clonogenic assay, we confirmed our previous findings  showing that genistein combined with radiation caused 80% inhibition in the cell survival fraction compared to 40% with genistein at 15 μmol/L and 65% with 3 Gy photon radiation. Furthermore, we have demonstrated that the combination of genistein and radiation caused greater cell killing in both human breast and renal cancer cell lines than each modality alone, suggesting that this effect was not restricted to PC-3 cells and that this combined modality may also be applied towards the treatment of other cancers. To get an optimal effect, continuous exposure of the cells to genistein before and after radiation was needed. Our data indicate that the sequence of genistein followed by radiation and continuous exposure of genistein result in the most effective conditions for the combined cancer treatment.
We have previously shown that genistein treatment of PC-3 cells resulted in G2/M cell cycle arrest and altered the expression of two cell cycle regulatory proteins, the cyclin-dependent kinase (CDK) inhibitor p21WAF1/Cip1 and cyclin B1 . Cell cycle analysis confirmed that either genistein or radiation alone promote a decrease in the percentage of cells in G0/G1 and a concomitant increase in the percentage of cells in G2/M. A more significant G2/M cell cycle arrest was induced by pre-treatment with genistein followed by radiation compared to each modality alone, an effect which was both dose- and time-dependent. Cells respond to DNA damaging agents by activating cell-cycle checkpoints. Both genistein and radiation were independently found to cause late G2/M cell accumulation measurable 4 days after treatment in the current study and in previous studies [23, 30]. Cells in the G2/M phase of the cell cycle have been shown to be more radiosensitive than cells in other phases of the cell cycle [30, 31]. Pre- treatment with genistein does arrest cells in G2/M phase and thus could increase their radiosensitivity resulting in increased cell killing in addition to the direct cytotoxic effects of genistein and radiation. This interaction is in direct agreement with our observation that increased killing is optimal with the sequence of genistein pre-treatment followed by radiation compared to the reverse sequence.
The cell cycle regulatory molecule p21WAF1/Cip1 is a member of Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) involved in cell cycle and apoptosis regulation [32–34]. Under cellular stress, p21WAF1/Cip1 expression is increased through p53-dependent and -independent pathways . As shown in our studies, genistein or radiation alone induced upregulation of p21WAF1/Cip1 protein in PC-3 cells, although they are p53 defective . Moreover, higher nuclear expression concomitant with decreased cytoplasmic p21WAF1/Cip1 expression were found in genistein pre-treated PC-3 cells exposed to radiation, suggesting nuclear translocation. Previous studies have shown that increased levels of p21WAF1/Cip1 in the nucleus led to inhibition of CDKs through its binding to the cyclin/CDK complexes including cyclin B/CDK complex [32–34]. The cyclin B1/CDK1 (also known as cdc2) complex is essential for progression of the cells through mitosis, therefore a decrease in cyclin B proteins can result in G2/M arrest . Our data showed a greater decrease in nuclear cyclin B1 in PC-3 cells treated with genistein combined with radiation, whereas radiation did not affect cyclin B1 (when measured at 1 hr post-radiation).
Our findings on G2/M arrest in response to radiation combined with genistein in PC-3 cells corroborate previous studies in DU145 human prostate cancer cells  and in cervical cancer cells . Our studies further address the role of the transcription factor NF-κB in the mechanism by which genistein enhances radiation-induced cell killing. Recent studies have shown that NF-κB, a major signaling molecule involved in the regulation of cellular proliferation and apoptosis [39–41], is constitutively activated in PCa and correlates with disease progression [42, 43]. NF-κB promotes malignant behavior by suppressing apoptosis and stimulating transcription of proteins involved in cell cycle progression. NF-κB activation and nuclear translocation can lead to the synthesis of molecules critical for cell survival in response to stress. We and others have demonstrated that genistein inhibits the activation of NF-κB in multiple cancer cell lines , including PC-3 and LNCaP prostate cancer cells . Furthermore, genistein pre-treatment abrogated the activation of NF-κB by the chemotherapeutic agents docetaxel or cisplatin . Such an effect is demonstrated in our current study showing that genistein pre-treatment also completely inhibited radiation-induced activation of NF-κB. We also observed an increase in NF-κB p65 protein in the cytoplasm of cells treated with genistein and radiation, suggesting that NF-κB may not be translocated into the nucleus as we have shown in previous studies with genistein alone . Recent studies have established a correlation between radioresistance of breast cancer cells and induction of both NF-κB and cyclin B1 and demonstrated that fractionated radiation induced cyclin B1 expression via an NF-κB-dependent mechanism . The cyclin B1 decrease that we observed following genistein combined with radiation could be related to the inhibition of NF-κB DNA binding activity.
Recent studies have also demonstrated that p21WAF1/Cip1 was induced in S/G2/M phases and correlated with NF-κB activation . Our findings on the effect of radiation alone causing upregulation of p21WAF1/Cip1, G2/M arrest and NF-κB activation could follow the same pathway. However, the effect of genistein combined with radiation on induction and nuclear translocation of p21WAF1/Cip1, in addition to the observed increase in cells arrested in G2/M phase, may not occur via an NF-κB-dependent mechanism, as NF-κB DNA binding activity was in fact inhibited by the combined treatment. The association between NF-κB inhibition and the upregulation and nuclear translocation of p21WAF1/Cip1 induced by genistein combined with radiation remains to be elucidated.
We propose a mechanism of increased cell killing by combined genistein and radiation treatment that is triggered by inhibition of NF-κB leading to altered transcription of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting apoptotic cell death. Increased apoptotic cell death was confirmed by the observation of significantly elevated expression levels of cleaved PARP protein in cells treated with genistein and radiation, compared to each modality alone, demonstrating increased apoptotic cell death.
Our current findings are consistent with the hypothesis that genistein pre-treatment sensitizes cancer cells to radiation-induced growth inhibition and apoptosis. We have also obtained similar results in recent studies demonstrating that genistein pre-treatment potentiates chemotherapy-induced tumor cell death both in vitro and in vivo [47–50]. Taken together, our studies demonstrate an important and novel strategy of combining conventional cancer treatment with nutrition and support the use of soy isoflavones in combination with radiation for the treatment of patients with prostate cancer.
We thank Dr. Michael C. Joiner for his technical advice with the clonogenic assay. This study was supported by the American Institute for Cancer Research (grant 03B108 to G.G.H.).
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