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

Abstract

Background: 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.

Methods: Tumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-kappaB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein.

Results: 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-kappaB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-kappaB 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.

Conclusion: A mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-kappaB, 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.

Figure 1

Effect of genistein and radiation treatment on the survival fraction of human tumor cell lines. (A) PC-3 prostate cancer cells were treated with 15 μmol/L genistein for 24 hr then irradiated with 2 or 3 Gy photons. After radiation, cells were plated in a clonogenic assay. (B) Cells from the PC-3 (prostate cancer), BR-231 (breast cancer), and KCI-18 and RC-2 (renal cell carcinoma) cell lines were treated with 15 μmol/L genistein for 24 hr then irradiated with 3 Gy photons and plated in a clonogenic assay. Colonies were stained with crystal violet and counted after 11–13 days incubation. The mean survival fraction ± S.E.M. of triplicate wells was normalized to control cells based on plating efficiency. (*): value statistically significant from control at p < 0.05. Combined treatment was significantly greater (p < 0.05) than genistein or radiation alone.

Figure 2

Determination of the most effective combined treatment sequence in PC-3 cells. PC-3 cells were analyzed by clonogenic assay to determine the most effective combined genistein + radiation treatment sequence. (A) PC-3 cells treated with 15 μmol/L genistein for 24 hr then irradiated with 3 Gy photons and plated in clonogenic assay. (B) PC-3 cells treated with 15 μmol/L genistein for 24 hr then irradiated with 3 Gy photons, followed by plating and continued treatment with 15 μmol/L genistein throughout clonogenic assay. (C) PC-3 cells irradiated with 3 Gy photons and 24 hr later treated with 15 μmol/L genistein for 24 hr then plated in clonogenic assay. (D) PC-3 cells irradiated with 3 Gy photons, and after 24 hr treated with 15 μmol/L genistein for 24 hr followed by plating and continued treatment with 15 μmol/L genistein throughout clonogenic assay. The colonies were stained and counted after 10 days incubation at 37°C in a 5% CO₂/5% O₂/90% N₂ incubator. Data are presented as the mean survival fraction (± S.E.M.) of triplicate wells as normalized to control cells based on plating efficiency. (*): value statistically significant from control at p < 0.05.

Figure 3

Cell cycle analysis of PC-3 cells treated with genistein and radiation. PC-3 cells were treated with either 15 μmol/L or 30 μmol/L genistein for 24 hr, 3 Gy photon radiation, or 15 μmol/L or 30 μmol/L genistein for 24 hr followed by 3 Gy photon radiation. On days 3 and 4 post-radiation, cells were processed for DNA content and cell cycle progression was analyzed by flow cytometry. (Arrow in Fig. 3L): indicates pre-G₀/G₁ apoptotic peak.

Figure 4

Effect of genistein and radiation treatment on p21^WAF1/Cip1 and cyclin B1 expression in PC-3 cells. PC-3 cells were treated with 30 μmol/L genistein for 24 hr then irradiated with 3 Gy photons. Nuclear and cytoplasmic proteins were extracted 1 hr post-radiation. The relative level of (A) p21^WAF1/Cip1 and (B) cyclin B1 protein in 20 μg of nuclear extract was determined by western blot analysis. Data are presented as the mean integrated density value (I.D.V.) of 3 separate experiments (± S.E.M.). (Con): control, untreated-cells; (Rad): cells irradiated with 3 Gy photons; (Gen): cells treated with 30 μmol/L genistein; (Gen + Rad): cells pre-treated with 30 μmol/L genistein for 24 hr then irradiated with 3 Gy photons. Retinoblastoma protein (Rb) was used as a nuclear protein loading control. (*): nuclear value statistically significant from control at p < 0.05; (**): cytoplasmic value statistically significant from control at p < 0.05.

Figure 5

Inhibition of radiation induced NF-κB activation and increased apoptosis by pre-treatment with genistein in PC-3 cells. (A) PC-3 cells were treated with 30 μmol/L genistein for 24 hr then irradiated with 3 Gy photons. At 30 min post-radiation, cells were processed for isolation of nuclear protein. NF-κB DNA binding activity in 10 μg of nuclear extract was determined using EMSA. The level of DNA binding activity is expressed as the mean integrated density value (I.D.V.) from 3 separate experiments (± S.E.M.). (B) The level of the 85-kDa cleaved PARP protein in 20 μg of nuclear extract was determined by western blot analysis. Data are presented as the mean integrated density value (I.D.V.) of 3 separate experiments (± S.E.M.). (Con): control, untreated-cells; (Rad): cells irradiated with 3 Gy photons; (Gen): cells treated with 30 μmol/L genistein; (Gen + Rad): cells pre-treated with 30 μmol/L genistein for 24 hr then irradiated with 3 Gy photons. Retinoblastoma protein (Rb) was used as a nuclear protein loading control; (*): value statistically significant from control at p < 0.05.