Effects of selenite and genistein on G2/M cell cycle arrest and apoptosis in human prostate cancer cells

Abstract

Combination of chemopreventive agents with distinct molecular mechanisms is considered to offer a potential for enhancing cancer prevention efficacy while minimizing toxicity. Here we report two chemopreventive agents, selenite and genistein, that have synergistic effects on apoptosis, cell cycle arrest, and associated signaling pathways in p53-expressing LNCaP and p53-null PC3 prostate cancer cells. We show that selenite induced apoptosis only, whereas genistein induced both apoptosis and G2/M cell cycle arrest. Combination of these two agents exhibited enhanced effects, which were slightly greater in LNCaP than PC3 cells. Selenite or genistein alone upregulated protein levels of p53 in LNCaP cells only and p21(waf1) and Bax in both cell lines. Additionally, genistein inhibited AKT phosphorylation. Downregulation of AKT by siRNA caused apoptosis and G2/M cell cycle arrest and masked the effects of genistein. Treatment with insulin-like growth factor I (IGF-I) elevated levels of total and phosphorylated AKT and suppressed the effects of genistein. Neither downregulation of AKT nor IGF-I treatment altered the cellular effects of selenite. Our study demonstrates that selenium and genistein act via different molecular mechanisms and exhibit enhanced anticancer effects, suggesting that a combination of selenium and genistein may offer better efficacy and reduction of toxicity in prostate cancer prevention.

FIG. 1

Induction of cell death, G₂/M cell cycle arrest, and apoptosis by selenite or genistein in LNCaP and PC3 cells. A: MTT assay of cell viability of LNCaP and PC3 cells treated with different doses of selenite for 5 days. B: The MTT assay of cell viability of LNCaP and PC3 cells treated with different doses of genistein for 5 days. C: Flow cytometric analysis of the G₂/M population in LNCaP and PC3 cells treated with different doses of selenite for 24 h. D: Flow cytometric analysis of the G₂/M population in LNCaP and PC3 cells treated with different doses of genistein for 24 h. E: Flow cytometric analysis of Annexin V-positive apoptotic cell population in LNCaP and PC3 cells treated with different doses of selenite for 48 h. F: Flow cytometric analysis of Annexin V-positive apoptotic cell population in LNCaP and PC3 cells treated with different doses of genistein for 48 h. Data were obtained from 3 independent experiments and the results shown are mean ± SD. *P < 0.05 compared with 0 µM.

FIG. 2

Induction of G₂/M cell cycle arrest and apoptosis by cotreatment with selenite and genistein in LNCaP and PC3 cells. A and B: The MTT assay of cell viability of LNCaP cells and PC3 cells treated with selenite (Sel), genistein (Gen), or in combination for 5 days. C and D: Flow cytometry analysis of the G₂/M population in LNCaP cells and PC3 cells treated with selenite, genistein, or in combination for 24 h. E and F: Flow cytometric analysis of Annexin V-positive apoptotic cell population in LNCaP cells and PC3 cells treated with selenite, genistein, or in combination for 48 h. Data were obtained from 3 independent experiments and the results shown are mean ± SD. Means with different letters above bars indicate significant differences (P < 0.05) in each panel. DMSO, dimethyl sulfoxide.

FIG. 3

Effects of genistein and selenite on Akt, p53, p21^waf1, and Bax in LNCaP and PC3 cells. A and B: Western blot analysis of total AKT, phosphorylated AKT at serine 473 [p-AKT(Ser 473)], total p53, phosphorylated p53 at serine 15 [p-p53(Ser 15)], p21^waf1, and Bax in LNCaP cells and PC3 cells. Cells were treated with selenite (Sel) or genistein (Gen) for 24 h, and 50 µg proteins were loaded for the assay. The results shown here is one representative of 3 repeated experiments. DMSO, dimethyl sulfoxide.

FIG. 4

Effects of downregulation of AKT on selenite- and genistein-induced cell cycle arrest and apoptosis in LNCaP and PC3 cells. A and B: Western blot analysis of effects of AKT siRNA transfection on protein levels of total AKT and phosphorylated AKT (p-AKT at Ser 473) in LNCaP and PC3 cells. Cells were transfected with 50 nM AKT siRNA for 24 h and then treated with selenite (Sel), genistein (Gen), or in combination for another 24 h. Fifty µg of protein were loaded for the assay. C and D: MTT assay of cell viability of LNCaP and PC3 cells treated with selenite, genistein or in combination for 5 days after transfection with 50 nM AKT siRNA for 24 h. E and F: Flow cytometric analysis of the G₂/M population of LNCaP cells and PC3 cells treated with selenite, genistein, or in combination after transfection with AKT siRNA. Cells were treated with selenite, genistein, or in combination for 24 h after transfection with 50 nM AKT siRNA for 24 h. G and H: Flow cytometric analysis of apoptosis in LNCaP and PC3 cells treated with selenite, genistein, or in combination for 48 h after transfection with 50 nM AKT siRNA for 24 h. Data were obtained from 3 independent experiments, and the results shown are mean ± SD. Group 1, control siRNA transfection. Group 2, AKT siRNA transfection. Means with different letters above bars indicate significant differences (P < 0.05) in each panel.

FIG. 5

Inhibition of genistein-induced cell cycle arrest and apoptosis by insulin-like growth factor-I (IGF-I). A and B: Western blot analysis of effects of IGF-I on expression of total AKT and p-AKT (Ser 473) in LNCaP and PC3 cells. Cells were treated with 20 nM IGF-I for 24 h followed by treatment with selenite, genistein or in combination for another 24 h. Fifty µg of protein were loaded for the assay. C and D: MTT assay of cell viability of LNCaP and PC3 cells treated with selenite, genistein, or in combination for 5 days after pretreatment with 20 nM IGF-I for 24 h. E and F: Flow cytometric analysis of the G₂/M population of LNCaP cells and PC3 cells treated with selenite, genistein, or in combination for 24 h after pretreatment with IGF-I for 24 h. G and H: Flow cytometric analysis of apoptosis in LNCaP cells and PC3 cells treated with selenite, genistein, or in combination for 48 h after pretreatment with IGF-I for 24 h. The data were obtained from 3 independent experiments and the results shown are mean ± SD. Group 1, without IGF-I pretreatment. Group 2, with IGF-I pretreatment. Means with different letters above bars indicate significant differences (P < 0.05) in each panel.