Differential effects of whole soy extract and soy isoflavones on apoptosis in prostate cancer cells

Abstract

Previous studies have suggested that soy isoflavones exert anticarcinogenic effects against prostate cancer. We propose that soy extracts, containing a mixture of soy isoflavones and other bioactive components, would be a more potent chemo-preventive agent than individual soy isoflavones. We compared the apoptotic effects of whole soy extracts and individual soy isoflavones, genistein and daidzein, on prostate cancer cells. The soy extract contained 50% w/w of total isoflavones with approximately 1:5.5:3.5 ratios of genistin, daidzin and glycitin, respectively. Benign prostate hyperplasia (BPH-1), LnCap and PC3 cells were treated with varying concentrations of soy extract, genistein or daidzein and analyzed for cell cycle alterations and induction of apoptosis. At equal concentrations (25 micromol/L), soy extract induced a significantly higher percentage of cells undergoing apoptosis than genistein or daidzein (P < 0.001). No significant changes in cell cycle arrest or apoptosis were observed in non-cancerous BPH-1 cells treated with soy extract, suggesting that the effects of soy extract may be tumor cell specific. On the contrary, both genistein and daidzein induced apoptosis in BPH-1 cells, suggesting that individual isoflavones may have cytotoxicity in non-cancerous cells. Soy extracts also increased Bax expression in PC3 cells, but no significant changes in nuclear factor kappaB (NF kappaB) activation were detected, suggesting that the induction of apoptosis was independent of the NF kappaB pathway. Food products that bear a combination of active compounds may be more efficacious and safer as chemo-preventive agents than individual compounds. This 'whole-food'-based approach is significant for the development of public health recommendations for prostate cancer prevention.

Figure 1

High doses of genistein induced cell cycle arrest and apoptosis in benign prostate hyperplasia (BPH-1), LnCap and PC3 cells. BPH-1, LnCap and PC3 cells were treated with varying concentrations (25, 50, 100 and 200 μmol/L) of genistein or daidzein for 48 h, and labeled with SR-VAD-FMK stain (fluorochrome-conjugated caspase inhibitor) to assess multicaspase activity by flow cytometry. Greater than 100 μmol/L of genistein and daidzein induced caspase activation in LnCap and PC3 cells, but ≥ 50 μmol/L of genistein and daidzein also activated caspase activation in BPH-1 cells. Control treatment consisted of media with <0.1% dimethyl sulfoxide. Values represent means ± SEM, n = 3. * P < 0.05, ** P < 0.01 compared with control

Figure 2

25 μmol/L of soy extract induced apoptosis in PC3 and LnCap cells with no effect at equal concentrations of individual soy isoflavones. Benign prostate hyperplasia (BPH-1), LnCap and PC3 cells were treated with 25 μmol/L of soy extract (soy), genistein (gen) or daidzein (daid) for 48 h. Multicaspase activity and cell cycle analysis were measured by flow cytometry. Soy extract induced higher caspase activation, indicated by more SR-VAD-FMK positively stained cells (caspase + ve cells), than genistein or daidzein at equal concentrations (25 μmol/L) in both LnCap and PC3 cells. Soy extract also resulted in more cell population in the sub-G1 stage of the cell cycle in PC3 cells. No significant changes in caspase activity or cell cycle were observed in BPH-1 cells treated with soy extract or individual soy isoflavones. Control treatment consisted of media with <0.1% dimethyl sulfoxide. Values represent means ± SEM, n = 3. * P < 0.05, ** P < 0.01 compared with control

Figure 3

Soy and soy isoflavones increased Bax expression. The protein expression levels of Bax in nuclear extracts of treated cells were measured by Western blotting. (a) LnCap and (b) PC3 cells were treated with 25 μmol/L of soy extract (soy), genistein (gen) or daidzein (daid). Control treatment consisted of media with <0.1% dimethyl sulfoxide. Soy extract, genistein and daidzein increased Bax compared with control in PC3 cells, but not in LnCap cells. Representative blot of triplicate experiments

Figure 4

No effect of soy extract on nuclear factor κB activation, but an increase with daidzein treatment in PC3 cells after 48 h. (a) Western blots of nuclear and cytosolic extracts from PC3 cells treated with dimethyl sulfoxide (con), 25 μmol/L of soy extract (soy), genistein (gen) or daidzein (daid), against p50 and p65 antibodies. Relative density of nuclear/cytosolic p50 was obtained by dividing optical density (OD) of nuclear p50 to cytosolic p50 after normalized to respective β-actin. (b) Cytosolic extract of treated PC3 cells was raised against IKKβ, p-IκBα and IκBα. The relative density of p-IκBα/IκBα was obtained by dividing the OD of p-IκBα to IκBα and normalized to β-actin. Daidzein increased nuclear p50 and cytosolic p-IκBα compared with control, but no significant changes were observed in cells treated with soy extract or genistein. Representative blot of triplicate experiments. Densitometry values represent means ± SEM, n = 3. P < 0.01 compared with control