Induction of apoptosis in prostate cancer by ginsenoside Rh2

Abstract

The therapeutic action of ginsenoside Rh2 on several cancer models has been reported. This study aimed to evaluate its apoptotic effect on prostate cancer and the underlying mechanism. Cultured DU145 cells were treated with Rh2 (5 × 10^-5 to 1 × 10^-4 M), peroxisome proliferator-activated receptor-delta (PPAR-delta) antagonist GSK0660 (1 × 10^-6 to 5 × 10^-6 M); or small interfering RNA (siRNA) of PPAR-delta. The treatment effects were evaluated with cell viability assay, life/death staining and flow cytometry for apoptosis. Immunostaining was used for reactive oxygen species (ROS) and superoxide detection. Western blot analysis for PPAR-delta and signal transducer and activator of transcription 3 (STAT3) protein expression were performed. The results showed that Rh2 significantly decreased DU145 cell survival and increased cell apoptosis. ROS and superoxide induction, PPAR-delta up-regulation and phosphorylated STAT3 (p-STAT3) down-regulation by Rh2 were demonstrated. GSK0660 partially but significantly inhibited the Rh2-induced apoptosis and restored cell viability. Treatment with siRNA reversed the Rh2-induced apoptosis as well as changes in PPAR-delta and p-STAT3 expression. In conclusion, our findings have demonstrated that ginsenoside Rh2 induces prostate cancer DU145 cells apoptosis through up-regulation of PPAR-delta expression which is associated with p-STAT3 up-regulation and ROS/superoxide induction. Rh2 may be potentially useful in the treatment of prostate cancer.

Keywords: apoptosis; ginsenoside Rh2; peroxisome proliferator-activated receptor; prostate cancer.

Figure 1. Cell viability assay showing the inhibitory effect of Rh2 on DU145 cell viability

DU145 cells were incubated with Rh2 (5 × 10^–5 to 1 × 10^–4 M) for 24 h. Human prostate cancer PC-3 cells and prostate stromal myofibroblast cell WPMY-1 were used as controls. Rh2 significantly reduced cell viability of DU145 and PC-3 cells in a concentration-dependent manner but not the WPMY-1 cell. The data are expressed as the means ± S.E.M. (n = 8 for each group). ^*P < 0.05 and ^**P < 0.01 compared with control DU145 cells; ^#P < 0.05 and ^##P < 0.01 compared with WPMY-1 cells treated with Rh2.

Figure 2. Live/dead cell staining showing GSK0660 and siRNA inhibition on Rh2 apoptotic effect

DU145 cells were incubated with Rh2 (1 × 10^–4 M) with/without GSK0060 (1–5 × 10^–6 M) for 24 h or transfected with PPAR-delta siRNA 48 h prior to Rh2 treatment. Live cells were stained green, whereas dead cells are stained red. (A) Co-incubation with GSK0660 (1–5 × 10^–6 M) inhibited the Rh2 apoptotic effect. (B) PPAR-delta siRNA (SiPPARδ) but not scramble siRNA (Scramble) inhibited the Rh2 apoptotic effect on DU145 cells.

Figure 3. Cell viability assay showing GSK0660 inhibition on Rh2 apoptotic effect

DU145 cells were incubated with Rh2 (1 × 10^–4 M) with/without GSK0060 (1–5 × 10^–6 M) for 24 hours. The bars depict the quantitative data of cell viability assay on DU145 cells. The data are expressed as the means ± S.E.M. (n = 8 for each group). ^*P < 0.05 and ^**P < 0.01 compared with control DU145 cells; ^#P < 0.05 and ^##P < 0.01 compared with DU145 cells treated with Rh2 only.

Figure 4. Flow cytometry showing GSK0660 and siRNA inhibition on Rh2 apoptotic effect

DU145 cells were incubated with Rh2 (1 × 10^–4 M) with/without GSK0060 (1–5 × 10^–6 M) for 24 h or transfected with PPAR-delta siRNA 48 h prior to Rh2 treatment. or PPAR-delta siRNA for 24 hours. (A) Rh2 significantly increased the percentage of apoptotic cells; the effect was inhibited by GSK0660. (B) Rh2 significantly increased the percentage of apoptotic cells; the effect was inhibited by PPAR-delta siRNA (SiPPARδ) but not scramble siRNA (Scramble).

Figure 5. Western blot showing Rh2 effects on PPAR-delta and p-STAT3/STAT3 protein expression

DU145 cells were incubated with Rh2 (5 × 10^–5 to 1 × 10^–4 M) for 24 h. Rh2 significantly increased PPAR-delta and decreased P-STAT3/STAT3 expression in a concentration-dependent manner. The data are expressed as the means ± S.E.M. (n = 8 for each group). ^*P < 0.05 and ^**P < 0.01 compared with control DU145 cells.

Figure 6. Western blot showing siRNA inhibition on Rh2-induced PPAR-delta and p-STAT3/STAT3 changes

DU145 cells were transfected with PPAR-delta siRNA 48 h prior to incubation with Rh2 (1 × 10^–4 M). Rh2 significantly increased PPAR-delta and decreased P-STAT3/STAT3 expression. PPAR-delta siRNA (SiPPARδ) but not scramble siRNA (Scramble) inhibited the Rh2-induced changes. The data are expressed as the means ± S.E.M. (n = 8 for each group). ^**P < 0.01 compared with control DU145 cells; ^##P < 0.01 compared with DU145 cells treated with Rh2 only.

Figure 7. Fluorescence microscopy showing intracellular ROS and superoxide formation

DU145 cells were incubated with Rh2 (1 × 10^–4 M) with/without GSK0060 (1–5 × 10^–6 M) for 24 h or transfected with PPAR-delta siRNA 48 h prior to Rh2 treatment. or PPAR-delta siRNA for 24 hours. (A) Rh2 significantly increased intracellular ROS (green) and superoxide (red); the effect was inhibited by GSK0660. (B) Rh2 significantly increased intracellular ROS and superoxide; the effect was inhibited by PPAR-delta siRNA (SiPPARδ) but not scramble siRNA (Scramble). The data are expressed as the means ± S.E.M. (n = 8 for each group). The ROS and superoxide level in the control DU145 cells are taken as 1 arbitrary unit. ^*P < 0.05 and ^**P < 0.01 compared with control DU145 cells; ^#P < 0.05 and ^##P < 0.01compared with DU145 cells treated with Rh2 only.