Sanggenol L Induces Apoptosis and Cell Cycle Arrest via Activation of p53 and Suppression of PI3K/Akt/mTOR Signaling in Human Prostate Cancer Cells

Abstract

Prostate cancer is the most common cancer in Western countries. Recently, Asian countries are being affected by Western habits, which have had an important role in the rapid increase in cancer incidence. Sanggenol L (San L) is a natural flavonoid present in the root barks of Morus alba, which induces anti-cancer activities in ovarian cancer cells. However, the molecular and cellular mechanisms of the effects of sanggenol L on human prostate cancer cells have not been elucidated. In this study, we investigated whether sanggenol L exerts anti-cancer activity in human prostate cancer cells via apoptosis and cell cycle arrest. Sanggenol L induced caspase-dependent apoptosis (up-regulation of PARP and Bax or down-regulation of procaspase-3, -8, -9, Bid, and Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), suppression of cell cycle (down-regulation of CDK1/2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B1 or up-regulation of p53 and p21), and inhibition of PI3K/Akt/mTOR signaling (down-regulation of PI3K, p-Akt, and p-mTOR) in prostate cancer cells. These results suggest the induction of apoptosis via suppression of PI3K/Akt/mTOR signaling and cell cycle arrest via activation of p53 in response to sanggenol L in prostate cancer cells.

Keywords: AIF; PI3K/Akt/mTOR; apoptosis; p53; prostate cancer; sanggenol L.

Figure 1

Sanggenol L inhibits cell growth in various human prostate cancer cell lines. (A) Chemical structure of sanggenol L. (B) Cell viabilities on DU145, LNCap, RC-58T, and PC-3 cells were tested after treatment with or without 10, 20, and 30 µM sanggenol L for 48 h. Cell viability was measured by SRB assay. Results were expressed as the percentage of control. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared to the control at * p < 0.05 and *** p < 0.001 using one-way ANOVA. (C) Cell viability of RC-58T cells was tested after treatment with or without 10, 20, and 30 µM sanggenol L for 24, 48, and 72 h by SRB assay. Results were expressed as the percentage of control. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared to the control at *** p < 0.001 using one-way ANOVA. (D) RC-58T cells were treated with or without 10, 20, or 30 µM sanggenol L for 48 h. Cell morphological changes were visualized by inverted microscopy (200×). Scale bar, 100 μm.

Figure 2

Sanggenol L induces apoptosis in RC-58T human prostate cancer cells. Cells were treated with or without 10, 20, and 30 µM sanggenol L for 48 h. (A) Apoptotic cells were evaluated by Annexin V staining assay. (B) Total apoptotic cells were quantified and Results were expressed as the percentage of control. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Dunnett’s test; *** p < 0.001. (C) DNA fragmentation was measured by performing 2% agarose gel electrophoresis. (D) Nuclear condensations were detected by Hoechst staining assay upon sanggenol L treatment.

Figure 3

Sanggenol L induces caspase-dependent apoptosis in RC-58T human prostate cancer cells. Cells were treated with or without 30 µM sanggenol L for 48 h. (A) The levels of procaspase-3, -8, -9, and cleaved-PARP, Bid, Bax, and Bcl-2 proteins in RC-58T prostate cancer cells. Total cell lysates were subjected to western blot analysis to measure the protein expression levels. (B) The influence of z-VAD-fmk (a caspase inhibitor) was measured by SRB assay. Results were expressed as the percentage of control. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Dunnett’s test; *** p < 0.001. (C) Total apoptotic proteins were quantified and data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Student’s t-test; ** p < 0.01 and *** p < 0.001.

Figure 4

Sanggenol L induces caspase-independent apoptosis through an apoptosis-inducing factor in RC-58T human prostate cancer cells. (A) The influence of N-PM (an AIF inhibitor) was measured by SRB assay. Significant differences were calculated using Dunnett’s test; *** p < 0.001. (B) The levels of AIF and Endo G proteins in RC-58T prostate cancer cells. Total cell lysates were subjected to western blot analysis to measure the protein expression levels. (C) Total apoptotic proteins were quantified and data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Student’s t-test; *** p < 0.001.

Figure 5

Sanggenol L induces cell cycle arrest in RC-58T cells. (A) Cells were treated with sanggenol L (10, 20, and 30 μM) for 48 h and cell cycle progression was analyzed by using the Muse™ cell cycle kit. (B) Cell populations at various cell cycle phases were quantified and data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Dunnett’s test; *** p < 0.001. (C) The levels of cell cycle-related proteins in RC-58T cells. (D) The levels of p53 and p21 proteins in RC-58T cells. Total cell lysates were subjected to western blot analysis to determine the protein expression levels. β-Actin, a housekeeping gene, was used as the denominator to quantify relative gene expression levels.

Figure 6

Sanggenol L inhibits cell growth of prostate cancer cells through suppression of the PI3K/Akt/mTOR signaling pathway. (A) The levels of phospho-PI3K, phospho-Akt, and phospho-mTOR proteins in RC-58T prostate cancer cells. Cells were treated with or without 10, 20, and 30 μM sanggenol L for 48 h. Total cell lysates were subjected to western blot analysis to measure the protein expression levels. β-Actin, a housekeeping gene, was used as the denominator to quantify relative gene expression levels. (B) The influence of LY294002 (a PI3K inhibitor) was determined by SRB assay. Results were expressed as the percentage of control. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were calculated using Student’s t-test; * p < 0.05, ** p < 0.01, and *** p < 0.001.