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Apigenin and silybinin induce apoptosis and cause cell cycle arrest in prostate epithelial cells
Apigenin and Silybinin induce Apoptosis and cause cell cycle arrest In Prostate Epithelial Cells Colm Morrissey, Amanda O’Neill, Barbara Spengler*, Volker Christoffel*, John M. Fitzpatrick and R. William. G. Watson Department of Surgery, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Mater Misericordiae University Hospital, Dublin, Ireland. *Bionorica AG, Kerschensteinerstr. 1 l-l 5, 92318, Neumarkt, Germany.
Introduction Phytoestrogens are non-steroidal plant-derived compounds possessing have recently been implicated in protecting against prostate cancer phytoestrogens inhibit cell growth and induce apoptosis in prostate reduce tumour volume in vivo. We screened five phytoestrogens for and apoptosis in two prostate epithelial cell lines, PWR-IE (benign) vitro.
Materials
oestrogenic activity and progression. A number of cancer cells in vitro and their effect on cell growth and PC-3 (metastatic) in
and Methods
PWR-1 E and PC-3 cells were treated with or without the purified phytoestrogens, and assessed for their effect on cell number using the crystal violet assay, cell cycle using flow cytometry, steady state protein levels by western analysis and apoptosis using prodidium iodide incorporation.
Results Two compounds apigenin and silybinin (O-100 PM) significantly reduced cell number in both cell lines. Apigenin (50 PM) inducing apoptosis in both cell lines, silybinin inducing apoptosis in the PWR-IE cells and cell cycle arrest in the PC-3 cell line maintaining a greater number of the cells in G2M phase at higher concentrations (100 PM). The metastatic PC-3 cell line is less susceptible to apigenin and silybinin treatment indicating their altered susceptibility to apoptosis. The induction of apoptosis by apigenin in the PWR-IE cells was inhibited by a pan-caspase inhibitor (Z-VAD-FMK) and a caspase 9 inhibitor (Z-LEHD-FMK). This suggests that apigenin induced apoptosis in the PWR-1 E cells is caspase dependent. The induction of apoptosis by apigenin in the PC-3 cells was not inhibited by Z-VAD-FMK, but was inhibited by Z-LEHD-FMK. The anti-apoptotic protein Bcl-2 and the inhibitor of apoptosis protein (clAP-2) steady state protein levels were higher and Bax levels were lower in the PC-3 compared to the PWR-1 E cells treated with apigenin. This suggests that apigenin induced apoptosis in the PC-3 cells is caspase 9 dependent, but may be blocked by the expression of anti-apoptotic proteins like Bcl-2 and c-IAP-2.
Conclusions The altered responsiveness of the two cell lines to the phytoestrogens demonstrate that determining the phenotype of the tumour may indicate the effectiveness of the treatment regime. These in vitro studies will lead to a better understanding of the phytoestrogens and their possible use in prostate cancer prevention or
Introduction Phytoestrogens are non-steroidal plant-derived compounds possessing have recently been implicated in protecting against prostate cancer phytoestrogens inhibit cell growth and induce apoptosis in prostate reduce tumour volume in vivo. We screened five phytoestrogens for and apoptosis in two prostate epithelial cell lines, PWR-IE (benign) vitro.
Materials
oestrogenic activity and progression. A number of cancer cells in vitro and their effect on cell growth and PC-3 (metastatic) in
and Methods
PWR-1 E and PC-3 cells were treated with or without the purified phytoestrogens, and assessed for their effect on cell number using the crystal violet assay, cell cycle using flow cytometry, steady state protein levels by western analysis and apoptosis using prodidium iodide incorporation.
Results Two compounds apigenin and silybinin (O-100 PM) significantly reduced cell number in both cell lines. Apigenin (50 PM) inducing apoptosis in both cell lines, silybinin inducing apoptosis in the PWR-IE cells and cell cycle arrest in the PC-3 cell line maintaining a greater number of the cells in G2M phase at higher concentrations (100 PM). The metastatic PC-3 cell line is less susceptible to apigenin and silybinin treatment indicating their altered susceptibility to apoptosis. The induction of apoptosis by apigenin in the PWR-IE cells was inhibited by a pan-caspase inhibitor (Z-VAD-FMK) and a caspase 9 inhibitor (Z-LEHD-FMK). This suggests that apigenin induced apoptosis in the PWR-1 E cells is caspase dependent. The induction of apoptosis by apigenin in the PC-3 cells was not inhibited by Z-VAD-FMK, but was inhibited by Z-LEHD-FMK. The anti-apoptotic protein Bcl-2 and the inhibitor of apoptosis protein (clAP-2) steady state protein levels were higher and Bax levels were lower in the PC-3 compared to the PWR-1 E cells treated with apigenin. This suggests that apigenin induced apoptosis in the PC-3 cells is caspase 9 dependent, but may be blocked by the expression of anti-apoptotic proteins like Bcl-2 and c-IAP-2.
Conclusions The altered responsiveness of the two cell lines to the phytoestrogens demonstrate that determining the phenotype of the tumour may indicate the effectiveness of the treatment regime. These in vitro studies will lead to a better understanding of the phytoestrogens and their possible use in prostate cancer prevention or