Involvement of p21 and FasL in induction of cell cycle arrest and apoptosis by neochamaejasmin A in human prostate LNCaP cancer cells

Neochamaejasmin A ( 1), a biflavonoid isolated from the roots of a traditional Chinese medicine, Stellera chamaejasme L., was shown to inhibit cellular (3)H-thymidine incorporation (IC 50 12.5 microg/mL) and subsequent proliferation of human prostate Cancer LNCaP cells. Treatment of LNCaP cells with low doses of 1 (< or =6.25 microg/mL) suppressed DNA-binding activities of the transcription factors NFkappaB and AP-1 to the promoter of cyclin D and also inhibited expression of the cell cycle regulatory proteins cyclin D, proliferating cell nuclear antigen, and nucleolin, thus arresting cells in G 1 phase of the cell cycle. A lengthy exposure with higher doses of 1 (> or =12.5 microg/mL) revealed the production of Reactive Oxygen Species, dissipation of the mitochondrial membrane potential, up-regulation of cyclin-dependent kinase inhibitor p21, and induction of cell Apoptosis. An aggregation of Fas-procaspase 8-procaspase 3 and p21-procaspase 3 proteins by coimmunoprecipitation, immunoblotting analysis, and MALDI-mass spectrometry indicated the involvement of Fas and p21 in 1-mediated cytotoxicity, and pretreatment of cells with antisense FasL Oligonucleotides partially abolished Apoptosis. Thus, 1 blocked cell cycle progression at the G 1 phase by activating the p21 protein and ultimately promoting the Fas-caspase 8-caspase 3 apoptotic machinery.