The treatment of the cells with various concentrations of biseugenol B compound was carried out for 180 minutes, followed by stimulation with 10 ng/mL of TNF- for half an hour. cytotoxicity toward PC3 with no toxicity toward normal prostate cells (RWPE-1), which indicates that biseugenol B has qualities that induce apoptosis in tumor cells. The treatment of PC3 cells with biseugenol B provoked apoptosis with cell-death-transducing signals. Downregulation of Bcl-2 and upregulation of Bax regulated the MMP, which in turn caused the release of cytochrome c from mitochondria into cytosol. The release of cytochrome c activated caspase-9, which consequently activated caspase-3/7 with the cleaved poly(ADP-ribose) polymerase protein, thereby resulting in apoptosis alteration. Involvement of an extrinsic apoptosis pathway was exhibited by the increase in caspase-8, while the increase in caspase-3/7 and caspase-9 demonstrated involvement of an intrinsic apoptosis pathway. Meanwhile, no significant increase was observed in caspases 3/7, 8 or 9 MELK-IN-1 in normal prostate cells (RWPE-1) after treatment with biseugenol B. Prevention of NF-B translocation from Akt1 the cytosol to the nucleus occurred in PC3 after treatment with biseugenol B. The results of our study reveal that biseugenol B triggers the apoptosis of PC3 cells via intrinsic and extrinsic apoptosis pathways and inhibition of NF-B signaling pathway. Our findings suggest that biseugenol B is a potentially useful agent for prostate cancer treatment. is considered as an evergreen genus distributed in tropical and subtropical Asia, as well as in North and South America.8 is used widely in Peoples Republic of China and Malaysia as a traditional medicine for influenza and stomachache.9 In addition, contains neolignans, a chemical compound in plants, which is used in traditional Chinese medicine to treat viral hepatitis and to protect the liver.10 Neolignans also exhibit pharmacological activity in MELK-IN-1 mammalian cells.11 Moreover, N6-isopentenyladenosine (iPA), isolated from and belongs to the main group of natural origin, neolignan and oxyneolignan, which possess anti-proliferative and anti-cancer properties.14C16 The chemical structure of 2,2-oxybis (4-allyl-1-methoxybenzene) or biseugenol B is shown in Figure 1.17 Open in a separate window Figure 1 Structures of compound 2,2-oxybis (4-allyl-1-methoxybenzene) or biseugenol B. In this study, we evaluated the apoptosis cell-death mechanism through a novel compound called biseugenol B using human prostate cancer cells (PC3) as an in vitro model. Methodology Cell culture Prostate cancer cells (PC3) and normal prostate cells (RWPE-1)18 were obtained from the American Type Cell Collection (Manassas, VA, USA) and incubated at 37C with 5% CO2.19 Prostate cancer cells (PC3) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% fetal bovine serum (FBS) and 1% of 100 unit/mL of penicillin and streptomycin,20 and normal prostate cells (RWPE-1) were cultured in a concentration of 4104 keratinocyte serum-free medium (K-SFM) supplemented with 0.2 ng/mL human epidermal growth factor (rhEGF) MELK-IN-1 and 25 g/mL bovine pituitary extract (BPE)21 and 1 antibiotic/antimycotic solution. Cultures were incubated at 37C in a humidified atmosphere containing 5% CO2 and passed weekly.22C24 Cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) By using MTT assay, viability assay was performed as described by Mohan.19 Briefly, 5104 cells were treated with biseugenol B at different concentrations in a 96-well plate and maintained in incubation for 24, 48 and 72 hours. At absorbance of 570 nm, the colorimetric assay was measured and recorded. The results were taken as a percentage of control giving percentage cell viability after 24, 48 and 72 hours exposure to test agent. The half maximal inhibitory concentration (IC50) value was measured as the potency of cell growth inhibition for test agent.19 Quantification of apoptosis using propidium iodide (PI) and acridine orange (AO) double staining The method of quantification of apoptosis was performed MELK-IN-1 by applying AO and PI double staining. Cell death induced by biseugenol B in.