Benincaside A Induces p53-Dependent Transactivation and Fas/CD95-Mediated Apoptosis in HCT 116 Human Colorectal Cancer Cells

An undescribed seco-kaurane diterpenoid, benincaside A (BA), was isolated from the seeds of Benincasa hispida. The seeds of B. hispida have been traditionally used in folk medicine and previous studies have reported anti-tumor potential in B. hispida seed extracts. Accordingly, we investigated the cytotoxicity and underlying mechanisms of BA in colorectal cancer cells. BA inhibited growth in HT29, Colo205, HCT116, and CT26 colorectal cancer cells, as determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while showing no toxicity toward normal human umbilical vein endothelial cells (HUVEC) and human fibroblast WS-1 cells. In HCT116 cells, BA-induced deoxyribonucleic acid (DNA) damage and apoptosis, as evidenced by morphological changes, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and assays of caspase-8 and caspase-3 activities. BA triggered apoptotic cell death via the extrinsic pathway, as indicated by elevated caspase-8 and caspase-3 activities. Intracellular reactive oxygen species (ROS) generation was observed in BA-treated HCT116 cells. The growth-inhibitory effects were significantly attenuated by pretreatment with N-acetylcysteine (NAC, an antioxidant), caffeine (an ATM kinase inhibitor), z-VAD-fmk (pan-caspase inhibitor), or z-IETD-fmk (caspase-8-specific inhibitor). Colorimetric assays confirmed increased caspase-8 and caspase-3 activities in BA-treated cells. This study is the first to report ROS-dependent signaling as a key mechanism underlying BA-induced cell death in HCT116 human colorectal cancer cells.