Death receptor 5-mediated TNFR family signaling pathways modulate γ-humulene-induced apoptosis in human colorectal cancer HT29 cells

A component from Emilia sonchifolia (L.) DC, gamma-humulene, was investigated. Significantly decreased cell viability of human colorectal cancer HT29 cells in a dose-dependent manner with IC50 53.67 +/- 2.99 mu M for 24-h treatment was found. gamma-Humulene induced apoptotic cell death and apoptosis was confirmed by morphological assessment. The staining with propidium iodide (PI) and flow cytometric analysis also showed that gamma-humulene significantly promoted the sub-G1 phase (an apoptotic population) in HT29 cells. Colorimetric assays indicated that pretreatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced activities of caspase-8 and caspase-3 in examined HT29 cells. gamma-Humulene stimulated the death receptor 5 (DR5), DR4, Fas-associated protein with death domain (FADD), the cleavage of caspase-8 and cleavage caspase-3, but reduced the protein levels of cellular Fas-associated death-domain-like IL-1 beta-converting enzyme inhibitory protein (c-FLIP) by Western blot analysis. Consequently, gamma-humulene-triggered cell death was significantly attenuated by DR5 siRNA and the caspase-8 inhibitor. Based on our results, we suggest that gamma-humulene induced apoptotic cell death in HT29 cells through a DR5-mediated caspase-8 and -3-dependent signaling pathway. Therefore, this agent might be useful for developing new therapeutic regimens for treatment of colorectal cancer in the future.