Mechanism of apoptosis induced by polyterpene from buna-shimeji (Hypsizigus marmoreus) in HL-60 cells

Hypsiziprenol Asub(9) is a polyterpene isolated from the fruiting body of the Japanese edible mushroom Buna-shimeji (Hypsizigus marmoreus). Although our recent studies revealed that hypsiziprenol Asub(9) has strong anti-tumor activity against tumor-bearing mice, its mechanism of action remains unclear. To elucidate further its anti-tumor action, we examined in detail hypsiziprenol Asub(9)-induced apoptosis in human cancer cell lines. We observed that hypsiziprenol Asub(9) strongly inhibited the growth of human promyelocytic leukemia HL-60 cells and several other cancer cell lines in a dose-dependent manner. Hoechst 33342 staining showed DNA fragmentation and condensation of chromatin in HL-60 cells treated with hypsiziprenol Asub(9). DNA laddering, a hallmark of apoptosis, was detected using agarose gel electrophoresis. In addition, flow cytometric analysis confirmed that hypsiziprenol Asub(9) increased sub-Gsub(1)/Gsub(0) populations in a time-dependent manner. Hypsiziprenol Asub(9) also increased activities of caspase-2, -3, -8 and -9 in a dose-dependent manner in the apoptotic HL-60 cell. Furthermore, JC-1 fluorescent staining determined that hypsiziprenol Asub(9)-induced apoptosis was associated with a loss of mitochondrial membrane potential. These results suggest that hypsiziprenol Asub(9) inhibits the growth of HL-60 cells by inducing apoptosis that is mediated through mitochondrial membrane potential loss and caspase activation.