Studies On The Mechanisms Of Oridonin-induced U937 Cell Apoptosis And -enhanced Phagocytosis Of Apoptotic Bodies

This dissertation reports mechanisms of oridonin-induced cell death in human histocytic lymphoma U937 cells as well as those of oridonin-enhanced phagocytosis of UV-irradiated apoptotic cells by U937 cell-derived macrophage-like cells.The studies demonstrate that oridonin significantly inhibited proliferation of U937 cells in a time- and dose-dependent manner. MTT assay, photomicroscopical observation, fluorescence microscopical observation, and DNA agarose gel electrophoresis showed that oridonin induced apoptotic cell death in U937 cells.U937 cells showed susceptible to apoptosis induced by 27 μM oridonin and an agonistic anti-Fas IgM mAb (CH-11) (500 ng/ml) as a Fas-sensitized positive control. Among inhibitors of initiator capases, caspase 8 inhibitor z-IETD, but neither caspasel inhibitor Ac-YVAD nor caspase 10 inhibitor z-AEVD, effectively blocked oridonin-induced cell death as well as DNA fragmentation. Western blot analysis showed the up-regulated expression of Fas, FasL, and FADD, and down-regulated expression of procaspase 8, suggesting that Fas/FasL pathway was activated in oridonin-induced cell apoptosis. Further, stimulation of U937 cells with oridonin and CH11 resulted in significant ERK MAPK activation. However, inhibition of ERK by PD98059 reversed oridonin-induced cell death as well as the activation of caspase 8, indicating that ERK-mediated control occured upstream of caspase 8. In addition, the expression of antiapoptotic mitochondrial protein Bcl-2 and Bcl-XL decreased, and that of proapoptotic protein Bax as well as the release of cytochrome c increased. Simultaneously, caspase 3 proenzyme decreased followed by the degradation of caspase-3 substrates, ICAD (inhibitor of caspase dependent DNase) and PARP (poly-(ADP-ribose) polymerase). However, PD98059 reversed the effect of oridonin on BcI-Xl, Bax as well as the release of cytochrome c, but failed to influence decreased Bcl-2 expression induced by oridonin. Taken together, these results suggest that Fas/FasL signaling pathway-mediated ERK activation sensitized U937 cells to mitochondrial pathway-mediated apoptosis induced by oridonin.On the other hand, preincubation of U937 cell-derived macrophages with 1.35 μM...