Abstract. Oridonin, a diterpenoid isolated from Rabdosia rubescences, could induce apoptosis through the generation of reactive oxygen species (ROS) in human hepatoma HepG2 cells. p53, a specific inhibitor of pifithrin α (PFT α), markedly inhibited ROS generation and apoptosis, showing that p53 was responsible for the cytotoxity of oridonin through mediation by ROS. Moreover, the ROS activated the p38 kinase, which in turn promoted the activation of p53, as verified by evidence showing that the ROS scavenger N-acetyl-cysteine (NAC) not only blocked the phosphorylation of p38 but also partially inhibited the activation of p53, and the p38 inhibitor SB203580 reduced the activation of p53 as well. Mitochondria were either the sources or the targets of ROS. This study showed that oridonin stimulated mitochondrial transmembrane permeabilization in a ROS-dependent manner because NAC almost thoroughly reversed the drop of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c from the mitochondrial inter-membrane space into cytosol. Furthermore, as a result of mitochondrial permeability transition, procaspases-9 and -3 were cleaved into 37-and 17-kDa proteolytic products, respectively, which acted as executors of oridonin-induced apoptosis.

Abstract. Natural products regulate cell growth in response to oncogene activation that induces cell cycle arrest and apoptosis in tumor cell lines. We investigated the mechanisms of caspase activation in human malignant melanoma, A375-S2 cells, by the natural product shikonin, which was isolated from the plant Lithospermum erythrorhizon SIEB. et ZUCC. Shikonin inhibited cell growth in a time-and dose-dependent manner, which might be mediated through up-regulation of p53 and down-regulation of cyclin-dependent protein kinase 4. Caspase activation was detected in shikonin-induced cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 protein levels and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Apoptosis-inducing factor, another apoptotic protein of mitochondria, partially contributed to shikonin-induced release of cytochrome c. Taken together, shikonin-induced DNA damage activates p53 and caspase-9 pathways.

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Oridonin is extracted and purified from traditional Chinese herbs, Rabdosia rubescens. It is a complicated compound containing diterpenoids and other ingredients. It is also one of the most important traditional Chinese herbs commonly used in clinical treatment nowadays. 1) Oridonin has been studied exclusively in China and has been shown to possess anti-tumor activity against murine sarcoma and leukemia cells. 2,3) Programmed cell death comprises several subtypes, as revealed by morphology. Autophagic cell death or type II programmed cell death is an evolutionary conserved membrane trafficking pathway which mediates the transport of cytosolic materials and superfluous organelles and long-lived proteins to the lysosome and occurs in all eukaryotic cells. 4,5) During autophagy, a cup-shaped structure, the pre-autophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes.6) The formation of the autophagosome is also dependent upon the enzyme phosphatidylinositol 3-phosphate kinase (class III PI3K) in mammalian cells 7) and its ortholog Vps34 in yeast.8) The first evidence for the implication of this enzyme stemmed from the discovery that 3-methyladenine is able to block the formation of autophagosomes. 9) In vitro studies showed that 3-MA inhibits PI3K activity.7,10) The inhibitory effect of 3-MA on intracellular trafficking of proteins from late endosomes to lysosomes 11) is compatible with the requirement of PI3K at this step. 12)Apoptotic cell death or type I programmed cell death was originally conceived on morphologic grounds, namely describing the condensation of chromatin at the nuclear membrane, prominent condensation of cytoplasm and the fragmentation of cell and nucleus, while organelles were well preserved and autophagocytosis was essentially absent. 13) It has been reported that oridonin was found to induce growth inhibition and apoptosis of human breast cancer cell line 14) and this cell line could be induced autophagy by tamoxifen.15) Therefore, in this study, we further investigated the relationship between apoptosis and autophagy specifically induced by oridonin in MCF-7 cells. MATERIALS AND METHODS MaterialsOridonin was obtained from the Kunming Institute of Botany, The Chinese Academy of Sciences (Kunming, China). Fetal bovine serum (FBS) was purchased from TBD Biotechnology Development (Tianjin, China); Monodansylcadervarine (MDC), 3-methyladenine (3-MA), propidium iodode (PI), P38 inhibitor SB203580, JNK inhibitor SP600125, ERK1/2 inhibitor PD98059, Hoechst 33258 and RNase A were purchased from Sigma Chemical (St. Louis, MO, U.S.A); Thiazolyl blue (MTT) was from Sino-American Biotechnology (Beijing, China); rabbit polyclonal antibodies against JNK, phosphorylated JNK, P38, phosphorylated P38, Bax and Bcl-2; mouse polyclonal antibodies against ERK and phosphorylated ERK, horseradish peroxidase-conjugated...

he dried, unripe fruit of Evodiae Fructus (Evodia rutaecarpa Benth., Rutaceae) is known as "Goshuyu" in Japanese. In traditional Chinese medicine, it has long been utilized for the treatment of headache, thoracicoabdominal pain, vomiting, cold and reduced blood circulation.