manumycin and oridonin

We have previously shown that oridonin isolated from Rabdosia rubescens augmented apoptosis while inhibiting autophagy within 24 h in HeLa cells. However, the mechanisms between apoptosis and autophagy induced by oridonin in A431 cells are largely unknown. Here, it was found that autophagic level is significantly upregulated when A431 cells are pretreated with manumycin A (Ras specific inhibitor) compared with oridonin alone treatment, whereas cells precultured with GW5074 (Raf inhibitor) or PD98059 (ERK inhibitor) did not exhibit such an effect. Ras, but not Raf or ERK, was engaged in the control of oridonin-induced autophagy. At the same time, manumycin A contributes to oridonin-induced downregulation of Ras protein expression. Treatment with the combination of oridonin and manumycin A downregulated phosphorylation of Akt, downstream of phosphatidylinositol 3-OH kinase (PI3-K). Preincubation with the PI3-K inhibitor wortmannin and Akt inhibitor KP372-1 enhanced oridonin-induced apoptosis, whereas it inhibited oridonin-induced autophagy. However, under oridonin treatment, the expression of Beclin-1, which has autophagy-inducing activity, was reduced, suggesting that Beclin-1 did not participate in the oridonin-induced autophagy. Morphologic observations, DNA fragmentation analysis, and LDH activity-based assay showed that 3-methyladenine (3-MA), an inhibitor of autophagy, increased the apoptotic sensitivity of A431 cells to oridonin. In addition, manumycin A contributed to oridonin-induced decrease of mitochondrial membrane potential (Deltapsim), consistent with the upregulation of Bax/Bcl-2 ratio. In conclusion, Ras negatively regulated autophagy in oridonin-treated A431 cells, which might be associated with activation of class I PI3-K. Downregulation of Deltapsim and increasing of the ratio of Bax/Bcl-2 might also be partially responsible for the initiation of the autophagic process.

Topics: Adenine; Androstadienes; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; bcl-X Protein; Beclin-1; Cell Line, Tumor; Cell Proliferation; Diterpenes; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; Indoles; Membrane Potential, Mitochondrial; Membrane Proteins; Microtubule-Associated Proteins; Molecular Structure; Phenols; Phosphatidylethanolamines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Polyenes; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-raf; ras Proteins; Wortmannin

In this study, we investigated autophagy induced by oridonin in HeLa cells. HeLa cells were exposed to oridonin, and the fluorescent changes, autophagic levels, and protein expressions were evaluated. Oridonin induced autophagy in HeLa cells in vitro in a dose- and time-dependent manner. Oridonin-treated HeLa cells, which had been prelabeled with the autophagosome-specific dye monodansylcadervarine (MDC), recruited more MDC-positive particles and had a significantly higher fluorescent density; and simultaneously, expressions of autophagy-related proteins, MAP-LC3 and Beclin 1, were increased by oridonin. In oridonin-induced Hela cells, pretreatment with 3-methyladenine (3-MA, the specific inhibitor of autophagy) dose-dependently decreased the autophagic ratio accompanied with downregulation of the protein expressions of MAP-LC3 and Beclin 1. Furthermore, when a Ras inhibitor was applied, the autophagic levels were augmented, whereas P38 and JNK inhibitors decreased the autophagic ratio significantly, indicating that this oridonin-induced autophagic process was negatively regulated by Ras, but positively regulated by P38 and JNK MAPKs. Raf-1 and ERK1/2 had no obvious correlation to these signaling pathways.

Topics: Adenine; Anthracenes; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Cadaverine; Diterpenes; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Flow Cytometry; HeLa Cells; Humans; Imidazoles; Isodon; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Microtubule-Associated Proteins; p38 Mitogen-Activated Protein Kinases; Polyenes; Polyunsaturated Alkamides; Pyridines; ras Proteins

Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFalpha and IL-1beta release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-kappaB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IkappaBalpha degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1beta precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IkappaBalpha degradation, resulting in NF-kappaB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1beta, which partially contribute to phagocytic activity of oridonin.

Topics: Apoptosis; Blotting, Western; Diterpenes; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Genes, ras; Humans; I-kappa B Proteins; Interleukin-1; Leupeptins; Macrophages; Phagocytosis; Polyenes; Polyunsaturated Alkamides; Proline; Proto-Oncogene Proteins c-raf; ras Proteins; Thiocarbamates; U937 Cells

Oridonin, isolated from Rabdosia rubescences, has been reported to exert cytotoxic effects on L929 cells. In this study, we investigated the mechanisms of FGF-2 protection of L929 cells from oridonin-induced apoptosis. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signal did not mediate this effect because the PI3K inhibitor wortmannin failed to reverse this protection and PKB activation was not observed in this process. In contrast, the extracellular signal-regulated kinase (ERK) was responsible for this rescue because its inhibition abolished the protective effect of fibroblast growth factor (FGF)-2. ERK had dual regulatory functions: mediating cell apoptosis or preventing cells from initiating the apoptotic response by phosphorylation or promoting expression of Bcl-2 in dependence of different stimuli. In L929 cells treated with oridonin alone, the activated ERK decreased the ratio of Bcl-2/Bax by mediating the phosphorylation of Bcl-2, resulting in apoptosis; the Ras inhibitor manumycin A and Raf inhibitor GW5074 failed to inhibit this apoptosis, indicating that there is a signal other than Ras/Raf pathway activated ERK. However, in the presence of FGF-2, Bcl-2 phosphorylation was blocked, and the Ras/Raf/ERK signal pathway was activated and protected against the oridonin-induced apoptosis by the alternative function of promoting of Bcl-2 expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Line, Tumor; Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Genes, bcl-2; Genes, ras; In Situ Nick-End Labeling; Indoles; Mice; Phenols; Phosphatidylinositol 3-Kinases; Polyenes; Polyunsaturated Alkamides; raf Kinases; Signal Transduction