manumycin and 3-methyladenine

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