gw-5074 and manumycin

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras-Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K-Akt signaling pathways.

Topics: Androstadienes; Animals; Apoptosis; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Fibrosarcoma; Heterocyclic Compounds, 4 or More Rings; Indoles; Mice; Microscopy, Electron, Transmission; Phenols; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plant Lectins; Polyenes; Polygonatum; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; raf Kinases; ras Proteins; Signal Transduction; Tetrazoles; Time Factors; Wortmannin

Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca(2+) influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca(2+) permeability, such as alpha5-containing alpha3 nAChR and alpha7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the alpha5 and alpha7 expression in KCs, which could be abolished by mecamylamine and alpha-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: alpha3(beta2/beta4) > alpha3(beta2/beta4)alpha5 > alpha7 > alpha7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the alpha3(beta2/beta4) to alpha3(beta2/beta4)alpha5 nAChR transition predominantly involved protein kinase C, alpha3(beta2/beta4)alpha5 to alpha7 nAChR transition-Ca(2+)/calmodulin-dependent protein kinase II and p38 MAPK, and alpha7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca(2+) permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha7 Nicotinic Acetylcholine Receptor; Bungarotoxins; Butadienes; Carbazoles; Conotoxins; Egtazic Acid; GATA2 Transcription Factor; Humans; Imidazoles; Indoles; Keratinocytes; Nerve Tissue Proteins; Nicotine; Nitriles; Phenols; Polyenes; Polyunsaturated Alkamides; Pyridines; Receptors, Nicotinic; RNA, Small Interfering; Signal Transduction; Smoking; Tobacco Smoke Pollution; Tyrphostins; Up-Regulation

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

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