dicumarol and thiazolyl-blue

β-Lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.

Topics: Antineoplastic Agents; Dicumarol; eIF-2 Kinase; Enzyme Activation; Humans; Immunoblotting; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Oligonucleotide Array Sequence Analysis; Protein Serine-Threonine Kinases; Reactive Oxygen Species; RNA, Small Interfering; Saccharomyces cerevisiae Proteins; Saccharomycetales; Tetrazolium Salts; Thiazoles; Transcriptome

Gastrodia elata (G. elata) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy. In this study, differential methanol (MeOH) extracts of G. elata were found to prevent serum-deprived rat pheochromocytoma (PC12) cell apoptosis by the MTT assay and Hoechst staining. A serine/threonine kinase inhibitor attenuated this protection. G. elata resulted in phosphorylation and dephosphorylation of ERK1/2 and JNK1/2-p38 MAPKs (members of the serine/threonine kinase family), respectively, as revealed by Western blot analysis. An upstream ERK inhibitor attenuated G. elata-induced ERK phosphorylation but not protective effect. Although JNK and p38 inhibitors attenuated their related enzyme activities during serum deprivation, only JNK inhibitor prevented serum-deprived apoptosis. Thus, G. elata prevents serum-deprived apoptosis through activation of the serine/threonine kinase-dependent pathway and suppression of JNK activity.

Topics: Analysis of Variance; Animals; Apoptosis; Blotting, Western; Dicumarol; Drugs, Chinese Herbal; Flavonoids; Imidazoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Neuroprotective Agents; PC12 Cells; Phosphorylation; Pyridines; Rats; Signal Transduction; Tetrazolium Salts; Thiazoles

Topics: Allopurinol; Amyloid beta-Peptides; Animals; Antimycin A; Antineoplastic Agents; Cisplatin; Coloring Agents; Deoxyglucose; Dicumarol; Doxorubicin; Humans; Mitochondria; Oxazines; Oxidation-Reduction; Oxygen Consumption; PC12 Cells; Peptide Fragments; Rats; Rotenone; Tetrazolium Salts; Thiazoles; Xanthenes