benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and herbimycin

Focal adhesion kinase (FAK) is a signaling molecule associated with cell survival. Previously, we showed that thimerosal, a reactive oxygen species (ROS) generator, can acutely induce FAK tyrosine phosphorylation (within minutes) and chronically induce apoptosis (within days) by redox modulation in HeLa S cells. In the present study, we report that a prolonged oxidative stress by thimerosal induces a remarkable cleavage of FAK, which is accompanied with apoptosis. In fact, the kinetics of FAK cleavage has a good correlation with and actually preceding the apoptosis that was independent of anoikis. The effects were almost completely blocked by the pretreatment with either N-acetyl-l-cysteine (ROS scavenger) or Z-VAD-FMK (pan-caspase inhibitor), suggesting ROS-induced caspase activation as a key mechanism. They could be also reproduced by hydrogen peroxide alone, which appeared to be responsible for thimerosal-mediated oxidative stress-induced apoptosis. Additionally, the down regulation of FAK with antisense oligonucleotide dramatically augmented thimerosal-induced apoptosis. We could observe similar results using human corneal epithelial cells. Taken together, our results show that FAK is a critical cellular target of caspases during oxidative stress (particularly by hydrogen peroxide), resulting in the acceleration of subsequent apoptosis regardless of the anchorage status of cells. From the present results, it is more likely that not cell detachment but the proteolytic cleavage (or inhibition) of FAK is a key modulator as well as a promising indicator of apoptosis in epithelial cells under oxidative stress.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Benzoquinones; Caspase 3; Caspase Inhibitors; Cell Line, Transformed; Chelating Agents; Cornea; Cysteine Proteinase Inhibitors; Egtazic Acid; Epithelial Cells; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; HeLa Cells; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Oxidative Stress; Protein Kinase Inhibitors; Reactive Oxygen Species; Rifabutin; RNA, Small Interfering; Salicylates; Sulfhydryl Compounds; Thimerosal

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Benzoquinones; Benzylisoquinolines; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Fusion Proteins, bcr-abl; Humans; Intracellular Membranes; Jurkat Cells; K562 Cells; Lactams, Macrocyclic; Lamin Type B; Lamins; Membrane Glycoproteins; Mitochondria; Neoplasm Proteins; Nuclear Proteins; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones; Rifabutin