Aplidins is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin s alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin s generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin s activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin s -induced JNK activation and cytotoxicity. Our results show that Aplidin s induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

The effects of reactive oxygen species (ROS) on different cellular types are variable. In some conditions they can be harmful metabolites, but they can also act as intracellular messengers that are able to activate different transcription factors. Based on previous reports in which ROS were shown to stimulate the proliferation of mesenchymal cells, this study was carried out to assess this effect on bovine aortic endothelial cells (BAECs). When cells were incubated with glucose oxidase (GO), an enzyme that generates H2O2 continuously, a significant increase in BAEC proliferation was detected. BAEC proliferation was measured by the incorporation of [3H]-thymidine in the DNA of BAECs, and also by an increase in the number of cells. The effect observed with GO was maximal at 8-24 h. Catalase abolishes proliferation. We also tested the ability of GO to phosphorylate tyrosine residues in endothelial cell proteins. A significant increase in tyrosine phosphorylation was found, which might constitute the molecular basis for proliferative effect of GO. In conclusion, these results demonstrate the ability of H2O2 to stimulate BAEC proliferation at least under certain experimental conditions. We suggest a general activation of the cascade of tyrosine phosphorylation as one of the possible cellular mechanisms responsible for GO-induced BAEC proliferation.