monensin and 3-methyladenine

Understanding the mechanisms by which anti-parasitic drugs alter the physiology and ultimately kill is an important area of investigation. Development of novel parasitic drugs, as well as the continued utilization of existing drugs in the face of resistant parasite populations, requires such knowledge. Here we show that the anti-coccidial drug monensin kills Toxoplasma gondii by inducing autophagy in the parasites, a novel mechanism of cell death in response to an antimicrobial drug. Monensin treatment results autophagy, as shown by translocation of ATG8 to autophagosomes, as well as causing marked morphological changes in the parasites' mitochondria. Use of the autophagy inhibitor 3-methyladenine blocks autophagy and mitochondrial alterations, and enhances parasite survival, in monensin-exposed parasites, although it does not block other monensin-induced effects on the parasites, such as late S-phase cell cycle arrest. Monensin does not induce autophagy in a parasite strain deficient in the mitochondrial DNA repair enzyme TgMSH-1 an enzyme that mediates monensin-induced late S-phase arrest. TgMSH-1 therefore either mediates cell cycle arrest and autophagy independently, or autophagy occurs downstream of cell cycle arrest in a manner analogous to apoptosis of cells arrested in G(2) of the cell cycle. Overall, our results point to autophagy as a potentially important mode of cell death of protozoan parasites in response to antimicrobial drugs and indicate that disruption of the autophagy pathway could result in drug resistance.

Topics: Adenine; Antiprotozoal Agents; Autophagy; DNA Repair Enzymes; Microtubule-Associated Proteins; Mitochondria; Monensin; S Phase Cell Cycle Checkpoints; Toxoplasma

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Caspase Inhibitors; Caspases; Cells, Cultured; Enzyme Inhibitors; HeLa Cells; Humans; Immediate-Early Proteins; Lysosomes; Mice; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Monensin; Phagosomes; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Viral Proteins

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.

Topics: Acridine Orange; Adenine; Animals; Aurothioglucose; Cadmium; Cell Death; Chloroquine; Copper; Endopeptidases; Enzyme Activation; Gentamicins; Leupeptins; Lipid Peroxidation; Liver; Lysosomes; Male; Methylamines; Monensin; Oxidation-Reduction; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Degradation of the amyloid precursor protein (APP) by lysosomes has been proposed to be the mechanism for generation of the beta/A4 polypeptide which is the major constituent of amyloid plaques. In this report, we use inhibitors to elucidate the steps involved in the lysosomal degradation of APP in PC12 cells. Monensin treatment significantly elevated the level of immature APP. Reducing the temperature to 17 degrees C, adding cytochalasin B and colchicine, or exchanging K+ for Na+ resulted in a substantial accumulation of both mature and immature APP isoforms. The inhibitor of autophagy, 3-methyladenine, had no effect on the level of APP isoforms. These results suggest that changes in ionic balance, membrane fluidity or vesicle fusion may affect APP processing.

Topics: Adenine; Amyloid beta-Protein Precursor; Animals; Chloroquine; Colchicine; Cytochalasin B; Endocytosis; Lysosomes; Monensin; Organelles; PC12 Cells; Rats