bcl-2 Protects SK-N-SH Cells From 6-Hydroxydopamine Induced Apoptosis by Inhibition of Cytochrome c Redistribution

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is, in part, due to the production of reactive oxygen/nitrogen species (RNOS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induce apoptosis of SK-N-SH cells. Western blots of cytosolic extracts from 6-OHDA treated cells reveal a translocation of cytochrome c from mitochondria into the cytosol. To further delineate the pathway by which 6-OHDA causes apoptosis, we investigated the effects on cell survival in bcl-2 overexpressing SK-N-SH cells and the relation to translocation of cytochrome c and free radical production. Overexpressing bcl-2 in SK-N-SH cells shifted the toxicity as well as the release of cytochrome c to higher concentrations of 6-OHDA. At concentrations of 6-OHDA in SK-N-SH-bcl-2 cells where a release of cytochrome c and subsequent cell death occurs, an increase of the oxidation of the fluorescent dye dihydrorhodamine-123 was detectable. This RNOS production paralleled the release of cytochrome c and was inhibited by bcl-2 as well. From these experiments, we hypothesize that cytochrome c might interact with 6-OHDA to promote an oxidative burst, which can be inhibited by bcl-2.