Caspase‐independent cell death by low concentrations of nitric oxide in PC12 cells: Involvement of cytochrome C oxidase inhibition and the production of reactive oxygen species in mitochondria

Abstract

We reported previously that low levels of nitric oxide (NO) induced cell death with properties of apoptosis, including chromatin fragmentation and condensation in undifferentiated PC12 pheochromocytoma cells. The present study demonstrates that cytotoxicity of low concentrations of NO is mediated by inhibition of mitochondrial cytochrome c oxidase and generation of reactive oxygen species (ROS). An NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) induced cell death even at low concentrations (10-100 microM), whereas peroxynitrite and a peroxynitrite generator, 3-(4-morpholinyl)-sydnonimine (SIN-1), did not have a significant effect on cell viability up to a concentration of 0.5 mM. The NOR3-induced cell death was unaffected by pretreatment with superoxide dismutase (SOD) or its mimetic peroxynitrite scavenger, manganese(III) tetrakis(benzoic acid)porphyrin chloride (Mn-TBAP), or with uric acid. These findings indicate that peroxynitrite does not contribute to this cell death. Furthermore, neither the release of cytochrome c from mitochondrial membranes, the cleavage of poly-ADP ribose polymerase (PARP), nor the activation of caspase-3-like activities was observed. Inhibitors of PARP, benzamide, and aminobenzamide, had no effect on the NOR3-induced cell death. In addition, pretreatment with general or selective caspase inhibitors, benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), and benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-Ch(2)-DCB) did not prevent NOR3-induced cell death. Taken together, these findings suggest that cell death induced by NOR3 occurs by a caspase-independent mechanism. In contrast, we found an early increase in mitochondrial H(2)O(2) production during NOR3 exposure using the fluorescent dye 2',7'-dichlorofluorescin-diacetate (DCFH-DA) and dihydrorohdamine123 (DHR123), and these events were accompanied by strong inhibition of cytochrome c oxidase activity in the cells. Furthermore, we observed that several antioxidants, such as ascorbate, glutathione (GSH), cysteine, tetrahydrobiopterin, and dithiothreitol (DTT), all effectively prevented the NOR3-induced cell death. NOR3 treatment decreased the level of total intracellular GSH, but did not affect the activities of antioxidant enzymes SOD, GSH-peroxidase (GPX), and catalase. These results suggest that cell death induced at physiologically low concentrations of NO is mediated by ROS production in mitochondria, most likely resulting from the inhibition of cytochrome c oxidase, with ROS acting as an initiator of caspase-independent cell death.

TNF-α/Cycloheximide-Induced Oxidative Stress and Apoptosis in Murine Intestinal Epithelial MODE-K Cells

“…The exact mechanism of the anti-apoptotic effect of CORM-A1 in MODE-K cells remains to be clarified, but our study shows that its anti-apoptotic effect correlates with a reduction in ROS production, which was more pronounced than that 19 observed with bilirubin, as well as an increase in GSH levels. Indeed, caspase-independent induction of apoptosis by ROS has been reported in PC12 pheochromocytoma cells so that inhibition of ROS production per se can be expected to reduce apoptosis [50]. The finding that CORM-A1 exerts significant protection against both TNF-α/CHX-induced oxidative stress and apoptosis supports the notion that CO can mediate the well-known anti-apoptotic effect of HO-1.…”

Protective Effect of Chlorogenic Acid against Aβ-Induced Neurotoxicity

“…Furthermore, the Aβ-induced apoptotic cell death in PC12 cells is associated with the decreased expression of the anti-apoptotic molecule Bcl-2 and the increased expression of the pro-apoptotic molecule Bax (Xiao et al, 2002;Yuyama et al, 2003). The activation of caspase-3 plays a role in Aβ-induced apoptosis (Allen et al, 2001) and an increase in activated caspase-3 has been reported in AD brains (Su et al, 2001).…”

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

“…concentration of oxygen, superoxide radical and thiols), may induce cell death via programmed mechanisms or by necrosis, is extensively documented (Borutaite and Brown, 2005) in several cell types (Bal-Price and Brown, 2000;Terwel et al, 2000;Yuyama et al, 2003). These reports differ not only in the cell type studied but also in the steady-state concentration of the NO, redox species involved and the chemistry it undergoes in a given biological milieu.…”