Differential Effects of Superoxide Dismutase Isoform Expression on Hydroperoxide-induced Apoptosis in PC-12 Cells

Abstract

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tertbutyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2-and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.

Activation of c-Jun-N-terminal kinase is required for apoptosis triggered by glutathione disulfide in neuroblastoma cells

“…6). These results, while in agreement with the role played by this enzyme in modulating the sensitivity of cancer cells to external or cytosolic-derived superoxide [19], may also be in line with the ineffectiveness of Cu,Zn SOD to counteract mitochondrial-mediated ROS production [20].…”

Decreased susceptibility of differentiated PC12 cells to oxidative challenge: relationship to cellular redox and expression of apoptotic protease activator factor-1

“…9, 24 We further found that an induction of GSH/GSSG imbalance per se directly initiated apoptosis in these cells without ROS involvement. 10 The current results show that TBH-induced apoptosis in dPC12 cells is similarly mediated by cellular GSH/GSSG redox, and that cellular redox play a causal role in apoptosis initiation.…”

“…Figure 8c shows that 20% apoptosis of dPC12 cells was observed at 300 mM TBH that was comparable to that after treatment of nPC12 cells with 100 mM TBH. 24 Accordingly, a greater magnitude of early decreases in cellular GSH with increases in GSSG and attenuated GSH-to-GSSG ratios were achieved with treatment of cells at 300 mM TBH (Figures 9a-c).…”

“…The fact that NAC pretreatment blocked apoptosis, but not its addition at 1 h post-TBH and at a time after peak redox shift (Figure 7a), agrees with this and previous interpretation. 24 Studies with diamide further showed that the induction of redox imbalance per se without ROS generation can elicit dPC12 cell apoptosis as in nPC12 cells, 10 suggesting that loss of redox balance may, in fact, be the mechanism by which oxidants like TBH mediate cell apoptosis. Finally, studies with ethacrynic acid and elevated TBH to modulate cellular redox demonstrated that a higher percentage of cell apoptosis was linked to a larger cellular redox imbalance (Figure 8).…”

SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

“…This protective ability of SOD2 in DRG in vitro is consistent with other studies using SOD2 overexpression. SOD2 overexpression eliminates pro-oxidantinduced mitochondrial ROS in PC-12 cells and decreases cell injury (Pias et al, 2003). Similarly, in retinal pigment epithelial cells, the gene dosage of SOD2 directly relates to the degree of mitochondrial injury and apoptosis induced by H 2 O 2 (Kasahara et al, 2005).…”