Abstract
We previously showed that tert-butyl hydroperoxide (TBH) induced apoptosis in naïve rat pheochromocytoma (nPC12) cells that correlated with cellular redox imbalance and mitochondrial apoptotic signaling. In this study, we tested the hypothesis that differentiation of nPC12 cells results in altered susceptibility to TBH utilizing a model of differentiated PC12 (dPC12) cells induced by nerve growth factor. TBH (100 μM) induced dPC12 apoptosis (12% at 24 h) at levels lower than naïve cells (35%). This resistance was associated with elevated GSH, NADPH (reduced nicotinamide adenine dinucleotide phosphate), TBH metabolism, redox enzyme activities, reduced cellular GSH/GSSG (glutathione disulfide) status and preservation of mitochondrial membrane potential. Altering cellular GSH with ethacrynic acid or N-acetylcysteine, respectively, exacerbated or protected against dPC12 apoptosis. dPC12 apoptosis was mediated by caspase-9 and -3 activation and apoptosis protease activator protein-1 (Apaf-1) expression. These results show that nPC12 transition to dPC12 cells afforded protection against oxidative challenge due to maintenance of reduced GSH/GSSG and decreased Apaf-1 expression.
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Abbreviations
- PC12:
-
pheochromocytoma cells
- nPC12:
-
naïve PC12
- dPC12:
-
differentiated PC12
- NGF:
-
nerve growth factor
- TBH:
-
tert-butyl hydroperoxide
- TNFα:
-
tumor necrosis factor-alpha
- NAC:
-
N-acetylcysteine
- EA:
-
ethacrynic acid
- ROS:
-
reactive oxygen species
- GSH:
-
reduced glutathione
- GSSG:
-
glutathione disulfide
- DAPI:
-
4′,6-diamidino-2-phenylindole
- F12 K:
-
Kaighn's modification of Ham's F-12
- HPLC:
-
high-performance liquid chromatography
- PBS:
-
phosphate-buffered saline
- TCA:
-
trichloroacetic acid
- IETD-CHO:
-
[acetyl-alanine-alanine-valine-alanine-leucine-leucine-proline-alanine-valine-leucine-leucine-alanine-leucine-leucine-alanine-proline-isoleucine-glutamate-threonine-aspartate-CHO]
- LEHD-CHO:
-
[acetyl-alanine-alanine-valine-alanine-leucine-leucine-proline-alanine-valine-leucine-leucine-alanine-leucine-leucine-alanine-proline-leucine-histidine-aspartate-CHO]
- NADH:
-
reduced nicotinamide adenine dinucleotide
- NADPH:
-
reduced nicotinamide adenine dinucleotide phosphate
- Apaf-1:
-
apoptosis protease activator factor-1
- JC-1:
-
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide
- PI:
-
propidium iodide
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Acknowledgements
We gratefully acknowledge Deborah Chervenak and Dr. Robert Chervenak for their assistance with flow cytometry, and Laura Coe for technical support. This study was supported by Grant DK 44510 from the National Institutes of Health.
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Ekshyyan, O., Aw, T. Decreased susceptibility of differentiated PC12 cells to oxidative challenge: relationship to cellular redox and expression of apoptotic protease activator factor-1. Cell Death Differ 12, 1066–1077 (2005). https://doi.org/10.1038/sj.cdd.4401650
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DOI: https://doi.org/10.1038/sj.cdd.4401650
