Related Experiment Videos
Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced
M-V Clément1, J L Hirpara, S Pervaiz
11Department of Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Abstract:
At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the beta-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.
Insights
Bcl-2 inhibits early cell acidification during apoptosis. Reducing superoxide levels enhances cancer cell sensitivity to apoptosis, suggesting a switch from mitochondria-dependent to independent death signaling for drug treatment.
Area of Science:
- Cellular Biology
- Apoptosis Signaling
- Cancer Research
Background:
- Two mechanisms of early cytosolic acidification in apoptosis are known: caspase 8 activation and mitochondria-derived hydrogen peroxide.
- Bcl-2 protein is a key regulator of apoptosis, often overexpressed in cancer cells.
Purpose of the Study:
- To investigate the role of Bcl-2 in regulating cytosolic acidification during apoptosis.
- To explore the interplay between superoxide levels, Bcl-2, and apoptotic pathways.
Main Methods:
- Overexpression of Bcl-2 in CEM leukemia cells.
- Inhibition of beta-nicotinamide adenine dinucleotide phosphate oxidase and Rac1 signaling.
- Assessment of caspase 8 activation, cytosolic pH, and apoptosis sensitivity.
Main Results:
- Bcl-2 inhibits both known mechanisms of early cytosolic acidification during apoptosis.
- Bcl-2 overexpression slightly elevates constitutive superoxide anion and pH.
- Decreasing superoxide levels significantly increases sensitivity to apoptosis via enhanced caspase 8 activation, independent of caspase 9 or cytochrome c.
Conclusions:
- Bcl-2 influences apoptotic signaling by modulating cytosolic acidification and superoxide levels.
- A mechanism exists for switching apoptotic pathways from mitochondria-dependent to independent signaling.
- Targeting superoxide levels could sensitize Bcl-2-expressing cancer cells to apoptosis, aiding drug treatment strategies.