Modulation of apoptosis by mitochondrial uncouplers: apoptosis-delaying features despite intrinsic cytotoxicity
Oliver J Stoetzer1, Alexei Pogrebniak, Renate Pelka-Fleischer
1Medizinische Klinik III, Department of Haematology and Oncology, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany.
Abstract:
Disruption of mitochondrial electron transport and opening of the so-called mitochondrial permeability transition pores (PTPs) are early events in apoptotic cell death and may be caused by the uncoupler of mitochondrial oxidation and phosphorylation, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We investigated the cellular toxicity of FCCP in HL60 and CCRF-CEM cells alone or in combination with the known apoptosis inducers such as inhibitor of serine/threonine protein kinases staurosporine (Sts) and protein kinase C inhibitor chelerythrine. FCCP induced apoptotic cell death in both cell lines in a dose-dependent manner, and we were able to demonstrate an appearance of caspase-3-dependent PARP cleavage fragments with Western blot and the appearance of large (15-50 kb) DNA fragments using pulsed-field gel electrophoresis. After 2 hr of incubation with Che or Sts more than half of the cells had died by apoptosis. We observed a statistically significant delay in Sts- and Che-induced apoptotic cell death in CCRF-CEM cells when the cells were preincubated with FCCP but not with zVAD-FMK: about 50% more cells survived after pre-treatment with FCCP, as compared to 1 hr treatment with Che alone (P<0.05), and 25% more cells were alive after 6 hr of treatment, as compared to 6 hr exposure to Sts alone (P<0.05). The protective effect of FCCP was, however, transient and lasted only 6 hr. Treatment with aurintricarboxylic acid completely prevented Che- and Sts-induced apoptotic cell death in CCRF-CEM and HL60 cells. Incubation with Che resulted in a drop in the intracellular ATP content, predominantly distinctive in HL60, and in NAD(+) content in CCRF-CEM cells. Both ATP and NAD(+) drop were prevented with ATA, but not with FCCP or zVAD. Our data suggest that treatment with uncouplers of oxidative phosphorylation can induce apoptotic cell death in haematopoietic cell lines. However, when used in combination with serine/threonine protein kinase inhibitors FCCP can even prevent apoptosis.
Insights
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) can induce apoptosis in cancer cells. However, FCCP can also delay apoptosis when combined with certain kinase inhibitors, offering a transient protective effect.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Mitochondrial permeability transition pores (PTPs) and electron transport disruption are early apoptotic events.
- Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) is a known uncoupler of mitochondrial oxidation and phosphorylation.
Purpose of the Study:
- To investigate the cellular toxicity of FCCP in HL60 and CCRF-CEM cells.
- To examine the combined effects of FCCP with apoptosis inducers staurosporine (Sts) and chelerythrine (Che).
Main Methods:
- Dose-dependent cytotoxicity assays.
- Western blot analysis for caspase-3-dependent PARP cleavage.
- Pulsed-field gel electrophoresis for DNA fragmentation.
- Cell survival assays following pre-treatment with FCCP.
Main Results:
- FCCP induced dose-dependent apoptotic cell death, evidenced by PARP cleavage and DNA fragmentation.
- Pre-incubation with FCCP delayed staurosporine- and chelerythrine-induced apoptosis in CCRF-CEM cells.
- Aurintricarboxylic acid (ATA) completely prevented apoptosis induced by Che and Sts.
- Chelerythrine caused a drop in intracellular ATP in HL60 cells and NAD+ in CCRF-CEM cells, which was prevented by ATA.
Conclusions:
- Uncouplers of oxidative phosphorylation, like FCCP, can induce apoptosis in hematopoietic cell lines.
- FCCP can exhibit a transient protective effect against apoptosis when combined with serine/threonine protein kinase inhibitors.
- ATA effectively prevents apoptosis induced by kinase inhibitors, suggesting a role for ATP/NAD+ depletion in this process.
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