Abstract
Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-κB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-κB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-κB in a variety of tumor cell lines. NF-κB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-κB activation involved increased IKK activity and phosphorylation of IκB-α at serine 32/36 followed by degradation of IκB-α. Reporter assays revealed that NF-κB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-κB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-κB inhibition by transient or stable expression of IκB-α super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IκB-α super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-κB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-κB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.
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Abbreviations
- AIF:
-
apoptosis-inducing factor
- BetA:
-
betulinic acid
- FACS:
-
fluorescence-activated cell-sorting
- GST:
-
gluthathione-S-transferase
- IAPs:
-
inhibitor of apoptosis proteins
- IκB:
-
inhibitor of κB
- IKK:
-
IκB kinase
- NF-κB:
-
nuclear factor-kappaB
- PDTC:
-
pyrrolidine dithiocarbamate
- Smac:
-
second mitochondria-derived activator of caspase
- TRAIL:
-
TNF-related apoptosis-inducing ligand
- XIAP:
-
X-linked inhibitor of apoptosis
- zVAD.fmk:
-
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
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Acknowledgements
We thank S Piater for expert technical assistance and B Baumann (Department of Physiological Chemistry, University of Ulm, Germany) for helpful discussions. This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe, the Bundesministerium für Forschung und Technologie, the Ministry of Science, Research and Arts of Baden-Württemberg, IZKF Ulm, Wilhelm-Sander-Stiftung and Else-Kröner-Stiftung (KMD and SF).
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Kasperczyk, H., La Ferla-Brühl, K., Westhoff, M. et al. Betulinic acid as new activator of NF-κB: molecular mechanisms and implications for cancer therapy. Oncogene 24, 6945–6956 (2005). https://doi.org/10.1038/sj.onc.1208842
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DOI: https://doi.org/10.1038/sj.onc.1208842
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