Addressing reported pro-apoptotic functions of NF-kappaB: targeted inhibition of canonical NF-kappaB enhances the apoptotic effects of doxorubicin

PLoS One. 2009 Sep 10;4(9):e6992. doi: 10.1371/journal.pone.0006992.


The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibiotics, Antineoplastic / pharmacology
  • Apoptosis*
  • Cell Line, Tumor
  • Doxorubicin / pharmacology*
  • Gene Expression Profiling
  • Gene Expression Regulation*
  • Humans
  • I-kappa B Kinase / metabolism
  • NF-kappa B / metabolism*
  • NF-kappa B p52 Subunit / metabolism
  • RNA, Small Interfering / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factor RelA / metabolism
  • Transcription, Genetic


  • Antibiotics, Antineoplastic
  • NF-kappa B
  • NF-kappa B p52 Subunit
  • RNA, Small Interfering
  • Transcription Factor RelA
  • Doxorubicin
  • I-kappa B Kinase