Addressing reported pro-apoptotic functions of NF-kappaB: targeted inhibition of canonical NF-kappaB enhances the apoptotic effects of doxorubicin.
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.
Bednarski
Brian K
BK
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Baldwin
Albert S
AS
Kim
Hong Jin
HJ
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D052003:NF-kappa B p52 Subunit / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic
D000903:Antibiotics, Antineoplastic; D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D004317:Doxorubicin; D051550:I-kappa B Kinase