Pharmacological inhibition and silencing of classical IKK-NF-κB pathway by siRNA sensitizes cancer cells to doxorubicin

Abstract

2059 Background: The IKK/NF-κB pathway (IKK1, IKK2 and NEMO; p65, p50, p52, c-Rel, RelB) is involved in chemoresistance. NF-κB is activable by either a classical or an alternative Ipathway. Here we studied whether NF-κB inhibitors sensitized cells to doxorubicin (dox), and assessed the role of individual IKK or NF-κB subunits by RNA interference [ small interfering RNA (siRNA)] on doxorubicin sensitization. Methods: We used BT474 and MDA-MB453 cells (breast) and HeLa cells (cervix). NF-κB inhibitors were bortezomib (Velcade, a proteasome inhibitor, which inhibits NF-κB by promoting accumulation of the inhibitory Iκ B-α) and a NEMO-Binding-Peptide (NBP, which prevents IKK proteins from complexing). NF-κB DNA binding activity was measured by EMSA. Western blot was used to assess nuclear translocation of individual NF-κB subunits (Iκ B-α, p65, c-Rel, RelB, p50 and p52). BT474 and MDA-MB453 cells were cultured with dox with/without NF-κB inhibitors and viability was assayed by MTT. siRNA was used to transiently down-regulate subunits that act preferentially on the classical NF-κ B (p65, p52, c-Rel, or NEMO) or on the alternative pathway (RelB) in HeLa cells. Chemosensitivity of transfected and control cells to dox was measured by MTT. Results: Dox increased NF-κB activity in BT474 and HeLa cells, as assayed by EMSA and/or by showing Iκ B-α degradation and phosphorylation of p65. Pretreatment of BT474 cells with bortezomib followed by dox resulted in a 30% reduction in cell viability compared to dox alone. Moreover, a 40% higher dox sensitivity was observed when cells were pretreated with a NEMO-Binding-Peptide. Similar data were obtained in MDA-MB453 cells. Transient down-regulation of key members of the classical IKK- NF-κB pathway (p65, p52, c-Rel and NEMO) by siRNA in HeLa cells lead to a 30% increase in dox cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no effect on dox cytotoxicity under our assay conditions. Conclusions: NF-κB inhibition sensitized cells to dox, implying directly p65, p52, c-Rel and NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the classical IKK-NF-κB pathway is sufficient to improve dox antitumor effects. [Table: see text]