The cellular and molecular effects of the proteasome inhibitor bortezomib on breast cancer cells are as yet poorly characterized.

The NFκB family is composed by five subunits (p65/RelA, c-Rel, RelB, p105-p50/ NFκB 1 , p100-p52/NFκB 2 ) and controls the expression of many genes that participate in cell cycle, apoptosis, and other key cellular processes. In a canonical pathway, NFκB activation depends on the IKK complex activity, which is formed by three subunits (IKKa and IKKb and IKKg/NEMO). There is an alternative NFκB activation pathway that does not require IKKb or IKKg/NEMO, in which RelB is a major player. We report in a panel of human breast cancer cells that the IKK/NFκB system is generally overexpressed in breast cancer cells and there is heterogeneity in expression levels of individual members between different cell lines. Doxorubicin, an anticancer agent used in patients with breast cancer, activated NFκB and appeared to be less effective in cells expressing predominantly members of the canonical IKK/NFκB. Two NFκB inhibitors, bortezomib and NEMO-Binding Domain Inhibitory Peptide, prevented doxorubicin-induced NFκB activation and increased doxorubicin antitumor effects in BT-474 cells. Transient down-regulation of members of the canonical pathway (p65, p52, c-Rel and IKKg/ NEMO) by siRNA in HeLa cells increased doxorubicin cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no evident effects on doxorubicin cytotoxicity. To conclude, NFκB inhibition sensitized cells to doxorubicin, implying directly p65, p52, c-Rel and IKKg/NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the canonical NFκB pathway is sufficient to improve doxorubicin antitumor effects.

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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]