The NF-κB pathway regulates sensitivity of the breast cancer line MDA-MB-436 to inhibition of proliferation by the calpain inhibitor ritonavir

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We have demonstrated that the HIV protease inhibitor, ritonavir, blocks the growth of breast cancer lines independent of EGFR, her2, ER or p53 status. Ritonavir inhibits the growth of lines MCF7, T47D, MDA-MB-231, MDA-MB-436 and SKBR3, with IC₅₀ values in the range of 10-45 μM. In all of these lines, phospho-Akt is down-regulated by ritonavir treatment. By fluorometric analysis of cancer lines, including MDA-MB-231, ritonavir blocks calpain activity in the range of 10-45 μM, but has significant effects on proteasome activity only at higher concentrations, of >60 μM. Because Akt has been implicated in calpain-mediated NF-κB activation, we determined whether NF-κB regulated genes are down-regulated by ritonavir treatment at the IC₅₀ for the MDA-MB-436 line. Affymetrix cDNA microarray analysis was performed, using the U133 chip set, assayed in quadruplicate. This line was chosen because of its constitutive nuclear NF-κB and our ability to down-regulate nuclear NF-κB by stable over-expression of the IκB-α superrepressor. RNA was isolated from MDA-MB-436 cells at 4 and 8 h of treatment with ritonavir or DMSO vehicle, at the same cell density as used for proliferation assays. The NF-κB regulated genes IL1-β, IL6 and IL8 were modestly down-regulated by 20 to 30% at 4 and 8 h of ritonavir treatment (P<0.01). To test whether constitutive nuclear NF-κB promotes resistance of the MDA-MB-436 line to ritonavir, we measured the ritonavir IC₅₀ of the parental MDA-MB-436, super-repressor expressing MDA-MB-436-IκBαSR, and vector control MDA-MB-436-LXSN lines and found the IC₅₀ for ritonavir to be 40, 42 and 26 μM, respectively. These findings indicate that calpain inhibition by ritonavir minimally blocks constitutive nuclear NF-κB and that instead constitutive NF-κB plays a role in resistance to ritonavir. We tested this hypothesis by determining whether ritonavir and the proteasome inhibitor PS-341 synergize to block the growth of breast cancer lines expressing constitutive nuclear NF-κB. By isobologram analysis, the combination of the proteasome inhibitor PS-341 and ritonavir resulted in a Chou-Talalay combination index of 0.15, indicating strong synergy. These results suggest that the combination of ritonavir and NF-κB inhibitors may be useful for the development of therapeutic strategies for breast cancers expressing constitutive nuclear NF-κB.