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The mechanisms by which the HIV protease inhibitor ritonavir inhibits cancer cell growth are poorly understood. We have previously demonstrated that ritonavir inhibits calpain and not the proteasome in the MDA-MB-231 line and that ritonavir inhibits the growth of the MDA-MB-231 line in vitro and in a mammary fat pad xenograft model. Ritonavir up-regulates the calpain target p27, and induces nuclear localization, associated with inhibition of cell cycle progression at the G1/S checkpoint. Apoptosis is induced 3-fold by ritonavir compared to the vehicle control at 24 h, from 5 to 18%, as measured by propidium iodide/Annexin-FITC staining. Akt levels are not affected at 24 of ritonavir exposure, while Akt phosphorylation is down-regulated by 80%. These results indicate that ritonavir inhibits two targets in the MDA-MB-231 line. Calpain inhibition by m-calpain siRNA or PD150606 does not inhibit Akt phosphorylation or induce apoptosis, but up-regulates p27, suggesting that ritonavir-induced apoptosis may involve a non-protease target. One candidate ritonavir target is Hsp90, which promotes Akt folding and phosphorylation. Surface plasmon resonance biosensor analysis revealed that ritonavir binds to human Hsp90, exhibiting a KD of 7.8 microM. Ritonavir inhibits Hsp90 /Akt and Hsp90/mutant p53 complex formation in the MDA-MB-231 line, suggesting that ritonavir blocks Hsp90 interaction with client proteins. These results indicate that ritonavir inhibits breast cancer growth, in part, by inhibiting Hsp90 function and associated Akt phosphorylation, resulting in apoptosis, and in part, by intracellular calpain inhibition and upregulation of p27, resulting in G1 cell cycle arrest.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]