We have observed that calpain activity is increased in ductal breast cancer, compared to adjacent and terminal ductal epithelia. Calpastatin over-expression in the breast cancer lines MCF7 and MDA-MB-231 results in down-regulation of phospho-Akt and cell cycle arrest and cell death, assayed by Hoechst dye 33342/pyronine and propidum iodide/annexin V flow cytometry methods. The HIV protease inhibitor, ritonavir, which we have demonstrated to block calpain-dependent cytoskeletal remodeling and calpain-dependent cleavage of cell permeant substrates, also down-regulates phospho-Akt by approximately 70%, by lowering total Akt protein levels. Ritonavir induces cell death in the MCF7, T47D, MDA-MB-231, MDA-MB-436, and SKBR3 breast cancer lines and the IC50 values for ritonavir, which range between 10 and 45 μM, are clinically attainable. Hoechst/pyronine staining reveals that ritonavir blocks cell cycle entry at the Go/G1 transition, as well as blocking G1/S in cycling cells. Transient ritonavir exposure at IC90 concentrations induces loss of normal colony formation of Go cells. Sustained exposure to the ritonavir IC50 results in loss of clonogenicity of Go cells. G1 and S/G2M cells are also made non-clonogenic by sustained ritonavir exposure. These findings suggest that ritonavir may induce cell death in non-cycling breast cancer cells independent of EGFR, her2 or p53 status, in part by down-regulating Akt signaling.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]