FIGURE 4.

Induction of cathepsin D contributes to lucanthone-mediated apoptosis. A, lucanthone induces cathepsin D and LC3-II expression. Breast cancer cell lines were treated with 10 μm lucanthone for 48 h. Protein levels were determined by immunoblotting. B, lucanthone induces apoptosis in breast cancer cell lines. Cells were treated with 5 or 10 μm lucanthone for 48 h. Apoptosis was determined by PI staining and flow cytometry. Mean ± S.D., n = 3. * indicates a significant difference from controls. p < 0.05. C, cathepsin D knockdown diminishes lucanthone-induced apoptosis. RNAi was performed as described under “Experimental Procedures.” MDA-MB-231 cells transfected with non-target or cathepsin D siRNA were treated with lucanthone for 48 h. Immunoblotting confirmed knockdown of cathepsin D, which was associated with reduced cleavage of caspase-3 following lucanthone treatment. Apoptosis was determined by PI staining and flow cytometry. Mean ± S.D., n = 3. * indicates significant difference from non-target siRNA transfected cells treated with lucanthone. p < 0.05. D, pepstatin A and Z-VAD-fmk blunt lucanthone-mediated apoptosis. Cells were treated with 10 μm lucanthone in the presence or absence of 100 μm pepstatin A or 20 μm Z-VAD-fmk for 48 h, and apoptosis was measured by PI-FACS analysis. Mean ± S.D., n = 3. * indicates a significant difference from lucanthone-treated cells. p < 0.05.