Figure 1.
(-)-Gossypol causes cell death and induces apoptosis through mitochondria pathway in human prostate cancer cell line PC-3. A. MTT-based cell viability assay of (-)-gossypol in PC-3 and LNCaP cells. Cells were seeded in 96-well plates and treated in triplicates. Results are presented as mean ± SEM and normalized to their respective controls (n=3). B. Western blot analysis showed the difference of Bcl-2, Bcl-xL, and Mcl-1 protein levels in three prostate cell lines (60μg/lane). C. Apoptotic cells were counted with DAPI staining. Cells were counted in five different fields, and plotted as mean ± SEM. D. Cytochrome c and AIF release from mitochondria to Cytosol. PC-3 cells were treated with (-)-gossypol for indicated times, and then subjected to a digitonin-based subcellular fractionation as described in Materials and Methods. Cytosolic fractions (~120 μg/lane) and mitochondria fractions (~60 μg/lane) were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The COX IV marker was used to determine cross-contamination of cytosolic fractions with mitochondrial proteins.