Impaired kinetics of Bax-GFP and Smac/DIABLO-GFP in caspase-8- and bid-silenced and Bcl-2 overexpressed breast cancer MCF-7 cells exposed to camptothecin.

Smac/DIABLO, a proapoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspases activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Present study is focused on the role of Bid in the control of Bax-GFP and Smac/DIABLO-GFP kinetics in breast cancer MCF-7 cells stimulated to apoptosis with camptothecin (CPT). Minute kinetics of proteins was examined by homeostatic confocal microscopy. The release of Smac/DIABLO-GFP from mitochondria comprised two phases: initial-rapid, lasting 20-30 min and subsequent 30 min-plateau phase, followed by the decrease of Smac/DIABLO-related fluorescence due to cell destruction. The kinetics of Bax-GFP aggregation on mitochondria coincided in time with Smac/DIABLO-GFP release from these organelles. Bid knock down and Bcl-2 overexpression delayed Bax-GFP aggregation and completely inhibited Smac/DIABLO-GFP release from mitochondria. Knock down of caspase 8 (activator of Bid) delayed both Bax-GFP aggregation and Smac/DIABLO-GFP release in CPT-treated cells. In conclusion, Bid protein is crucial for the control of the release of Smac/DIABLO from mitochondria in breast cancer MCF-7 stimulated to apoptosis with CPT.

Apoptosis and autophagy in mammary gland remodeling and breast cancer chemotherapy.

Apoptosis - programmed cell death (PCD) type I is physiological process responsible for cell loss during mammary gland involution after natural weaning or litter removal in rodents, after weaning in sow and during drying off in goat and cow. The regulation of mammary epithelial cell (MEC) apoptosis in bovine mammary gland occurs at three levels. The first level comprises intracellular regulatory proteins, e.g. Bcl-2 family death promoters and inhibitors. The second level is represented by intramammary inductors of apoptosis, e.g. FIL, IGFBPs, Fas ligand, TGF-betas. The expression and activity of these auto/paracrine inductors of apoptosis is controlled and modulated by the third level factors, e.g. systemic galactopoetic hormones, nutrition, reproductive status and milking management. Our recent study proved that apoptosis in involuting bovine mammary gland is accompanied by increased intensity of autophagy, regarded as a cytoprotective process but in advanced stage as a PCD type II. Moreover, we have reported for the first time the ability of TGF-beta(1) to induce both apoptosis and autophagy in bovine BME-UV1 MEC. Much more pronounced heterogeneity of PCD was observed when breast cancer cells were exposed to anticancer drugs. The primary responses of breast cancer MCF-7 cells to camptothecin (CPT) are apoptosis and autophagy (as a cytoprotective process). In this case autophagy occurs in cells which are resistant to apoptosis as a tool of cancer cell survival. The fail-safe responses of breast cancer cells to persisting CPT-induced stress are apoptosis accompanied by morphological and biochemical features of autophagy or type II PCD with advanced subcellular degradation. The threshold between autophagy as a cytoprotective process (reversible) or PCD (irreversible) is difficult to establish and probably depends on the extent of degradation of cellular components. Proapoptotic protein Bid may serve as a molecular switch between apoptosis and autophagy. Bid knock down in MCF-7 cells exposed to CPT leads to a shift of cell death from apoptosis to autophagy. Since bid and other proapoptotic genes undergo mutations in malignant cells, the ability of cancer cells commitment to autophagy may have important therapeutic implications.

Cathepsins and BID are involved in the molecular switch between apoptosis and autophagy in breast cancer MCF-7 cells exposed to camptothecin.

The details of molecular switching points between apoptosis and autophagy in tumor cells have still not been fully elucidated. This study focused on the role of cathepsin B and its substrate, BID as molecular links between apoptosis and autophagy in human breast cancer MCF-7 cells exposed to camptothecin. Apoptosis occurred rapidly with a peak in 60 min after drug administration, whereas autophagy developed at a much slower rate with continuous progression during 24 h of cell exposure to the drug. CPT induced very rapid activation of cathepsin B. Inhibition of cathepsins by E64d prevented CPT-induced BAX and BID aggregation on mitochondria and reduced significantly reduced apoptotic cell number. The above effects were accompanied by an increase in autophagosome formation, measured by expression of MAP I LC3. BID knock down resulted in strong suppression of CPT-induced apoptosis and a shift of cell death towards autophagy, manifesting with an increase of Beclin 1 and MAP I LC3 cellular content.