α‐Fetoprotein positively regulates cytochrome c‐mediated caspase activation and apoptosome complex formation

Previous results have shown that the oncoembryonic marker α‐fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas‐dependent and tumor necrosis factor receptor‐dependent signaling. In this study we further investigate the molecular interactions involved in the AFP‐mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell‐free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low‐dose cytochrome c‐mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high‐resolution chromatography, we show that AFP positively regulates cytochrome c/dATP‐mediated apoptosome complex formation, enhances recruitment of caspases and Apaf‐1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein–protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP‐2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP‐2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex.