Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xL in a strictly caspase-3-dependent manner in human carcinoma cells

• Published in: Oncogene Vol. 23; no. 26; pp. 4523 - 4535
• Main Authors: HASENJÄGER, Anne, GILLISSEN, Bernhard, MÜLLER, Antje, NORMAND, Guillaume, HEMMATI, Philipp G, SCHULER, Martin, DÖRKEN, Bernd, DANIEL, Peter T
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 03.06.2004; Nature Publishing Group
• Subjects: Ageing, cell death; Apoptosis; Bax protein; Bcl-x protein; Biological and medical sciences; Cancer; Caspase-3; Caspase-9; Cell death; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cytochrome; Cytochrome c; Cytosol; DIABLO protein; Fundamental and applied biological sciences. Psychology; Hematology; IAP protein; Membrane potential; Mitochondria; Molecular and cellular biology; Oncology; Peptides; Proteins; Therapeutic targets; Tumor cells; Berlin Germany; Germany; Human; Carcinoma; Enzyme; caspase-9; Caspase; Malignant tumor; Carcinogenesis; Cytochrome c; Peptidases; Bcl-xL; Bax; Hydrolases; Smac; Release; Apoptosis; caspase-3
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1207594

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.