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

Oncogene volume 23, pages 4523–4535 (2004)Cite this article

Abstract

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-xL. 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-xL during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-xL-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-xL that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

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Acknowledgements

The technical assistance of Antje Richter is gratefully acknowledged. We wish to thank Dr. Bert Vogelstein, Johns Hopkins Cancer Center, Baltimore, for the kind gift of Bax-deficient HCT116 cells. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SFB 506, Da238/4-1 and Da238/4-2), and the European Union RTN network ‘Oncodeath’. AH was a recipient of a fellowship from the DFG graduate college 331. PTD, PGH and MS are supported by the Deutsche Krebshilfe.

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  1. Department of Hematology Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany

    Anne Hasenjäger, Bernhard Gillissen, Antje Müller, Guillaume Normand, Philipp G Hemmati, Bernd Dörken & Peter T Daniel

  2. Department of Medicine III, Johannes Gutenberg University, Mainz, Germany

    Martin Schuler

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  1. Anne Hasenjäger
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Correspondence to Peter T Daniel.

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Hasenjäger, A., Gillissen, B., Müller, A. et al. Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xL in a strictly caspase-3-dependent manner in human carcinoma cells. Oncogene 23, 4523–4535 (2004). https://doi.org/10.1038/sj.onc.1207594

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