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

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-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.

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway

“…While As 2 O 3 -mediated cell death involved the mitochondrial death pathway, we consistently observed only rather limited activation of the caspase-3 upon As 2 O 3 -treatment when compared with other chemotherapeutic drugs Radetzki et al, 2002;von Haefen et al, 2003;Prokop et al, 2003) or death receptor ligation Schmelz et al, 2004), ceramide , Smac/ Diablo (Hasenja¨ger et al, 2004) or endogenous activators of the intrinsic apoptosis pathway such as p14 ARF (Hemmati et al, 2002) or BH3-only proteins such as Nbk (Gillissen et al, 2003). This rather weak activation of the effector caspase-3 by arsenite was, however, not due to a direct inhibitory effect of As 2 O 3 on caspases.…”

Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells

“…Control vector (pcDNA3-transfected) and pcDNA3-caspase-3-transfected MCF-7 Hasenja¨ger et al, 2004) and HCT116 wild-type or p21 À/À cells (Waldman et al, 1996) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 0.56 g/l L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all from GIBCO-BRL, Karlsruhe, Germany) at 371C with 5% CO 2 in a fully humidified atmosphere. Cells were infected with recombinant, replication-deficient adenovirus diluted in cell culture medium in the absence of FCS or antibiotics at the indicated MOI for 1.5 h at 371C or were mock treated with serum-free medium only.…”

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway

“…Active caspase-9 then mediates cleavage of the effector caspases-3, -6, and -7. Cytosolic Smac was demonstrated to alleviate IAPmediated repression of caspase activation and to facilitate activation of caspases-9 and -3 (Wu et al, 2000;Hasenja¨ger et al, 2004).…”