Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-x(L) in a strictly caspase-3-dependent manner in human carcinoma cells.

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.

Hasenjäger A ，Gillissen B ，Müller A ，Normand G ，Hemmati PG ，Schuler M ，Dörken B ，Daniel PT ... -  《ONCOGENE》

Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.

Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL-induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 microg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL-induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL-induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.

Chen J ，Mehta JL ，Haider N ，Zhang X ，Narula J ，Li D ... -  《-》

Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria.

Yamaguchi H ，Bhalla K ，Wang HG 《CANCER RESEARCH》

Effects of antioxidants and caspase-3 inhibitor on the phenylethyl isothiocyanate-induced apoptotic signaling pathways in human PLC/PRF/5 cells.

Phenylethyl isothiocyanate (PEITC) is a well recognized potential chemopreventive compound against human cancers. In this study, the molecular mechanism of PEITC-induced apoptosis was examined with two antioxidants (N-acetyl-cysteine and vitamin E) and a caspase-3 inhibitor (z-DEVD-fmk). Results demonstrated that PEITC significantly induced human hepatoma PLC/PRF/5 (CD95-negative) cells undergoing apoptosis. Treatment with 0 approximately 10 microM PEITC-triggered cell apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and the subsequent appearance of sub-G1 population. Results also displayed that PEITC-induced apoptosis involves the up-regulation of p53 and Bax protein, down-regulation of the XIAP, Bcl-2, Bcl-(XL) and Mcl-1 proteins, cleavage of Bid, and the release of cytochrome c and Smac/Diablo, which were accompanied by the activation of caspases -9, -3 and -8. PEITC-induced the generation of reactive oxygen species and the decrease of mitochondrial membrane potential (Deltapsim) in a time-dependent pattern. N-acetyl-cysteine and vitamin E at 100 microM, and z-DEVD-fmk at 50 microM markedly blocked PEITC-induced apoptosis, which was demonstrated by a decline in the reactive oxygen species generation and the release of the cytochrome c and Smac/Diablo from mitochondria to the cytosol. N-acetyl-cysteine, vitamin E and z-DEVD-fmk also prevented the PEITC in inducing the loss of Deltapsim. They also affected the activity of XIAP and Bax proteins. Taken together, these studies suggest that PEITC is an apoptotic inducer that acts on the mitochondria and the feedback amplification loop of caspase-8/Bid pathways in PLC/PRF/5 cells.

Wu SJ ，Ng LT ，Lin CC 《EUROPEAN JOURNAL OF PHARMACOLOGY》

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.

Izeradjene K ，Douglas L ，Tillman DM ，Delaney AB ，Houghton JA ... -  《CANCER RESEARCH》