SMAC/Diablo -dependent apoptosis induced by nonsteroidal antiinflammatory drugs (NSAIDs) in colon cancer cells

Abstract

Nonsteroidal antiinflammatory drugs (NSAIDs) form a paradigm for the chemoprevention of cancer, preventing colonic tumor progression in both experimental animals and humans. However, the mechanisms underlying the antineoplastic effects of NSAIDs are currently unclear. We found that the mitochondrial second mitochondrial-derived activator of caspase (SMAC)͞direct inhibitor of apoptosis protein-binding protein with low pI (Diablo) protein translocates into the cytosol during NSAID-induced apoptosis in colon cancer cells. When SMAC͞Diablo is disrupted by homologous recombination and RNA interference in these cells, the NSAIDinduced apoptosis is abrogated. Biochemical markers of apoptosis, such as caspase activation, cytosolic release of cytochrome c and apoptosis-inducing factor, and mitochondrial membrane potential change, are accordingly decreased. These results establish that SMAC͞Diablo is essential for the apoptosis induced by NSAIDs in colon cancer cells.

SMAC/Diablo mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events

“…PUMA-induced apoptosis is inhibited in SMAC-deficient cells, but not in AIF-deficient cells To determine whether SMAC release is relevant, or just a coincidental event in PUMA-induced apoptosis, we analysed SMAC-knockout (SMAC-KO) HCT116 cells that we previously created by homologous recombination (Kohli et al, 2004). After Ad-PUMA infection, significantly less apoptosis was detected by nuclear staining in the SMAC-KO cells compared with that in the parental cells 48 h after treatment (18 vs 68%; Figure 2a and b).…”

“…Therefore, a SMAC-reconstituted cell line (SMAC-R) we previously generated was analysed for its response to PUMA (Kohli et al, 2004). PUMA-induced apoptosis was found to be completely restored by the reconstituted SMAC in the SMAC-KO cells .…”

“…PUMA-induced apoptosis was found to be completely restored by the reconstituted SMAC in the SMAC-KO cells . To rule out the possibility that these observations are due to the specific genetic background of HCT116 cells, we analysed SMAC-deficient DLD1 colon cancer cells (SMAC-KD) generated by stable transfection of small interference RNA (siRNA) (Kohli et al, 2004). Knockdown of SMAC in DLD1 cells led to a significant decrease in PUMA-induced apoptosis, as determined by nuclear staining, Annexin V staining and cell cycle analysis ( Figure 3 and data not shown).…”

“…Activation of caspase-3 and caspase-9 was also decreased in SMAC-KO cells undergoing apoptosis induced by the DNA-damaging agent etoposide (Figure 5b). In contrast, treating the cells with N-acetylleu-leu-norleucinal (ALLN), a proteasome inhibitor, which does not rely on SMAC to induce apoptosis (Kohli et al, 2004), led to similar extent of caspase-3 and caspase-9 activation in the parental HCT116 and SMAC-KO cells (Figure 5b), suggesting that the role of SMAC in apoptosis is limited to certain stimuli.…”

“…Our previous study in colon cancer cells demonstrated the involvement of SMAC in apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) (Kohli et al, 2004). Studies by other groups suggest the existence of a caspase-mediated feedback amplification loop in regulating cytochrome c release and other mitochondrial events in apoptosis (Chen et al, 2000;Lakhani et al, 2006).…”