The intrinsic apoptosis pathway represents an important mechanism of stress-induced death of
cancer cells. To gain insight into the functional status of the
apoptosome apparatus in
non-small cell lung carcinoma (NSCLC), we studied its sensitivity to activation, the assembly of
apoptosome complexes and stability of their precursors, and the importance of X-linked inhibitor of apoptosis (XIAP) in the regulation of
apoptosome activity, using cell-free cytosols from NSCLC cell lines and NSCLC tumours and lungs from 62 surgically treated patients. Treatment of cytosol samples with
cytochrome c (cyt-c) and dATP induced proteolytic processing of
procaspase-9 to
caspase-9, which was followed by
procaspase-3 processing to
caspase-3, and by generation of caspase-3-like activity in 5 of 7 studied NSCLC cell lines. Further analysis demonstrated formation of high-Mr Apaf-1 complexes associated with cleaved
caspase-9 in the (cyt-c + dATP)-responsive COLO-699 and CALU-1 cells. By contrast, in A549 cells, Apaf-1 and
procaspase-9 co-eluted in the high-Mr fractions, indicating formation of an
apoptosome complex unable of
procaspase-9 processing. Thermal pre-treatment of cell-free cytosols in the absence of exogenous cyt-c and dATP lead to formation of Apaf-1 aggregates, unable to recruit and activate
procaspase-9 in the presence of cyt-c and dATP, and to generate caspase‑3‑like activity. Further studies showed that the treatment with cyt-c and dATP induced a substantially higher increase of caspase-3-like activity in cytosol samples from NSCLC tumours compared to matched lungs. Tumour histology, grade and stage had no significant impact on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Upon addition into the cytosol, the XIAP-neutralizing
peptides AVPIAQK and ATPFQEG only moderately heightened the (cyt-c + dATP)-induced caspase‑3‑like activity in some NSCLC tumours. Taken together, the present study provides evidence that the
apoptosome apparatus is functional in the majority of NSCLCs and that its sensitivity to the (cyt-c + dATP)-mediated activation is often enhanced in NSCLCs compared to lungs. They also indicate that XIAP does not frequently and effectively suppress the activity of
apoptosome apparatus in NSCLCs.