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. 2014 May;44(5):1443-54.
doi: 10.3892/ijo.2014.2333. Epub 2014 Mar 10.

Differential sensitivity to apoptosome apparatus activation in non-small cell lung carcinoma and the lung

Erika Moravcikova  1 , Evzen Krepela  1 , Jan Prochazka  1 , Kamila Benkova  2 , Norbert Pauk  3
Affiliations

Differential sensitivity to apoptosome apparatus activation in non-small cell lung carcinoma and the lung

Erika Moravcikova et al. Int J Oncol. 2014 May.

Abstract

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.

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Figures

Figure 1.
Figure 1.
Analysis of the (cyt-c + dATP)-induced procaspase-9 processing in cell-free cytosols from NSCLC cell lines. (A) Incubation of cell-free cytosol samples from CALU-1, SKMES-1 and LXF-289 cells with exogenous cyt-c and dATP induced proteolytic processing of procaspase-9 (p46) to the p35 and p37 cleaved caspase-9 forms. (B) Procaspase-9 (p46) in cell-free cytosol samples from A549 and SKLU-1 cells was resistant to the (cyt-c + dATP)-mediated proteolytic processing. In SKLU-1 cells, the p37 caspase-9 cleaved forms was regularly detected in both the (cyt-c + dATP)-untreated and -treated cysosol samples.
Figure 2.
Figure 2.
Analysis of the (cyt-c + dATP)-induced procaspase-9 processing and caspase-9 activity in NCI-H520 cell-free cytosol. In the absence of the caspase inhibitor Ac-DEVD-CHO, the (cyt-c + dATP)-induced processing of procaspase-9 (p46) to the p35 and p37 caspase-9 cleaved forms (A, left panel) was followed by procaspase-3 (p32) processing to the p20 and p17 caspase-3 cleaved forms (B, left panel). In the presence of Ac-DEVD-CHO (1 μM), procaspase-9 (p46) was processed to the p35 caspase-9 cleaved form only (A, right panel) and the processing of procaspase-3 (p32) was completely blocked (B, right panel).
Figure 3.
Figure 3.
Analysis of the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity in cell-free cytosols from NSCLC cell lines. The endogenous (E), the total (cyt-c + dATP)-induced (T), and the incremental (cyt-c + dATP)-induced (I=T–E) caspase-3-like activities were measured using Ac-DEVD-AFC as the substrate in cell-free cytosol samples from a panel of seven NSCLC cell lines. The cell-free cytosol from SKLU-1 cells regularly contained a high endogenous caspase-3-like activity. Data are indicated as mean ± SEM from three independent experiments.
Figure 4.
Figure 4.
Analysis of the formation of apoptosome complexes in cell-free cytosol from COLO-699 cells. (A) Non-incubated control cell-free cytosol: both Apaf-1 and unprocessed procaspase-9 (p46) eluted in low Mr-fractions. (B) Cell-free cytosol after incubation without exogenous cytochrome c and dATP: Apaf-1 eluted in both high and low Mr-fractions, while procaspase-9 (p46) eluted in low Mr-fractions only. (C) Cell-free cytosol after incubation with exogenous cytochrome c and dATP: Apaf-1 eluted in both high and low Mr-fractions, while procaspase-9 (p46) was completely converted to the p35 caspase-9 cleaved form, which coeluted with Apaf-1 in high Mr-fractions and also eluted in low Mr-fractions. The positions of Mr markers are indicated with arrows. For further experimental details see Materials and methods.
Figure 5.
Figure 5.
Analysis of the formation of apoptosome complexes in cell-free cytosol from A549 cells. After incubation of A549 cell-free cytosol with exogenous cytochrome c and dATP, Apaf-1 and procaspase-9 (p46) co-eluted in high-Mr fractions as well as in low-Mr fractions. Small amounts of p35 and p37 caspase-9 cleaved forms were found in low-Mr fractions. The positions of Mr markers are indicated with arrows. For further experimental details see Materials and methods.
Figure 6.
Figure 6.
Thermal pre-treatment of cell-free cytosol causes loss of the (cyt-c + dATP)-mediated induction of caspase-3-like activity: dependency on temperature and time, and its effect in different cell lines and NSCLC tumours and lung parenchyma. Cell-free cytosol from NCI-H520 cells was pre-incubated without cyt-c and dATP at different temperatures (A) and for different times (B), following incubation with exogenous cyt-c and dATP at 37°C for 30 min. Cytosol samples without pre-incubation (kept on ice) that were incubated without and with cyt-c plus dATP served as negative and positive controls, respectively. Cell-free cytosols from different cell lines (C) and NSCLC tumours of different histopathological types and lung parenchyma (D) were kept on ice or were pre-incubated at 37°C for 10 min following incubation without or with cyt-c and dATP at 37°C for 30 min. After completing the indicated treatments, the endogenous and the total (cyt-c + dATP)-induced caspase-3-like activities were measured using Ac-DEVD-AFC as the substrate. One representative data set of three independent experiments is shown in (A) and (B). Data in (C) and (D) represent means ± SEM from three independent experiments for each examined cell line and tissue. Note the logarithmic scale on the y-axis in (C) and (D).
Figure 7.
Figure 7.
Effect of XIAP-neutralizing peptides on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity in cell-free cytosols from NSCLC tumours. (A and B) Caspase-3-like reactions in cell-free cytosol sample aliquots from an SQCLC tumour and a UNDIF tumour, respectively, that had been preincubated without (a control, C) or with cyt-c + dATP (CydA) and without or with 500 μM of the XIAP-neutralizing peptide AVPIAQK (P1) or ATPFQEG (P2). In (A), the rates of the caspase-3-like reactions labelled C, P1, CydA and CydA + P1 were, respectively, 0.23, 0.29, 2.4 and 4.7 nkat. In (B), the rates of the caspase-3-like reactions labelled C, P2, CydA and CydA + P2 were, respectively, 0.28, 0.28, 7.6 and 13.0 nkat. (C and D) depict the responses of the endogenous and the (cyt-c + dATP)-induced caspase-3-like activities, respectively, in cell-free cytosols from 21 NSCLC tumours to the peptides AVPIAQK (P1, 500 μM) and ATPFQEG (P2, 500 μM). The incremental (cyt-c + dATP)-induced caspase-3-like activities in the absence of the peptides (a control, I) and in their presence (I-P1 and I-P2) were calculated as described in Materials and methods. Data indicated as mean ± SEM from three independent experiments. The caspase-3-like activity ratios I-P1/I and I-P2/I higher than 2 (+) and higher than 1.5 (*) but lower than 2 are indicated.
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