Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps.
Dysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of...
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0198203&type=printable |
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| author | Lubna Danish Dirke Imig Frank Allgöwer Peter Scheurich Nadine Pollak |
| author_facet | Lubna Danish Dirke Imig Frank Allgöwer Peter Scheurich Nadine Pollak |
| author_sort | Lubna Danish |
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| description | Dysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of apoptosis using the highly bioactive TRAIL derivative Db-scTRAIL. NCI-H460 cells were TRAIL sensitive but an only about 3 fold overexpression of Bcl-2 was sufficient to induce a highly TRAIL resistant phenotype, confirming that the mitochondrial pathway is crucial for TRAIL-induced apoptosis induction. TRAIL resistance was paralleled by a strong inhibition of caspase-8, -9 and -3 activities and blocked their full processing. Notably, especially the final cleavage steps of the initiator caspase-8 and the executioner caspase-3 were effectively blocked by Bcl-2 overexpression. Caspase-9 knockdown failed to protect NCI-H460 cells from TRAIL-induced cell death, suggesting a minor role of this initiator caspase in this apoptotic pathway. Rather, knockdown of the XIAP antagonist Smac resulted in enhanced caspase-3 degradation after stimulation of cells with TRAIL. Of note, downregulation of XIAP had only limited effects on TRAIL sensitivity of wild-type NCI-H460 cells, but resensitized Bcl-2 overexpressing cells for TRAIL-induced apoptosis. In particular, XIAP knockdown in combination with TRAIL allowed the final cleavage step of caspase-3 to generate the catalytically active p17 fragment, whose production was otherwise blocked in Bcl-2 overexpressing cells. Together, our data strongly suggest that XIAP-mediated inhibition of final caspase-3 processing is the last and major hurdle in TRAIL-induced apoptosis in NCI-H460 cells, which can be overcome by Smac in a Bcl-2 level dependent manner. Quantitative investigation of the XIAP/Smac interplay using a mathematical model approach corroborates our experimental data strengthening the suggested roles of XIAP and Smac as critical determinants for TRAIL sensitivity. |
| format | Article |
| id | doaj-art-9e232127ea1d4ef3932132940bc1d1b5 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-9e232127ea1d4ef3932132940bc1d1b52025-08-20T02:03:58ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-01136e019820310.1371/journal.pone.0198203Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps.Lubna DanishDirke ImigFrank AllgöwerPeter ScheurichNadine PollakDysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of apoptosis using the highly bioactive TRAIL derivative Db-scTRAIL. NCI-H460 cells were TRAIL sensitive but an only about 3 fold overexpression of Bcl-2 was sufficient to induce a highly TRAIL resistant phenotype, confirming that the mitochondrial pathway is crucial for TRAIL-induced apoptosis induction. TRAIL resistance was paralleled by a strong inhibition of caspase-8, -9 and -3 activities and blocked their full processing. Notably, especially the final cleavage steps of the initiator caspase-8 and the executioner caspase-3 were effectively blocked by Bcl-2 overexpression. Caspase-9 knockdown failed to protect NCI-H460 cells from TRAIL-induced cell death, suggesting a minor role of this initiator caspase in this apoptotic pathway. Rather, knockdown of the XIAP antagonist Smac resulted in enhanced caspase-3 degradation after stimulation of cells with TRAIL. Of note, downregulation of XIAP had only limited effects on TRAIL sensitivity of wild-type NCI-H460 cells, but resensitized Bcl-2 overexpressing cells for TRAIL-induced apoptosis. In particular, XIAP knockdown in combination with TRAIL allowed the final cleavage step of caspase-3 to generate the catalytically active p17 fragment, whose production was otherwise blocked in Bcl-2 overexpressing cells. Together, our data strongly suggest that XIAP-mediated inhibition of final caspase-3 processing is the last and major hurdle in TRAIL-induced apoptosis in NCI-H460 cells, which can be overcome by Smac in a Bcl-2 level dependent manner. Quantitative investigation of the XIAP/Smac interplay using a mathematical model approach corroborates our experimental data strengthening the suggested roles of XIAP and Smac as critical determinants for TRAIL sensitivity.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0198203&type=printable |
| spellingShingle | Lubna Danish Dirke Imig Frank Allgöwer Peter Scheurich Nadine Pollak Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. PLoS ONE |
| title | Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. |
| title_full | Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. |
| title_fullStr | Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. |
| title_full_unstemmed | Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. |
| title_short | Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps. |
| title_sort | bcl 2 mediated control of trail induced apoptotic response in the non small lung cancer cell line nci h460 is effective at late caspase processing steps |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0198203&type=printable |
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