TUCAN/CARDINAL/CARD8 and apoptosis resistance in non-small cell lung cancer cells.
BACKGROUND
Activation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in NSCLC cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor. The aim of the present study was to investigate the mechanism of caspase-9 inhibition, with a focus on a possible role of TUCAN as caspase-9 inhibitor and a determinant of chemosensitivity in NSCLC cells.


METHODS
Caspase-9 processing and activation were investigated by Western blot and by measuring the cleavage of the fluorogenic substrate LEHD-AFC. Proteins interaction assays, and RNA interference in combination with cell viability and apoptosis assays were used to investigate the involvement of TUCAN in inhibition of caspase-9 and chemosensitivity NSCLC.


RESULTS
Analysis of the components of the caspase-9 activation pathway in a panel of NSCLC and SCLC cells revealed no intrinsic defects. In fact, exogenously added cytochrome c and dATP triggered procaspase-9 cleavage and activation in lung cancer cell lysates, suggesting the presence of an inhibitor. The reported inhibitor of caspase-9, TUCAN, was exclusively expressed in NSCLC cells. However, interactions between TUCAN and procaspase-9 could not be demonstrated by any of the assays used. Furthermore, RNA interference-mediated down-regulation of TUCAN did not restore cisplatin-induced caspase-9 activation or affect cisplatin sensitivity in NSCLC cells.


CONCLUSIONS
These results indicate that procaspase-9 is functional and can undergo activation and full processing in lung cancer cell extracts in the presence of additional cytochrome c/dATP. However, the inhibitory protein TUCAN does not play a role in inhibition of procaspase-9 and in determining the sensitivity to cisplatin in NSCLC.
Checinska
Agnieszka
A
Department of Medical Oncology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands. a.checinska@vumc.nl
Giaccone
Giuseppe
G
Hoogeland
Bas S J
BS
Ferreira
Carlos G
CG
Rodriguez
Jose A
JA
Kruyt
Frank A E
FA
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000032:analysis / Q000378:metabolism; D053468:CARD Signaling Adaptor Proteins; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D045304:Cytochromes c / Q000494:pharmacology; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D015536:Down-Regulation; D020869:Gene Expression Profiling; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D034622:RNA Interference; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; C455623:CARD8 protein, human; D061945:Caspase Inhibitors; D003838:Deoxyadenine Nucleotides; D009363:Neoplasm Proteins; D045304:Cytochromes c; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate; D002945:Cisplatin