Abstract
BackgroundActivation 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.MethodsCaspase-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.ResultsAnalysis 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.ConclusionThese 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.
Highlights
Activation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in Non-small cell lung cancer (NSCLC) cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor
Our results show that TUCAN is expressed at high level in NSCLC cells when compared to small cell lung cancer (SCLC) cells
Analysis of components of the apoptosome and processing of caspase-9 induced by exogenous cytochrome c and dATP in lung cancer cells Given our previous finding that procaspase-9 is inhibited in NSCLC H460 cells [15], we started our investigation by examining the expression levels of the main apoptosome
Summary
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. Resistance to apoptosis in tumor cells can hamper the curative effect of chemotherapy, and several studies have demonstrated apoptosis resistance in NSCLC [3]. The caspases are responsible for the execution of apoptosis [4,5], and the efficacy of caspase-activation in tumor cells in response to treatment will, at least in part, determine the therapeutic effect [6]. The intrinsic pathway is triggered upon disruption of the mitochondria, leading to the release of cytochrome c into the cytosol where it induces apoptosome formation and caspase-9 activation [9]. The initiator caspases, activated in the apoptosome (caspase9) or DISC (caspase-8) can, in turn, cleave and activate the executioner caspases-3, -6 and -7, causing irreversible apoptosis
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