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Enzymaticky aktivní katepsin D zvyšuje citlivost prsní nádorové linie MDA-MB-231 k TRAILu

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JANČEKOVÁ Blanka BENEŠ Petr ONDROUŠKOVÁ Eva KNOPFOVÁ Lucia ŠMARDA Jan

Rok publikování 2015
Druh Další prezentace na konferencích
Citace
Popis Cathepsin D (CTSD), a ubiquitously expressed lysosomal aspartic protease, is up-regulated in human breast carcinoma and many other tumour types. Its overexpression was reported to increase invasiveness and metastatic potential of tumour cells and is correlated with poor prognosis in patients. However, CTSD has also been suggested to act as key mediator of apoptosis induced by some chemotherapeutics and its proteolytic activity has often been shown to be involved in this event. Thus, to elucidate the mechanism of action of CTSD in chemotherapy-induced cell death is crucial to develop an appropriate strategy to target this protease in cancer treatment. Cathepsin D down-regulation using cathepsin D small interfering RNA was shown to partially inhibit cell death resulting from treatment of cells with Tumour necrosis factor-related apoptosis inducing ligand (TRAIL) (1). Nevertheless, the role of cathepsin D enzymatic activity in the TRAIL-induced apoptosis remains unknown. The objective of this study is to investigate the molecular mechanism behind the CTSD-mediated regulation of TRAIL-induced cell death and the role of its proteolytic activity in this regulation. For this purpose MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (?CD) were prepared using stable transfection. Parental cell line and selected CD/?CD overexpressing stable transfected clones were than subjected to TRAIL and frequency of apoptotic cell death was determined by analysis of nuclear morphology, annexin V binding assay and cleavage of poly (ADP-ribose) polymerase (PARP). Our results demonstrate that CTSD facilitates the TRAIL-induced apoptosis of breast cancer MDA-MB-231 cells in enzymatic activity-dependent manner. Moreover, activation of caspases was documented and the importance of lysosomal acidification in this process was evaluated. These experiments bring recent findings to the identification of new biomarkers affecting sensitivity of tumours to TRAIL that may therefore improve the effectiveness of therapeutic strategies targeting the TRAIL-signalling pathway in the future.
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