Eva Ondroušková

Enzymatically active cathepsin D sensitizes breast carcinoma cells to TRAIL

Cathepsin D (CD), a ubiquitously expressed lysosomal aspartic protease, is upregulated in human breast carcinoma and many other tumor types. CD has been repeatedly reported to act as key mediator of apoptosis induced by various chemotherapeutics. However, there is still controversy over the role of enzymatic/proteolytic versus protein-protein interaction activities of CD in apoptotic signaling. The elucidation of molecular mechanism responsible for the effect of CD in the chemotherapy-induced cell death is crucial for development of an appropriate strategy to target this protease in cancer treatment. Therefore, the objective of this study was to investigate the molecular mechanism behind the CD-mediated regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. For this purpose, MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (ΔCD) were subjected to TRAIL and the frequency of apoptosis was determined. Our results show that CD facilitates the TRAIL-induced apoptosis of MDA-MB-231 breast cancer cells in enzymatic activity-dependent manner. Moreover, the importance of endosomal/lysosomal acidification in this process was documented. Analysis of the potential substrates specifically cleaved by CD during the TRAIL-induced apoptosis confirmed caspase-8 and Bid proteins as the CD targets. Moreover, in search for protein regulators of apoptosis that can be cleaved by CD at physiologically relevant pH, we identified the Bcl-2 protein as a suitable candidate. The modulatory role of CD in cell response to TRAIL was also confirmed in another breast cancer cell line SKBR3. These experiments identified the CD enzymatic activity as a new factor affecting sensitivity of breast cancer cells to TRAIL.

Heavy metals induce phosphorylation of the Bcl-2 protein by Jun N-terminal kinase.

Abstract The Bcl-2 protein is one of the key components of biochemical pathways controlling programmed cell death. The function of this protein can be regulated by posttranslational modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by defects of microtubule dynamics. This study shows that phosphorylation of Bcl-2 can be induced by heavy metals due to activation of the Jun N-terminal kinase pathway that is not linked to the G2/M cell cycle arrest. Furthermore, we demonstrate that hyperphosphorylated Bcl-2 protein is a more potent inhibitor of zinc-induced cell death than its hypophosphorylated mutant form. These data suggest that regulation of Bcl-2 protein function by phosphorylation is an important part of cell responses to stress.