Tumor necrosis factor-related apoptosis-inducing ligand-TRAIL-is a protein operating as a ligand capable of inducing apoptosis particularly in cancerously transformed cells, while normal healthy cells are typically nonresponsive. We have previously demonstrated that pluripotent human embryonic stem cells (hESC) are also refractory to TRAIL, even though they express all canonical components of the death receptor-induced apoptosis pathway. In this study, we have examined a capacity of DNA damage to provoke sensitivity of hESC to TRAIL. The extent of DNA damage, behavior of molecules involved in apoptosis, and response of hESC to TRAIL were investigated. The exposure of hESC to 1 μM and 2 μM concentrations of cisplatin have led to the formation of 53BP1 and γH2AX foci, indicating the presence of double-strand breaks in DNA, without affecting the expression of proteins contributing to mitochondrial membrane integrity. Interestingly, cisplatin upregulated critical components of the extrinsic apoptotic pathway-initiator caspase 8, effector caspase 3, and the cell death receptors. The observed increase of expression of the extrinsic apoptotic pathway components was sufficient to sensitize hESC to TRAIL-induced apoptosis; immense cell dying accompanied by enhanced PARP cleavage, processing of caspase 8, and full activation of caspase 3 were all observed after the treatment combining cisplatin and TRAIL. Finally, we have demonstrated the central role of caspase 8 in this process, since its downregulation abrogated the sensitizing effect of cisplatin.

• Publication type
• Journal Article MeSH

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.

• Keywords
• Apoptosis, Bcl-2, Breast cancer, Caspases, Cathepsin D, TRAIL,
• MeSH
• Adenocarcinoma enzymology   pathology   MeSH
• Enzyme Activation MeSH
• Apoptosis drug effects   MeSH
• Drug Resistance, Neoplasm MeSH
• Endosomes metabolism   MeSH
• Caspase 8 metabolism   MeSH
• Cathepsin D antagonists & inhibitors   genetics   physiology   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Lysosomes metabolism   MeSH
• RNA, Small Interfering genetics   MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins antagonists & inhibitors   genetics   physiology   MeSH
• Breast Neoplasms enzymology   pathology   MeSH
• BH3 Interacting Domain Death Agonist Protein metabolism   MeSH
• TNF-Related Apoptosis-Inducing Ligand pharmacology   MeSH
• Proto-Oncogene Proteins c-bcl-2 metabolism   MeSH
• Recombinant Proteins metabolism   pharmacology   MeSH
• RNA Interference MeSH
• Transfection MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• BID protein, human MeSH Browser
• CASP8 protein, human MeSH Browser
• CTSD protein, human MeSH Browser
• Caspase 8 MeSH
• Cathepsin D MeSH
• RNA, Small Interfering MeSH
• Neoplasm Proteins MeSH
• BH3 Interacting Domain Death Agonist Protein MeSH
• TNF-Related Apoptosis-Inducing Ligand MeSH
• Proto-Oncogene Proteins c-bcl-2 MeSH
• Recombinant Proteins MeSH
• TNFSF10 protein, human MeSH Browser

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.

• MeSH
• fas Receptor genetics   metabolism   MeSH
• Apoptosis drug effects   MeSH
• Cell Differentiation MeSH
• Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• CASP8 and FADD-Like Apoptosis Regulating Protein antagonists & inhibitors   genetics   metabolism   MeSH
• Harringtonines pharmacology   MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors pharmacology   MeSH
• Caspase 3 genetics   metabolism   MeSH
• Caspase 8 genetics   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• Pluripotent Stem Cells cytology   drug effects   metabolism   MeSH
• Cell Proliferation MeSH
• Myeloid Cell Leukemia Sequence 1 Protein genetics   metabolism   MeSH
• TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   pharmacology   MeSH
• Receptors, Tumor Necrosis Factor, Type I genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Drug Synergism MeSH
• Receptors, TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• fas Receptor MeSH
• FAS protein, human MeSH Browser
• CASP8 and FADD-Like Apoptosis Regulating Protein MeSH
• Harringtonines MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors MeSH
• Caspase 3 MeSH
• Caspase 8 MeSH
• RNA, Small Interfering MeSH
• MCL1 protein, human MeSH Browser
• Myeloid Cell Leukemia Sequence 1 Protein MeSH
• TNF-Related Apoptosis-Inducing Ligand MeSH
• Receptors, Tumor Necrosis Factor, Type I MeSH
• TNFRSF10A protein, human MeSH Browser
• TNFSF10 protein, human MeSH Browser
• Receptors, TNF-Related Apoptosis-Inducing Ligand MeSH