Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4-/DR5-specific signaling in colorectal and pancreatic cancer cells.

• MeSH
• apoptóza genetika   MeSH
• buňky HT-29 MeSH
• kaspasa 8 genetika   MeSH
• kolorektální nádory genetika   patologie   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• nádory slinivky břišní genetika   patologie   MeSH
• nekróza genetika   patologie   MeSH
• NF-kappa B genetika   MeSH
• pankreas metabolismus   patologie   MeSH
• proliferace buněk genetika   MeSH
• protein TRAIL genetika   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce genetika   MeSH
• TRAIL receptory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chloroquine (CQ), an anti-malarial drug, has immune-modulating activity and lysosomotropic activity. In this study, we investigated CQ sensitizes TRAIL-mediated apoptosis in human renal cancer Caki cells. Combination of CQ and TRAIL significantly induces apoptosis in human renal cancer Caki cells and various human cancer cells, but not in normal mouse kidney cells (TMCK-1) and human mesangial cells (MC). CQ up-regulates DR5 mRNA and protein expression in a dose- and time- dependent manner. Interestingly, CQ regulates DR5 expression through the increased stability in the mRNA and protein of DR5, rather than through the increased transcriptional activity of DR5. Moreover, we found that CQ decreased the expression of Cbl, an E3 ligase of DR5, and knock-down of Cbl markedly enhanced DR5 up-regulation. Other lysosomal inhibitors, including monensin and nigericin, also up-regulated DR5 and sensitized TRAIL-mediated apoptosis. Therefore, this study demonstrates that lysosomal inhibition by CQ may sensitize TRAIL-mediated apoptosis in human renal cancer Caki cells via DR5 up-regulation.

• MeSH
• apoptóza účinky léků   MeSH
• chlorochin aplikace a dávkování   MeSH
• genový knockdown MeSH
• lidé MeSH
• lyzozomy účinky léků   genetika   MeSH
• messenger RNA biosyntéza   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory ledvin farmakoterapie   genetika   patologie   MeSH
• protein TRAIL biosyntéza   genetika   MeSH
• protoonkogenní proteiny c-cbl genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• TRAIL receptory biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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
• antigeny CD95 genetika   metabolismus   MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• FLIP (buněčný) antagonisté a inhibitory   genetika   metabolismus   MeSH
• harringtoniny farmakologie   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• kaspasa 3 genetika   metabolismus   MeSH
• kaspasa 8 genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• proliferace buněk MeSH
• protein MCL-1 genetika   metabolismus   MeSH
• protein TRAIL genetika   metabolismus   farmakologie   MeSH
• receptory TNF - typ I genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• synergismus léků MeSH
• TRAIL receptory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• antitumorózní látky terapeutické užití   MeSH
• apoptóza genetika   imunologie   účinky léků   MeSH
• cyklin-dependentní kinasy genetika   škodlivé účinky   MeSH
• genetická transkripce genetika   MeSH
• hematologické nádory etiologie   krev   patofyziologie   MeSH
• inhibiční proteiny cyklin-dependentních kinas genetika   škodlivé účinky   MeSH
• inhibitory apoptózy analýza   genetika   MeSH
• lidé MeSH
• protein TRAIL genetika   škodlivé účinky   MeSH
• proteiny tepelného šoku genetika   škodlivé účinky   MeSH
• Check Tag
• lidé MeSH