title: Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells.
abstract: For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary. zsuzsanna.dunai@gmail.com
authors.forename: Zsuzsanna A
authors.initials: ZA
authors.lastname: Dunai
authors.forename: Gergely
authors.initials: G
authors.lastname: Imre
authors.forename: Gabor
authors.initials: G
authors.lastname: Barna
authors.forename: Tamas
authors.initials: T
authors.lastname: Korcsmaros
authors.forename: Istvan
authors.initials: I
authors.lastname: Petak
authors.forename: Pal I
authors.initials: PI
authors.lastname: Bauer
authors.forename: Rudolf
authors.initials: R
authors.lastname: Mihalik
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D009336:Necrosis; D019311:Staurosporine / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000502:physiology; D020298:U937 Cells
chemical_list: D061945:Caspase Inhibitors; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D020169:Caspases; D019311:Staurosporine