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.

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“…Cells that are stuck favoring the TCA cycle and unable to meet increased energy demands are more likely to die from necroptosis (45). Necroptosis is another form of programmed cell death often mediated by the protein kinases RIPK1 and RIPK3 (46,47) and enhanced by the presence of reactive oxygen species. Importantly, necroptotic pathways have been shown to be in direct opposition to apoptotic and pyroptotic cell death.…”

A Receptor-interacting Protein 1 (RIP1)-independent Necrotic Death under the Control of Protein Phosphatase PP2A That Involves the Reorganization of Actin Cytoskeleton and the Action of Cofilin-1

“…2G). Finally we used staurosporine, a recently suggested necroptotic trigger (27). Staurosporine induced both apoptosis and necrosis in U87MG/Bcl-xL cells (Fig.…”

New Insights into the Molecular Mechanisms of Selected Anticancer Metal Compounds through Bioinformatic Analysis of Proteomic Data

“…Moreover, the cellular senescence has been found associated with changes of eEF1 levels in colon and lung cancer cell lines [31]. The A and B classes of proteins are connected together by an intermed i ate node: Receptor-like protein kinase1 (RIPK1-Q13546) a Serine-threonine kinase, which transduces inflammatory and celldeath signals (necroptosis) and DNA damage [32].…”