Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells

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

Schematic diagram about the action of inhibitors on TRAIL and STS- induced cell death pathways.

<p>Both TRAIL and STS triggered necroptosis in caspase depleted U937 cells upon prolonged incubation time. RIPK1 inhibitors Nec and GA could completely inhibit the TRAIL-evoked necroptosis, but had partial inhibitory potential to the STS-provoked process. Contrarily CA and NSA abolished both the TRAIL and STS-induced necroptosis. In absence of caspase inhibitor TRAIL and STS induced apoptosis which is followed by secondary necrosis. PJ-34 delayed the necrotic plasma membrane disruption during secondary necrosis, but failed to inhibit necroptosis.</p

STS induces primary necrosis in the presence of caspase inhibitor.

<p>(A-B) Nec (10 µM) and (C) GA (1 µM) significantly inhibited the STS-triggered necroptosis. Cells were exposed to STS (1 µM) in the presence or absence of zVD (5 µM) for 12 hrs. Percentage of PI positive cells was determined by flow cytometry (A, C). (n = 4) and by Hoechst/PI double staining technique (B) (representative of n = 2), (400x). Scale bar on the first subfigure applies to all the figures in the panel. (D) Nec (10 µM) arrested the STS-induced (1 µM) necroptosis in the presence of zVD (5 µM) after 20 hrs incubation. The mitochondrial transmembrane potential and plasma membrane integrity is shown in representative dot plots of DiOC₆(3) and PI stained, unfixed cells. The values indicate the percentage of cells in the marked regions (n = 13). (E-G) Nec (10 µM) (E, F) and GA (1 µM) (G) partially inhibited the STS-triggered necroptosis. Cells were exposed to STS (1 µM) in the presence or absence of zVD (5 µM) for 20 hrs. Percentage of PI positive cells was determined by flow cytometric analysis (E, G) (n = 13 for Nec and n = 4 for GA), and by Hoechst/PI double staining technique (F) (n = 2). Scale bar on the first subfigure applies to all the figures in the panel. Values are mean±SD. *, P<0.05, **, P<0.01 and ***, P<0.001 calculated by Student’s t-probe.</p