Necroptosis, a recently discovered form of non-apoptotic programmed cell death, can be implicated in many pathological conditions including neuronal cell death. that the mechanisms of neuroprotective effect of Nec-1 against H2O2-induced cell damage in SH-SY5Y cells involved the inhibition of lysosomal protease, cathepsin D, but not caspase-3 or calpain activities. In HT-22 cells, (S,R,S)-AHPC-PEG2-NH2 Nec-1 was protective in two models of oxidative stress (H2O2 and glutamate) and that effect was blocked by a caspase inhibitor. Our data showed neuroprotective effects of the necroptosis inhibitor, Nec-1, against oxidative stressCinduced cell damage and pointed to involvement of cathepsin D inhibition in the mechanism of its action. Moreover, a cell typeCspecific interplay between necroptosis and apoptosis has been demonstrated. test was also used for comparison of basal activities of caspase-3 or cathepsin D in UN- and RA-SH-SY5Y cells. Results Neuroprotective Effects of Nec-1 Against H2O2- and 6-OHDA-Induced Cell Damage in UN- and RA-SH-SY5Y Cells: the Impact of Cell Differentiation State Twenty-four hours of treatment with Nec-1 at up to 40?M was safe for UN- or RA-SH-SY5Y cells as confirmed by cell viability assay (Fig.?1a, d). Nec-1 (10C40?M) attenuated the cell damage induced by H2O2 in UN- and (S,R,S)-AHPC-PEG2-NH2 RA-SH-SHY5Y cells (Fig. 1a, d) with a significantly higher protection (measured as a mean area under the curve (AUC)) (S,R,S)-AHPC-PEG2-NH2 mediated in the former cell phenotype (AUC?=?95.26??5.74 and AUC?=?44.82??4.34 for UN- and RA-SH-SY5Y, respectively; test, (DIC) images (Fig.?2) and by CalceinAM staining (Fig.?3a). Additionally, we showed a significant increase in the number of pyknotic nuclei after treatment of (S,R,S)-AHPC-PEG2-NH2 UN-SH-SY5Y cells (after 9?h) and RA-SH-SY5Y cells (after 9 and 18?h) with H2O2 which was not changed by Nec-1 (20?M) pre-treatment at any of the tested time points (Fig. ?(Fig.3b).3b). However, we observed that Nec-1 partially protected the cells against H2O2-induced reduction in the number of healthy nuclei which was observed after 18?h but not after 9?h of treatment in both cell phenotypes (Fig. ?(Fig.3c).3c). Next, we measured the impact of Nec-1 pre-treatment on H2O2-evoked neurite shortening after 9 and 18?h of treatment. In UN-SH-SY5Y cells, we found a significant reduction in this parameter after 18?h of treatment with H2O2 which was completely blocked by Nec-1 pre-treatment (Fig. ?(Fig.3d,3d, left panel). In the case of RA-SH-SY5Y cells, the H2O2 evoked reduction in neurite length after 9 and 18?h of treatment which was significantly reduced by Nec-1 (Fig. ?(Fig.3d,3d, right panel). Open in a separate window Fig. 1 The effect of necrostatin-1 on H2O2-induced cell damage in UN- and RA-SH-SY5Y cells. UN- and RA-SH-SY5Y cells (aCc and dCf, respectively) were pre-treated for 30?min with necrostatin-1 (Nec-1; 1C40?M) followed by 24?h of treatment with H2O2 (0.25 and 0.5?mM for UN- and RA-SH-SY5Y, respectively). As a positive control for the assays, we Mouse monoclonal antibody to MECT1 / Torc1 used antioxidant N-acetylcysteine (NAC, 1?mM) which was given concomitantly with the cell damaging factor. a, d Results of cell viability assessment in UN-(a) and RA-(d) SH-SY5Y cells measured by the MTT reduction assay. Data were normalized to vehicle-treated cells (control) and are presented as the mean SEM from 3 to 11 separate experiments with 5 repetitions each. (b, e) Results of cell toxicity assessment in UN-(b) and RA-(e) SH-SY5Y cells measured by the LDH release assay. Data were normalized to vehicle-treated cells (control) and are presented as the mean SEM.