Staurosporine induces apoptosis and necroptosis in cultured rat astrocytes

Abstract

Apoptosis and necroptosis are highly regulated, interconnected forms of a cell death. The distinction between them is critical, because necroptosis may cause significant cell loss and local inflammation, whereas apoptosis is essential for tissue homeostasis. The same stimulus can induce both apoptosis and necroptosis. Both forms of a cell death were detected in various pathologies, including pathologies in the central nervous system. Astrocytes are a large, heterogeneous cell population in the central nervous system, with many supportive, developmental functions. Although their demise may seriously impair normal functions of the central nervous system, it is still poorly understood. In this study, apoptosis and necroptosis were induced in cultured rat astrocytes by staurosporine. When a low concentration (10(-7) M) of staurosporine was applied, a significantly increased proportion of early apoptotic cells was detected after regeneration in a staurosporine free medium. The proportion of necroptotic cells was already increased without regeneration after 3 hours of exposure to staurosporine. When a higher (10(-6) M) concentration of staurosporine was applied, further significantly increased necroptosis was detected after regeneration in a staurosporine free medium. Necroptosis was significantly reduced when RIP1 kinase was inhibited by necrostatin-1, whereas inhibition of caspases with z-vad-fmk, an irreversible pan-caspase inhibitor, did not prevent necroptosis. This report of necroptosis induced by staurosporine represents a simple approach for the in vitro induction and detection of apoptosis and necroptosis.

The Predominant Protective Effect of Tianeptine Over Other Antidepressants in Models of Neuronal Apoptosis: The Effect Blocked by Inhibitors of MAPK/ERK1/2 and PI3-K/Akt Pathways

“…Exploring further the caspase-3-independent targets for the neuroprotective action of Tian, we tested the engagement of necroptosis, a programmed necrotic cell death pathway (Christofferson and Yuan 2010). It is well known that the same harmful stimulus can induce both apoptosis and necroptosis in in vitro and in vivo conditions (Ji et al 2011; Šimenc and Lipnik-Štangelj 2012; Wang et al 2012). Necroptosis was firstly discovered in immunology and oncology field, however, recently it is also very often described in relation to neurodegeneration, e.g., in the retinal ischemia–reperfusion injury model, neonatal ischemia, and glutamate toxicity (Chavez-Valdez et al 2012; Degterev et al 2005, 2008; Northington et al 2011; Rosenbaum et al 2010; Wang et al 2012; Xu et al 2007; Zhang et al 2013).…”

“…Necroptosis was firstly discovered in immunology and oncology field, however, recently it is also very often described in relation to neurodegeneration, e.g., in the retinal ischemia–reperfusion injury model, neonatal ischemia, and glutamate toxicity (Chavez-Valdez et al 2012; Degterev et al 2005, 2008; Northington et al 2011; Rosenbaum et al 2010; Wang et al 2012; Xu et al 2007; Zhang et al 2013). The participation of necroptosis was also reported to be involved in St-evoked cell damage in glia cells and U937 cells as well as in cisplatin-induced kidney injury (Dunai et al 2012; Šimenc and Lipnik-Štangelj 2012; Tristão et al 2012). In our study, we showed that the inhibitor of necroptosis, necrostatin-1 (Nec-1) was able to attenuate the extent of cell damage evoked by St and Dox, however the most significant effect was achieved at its low concentration (1 μM) and this effect was comparable with protection mediated by Tian (about 30 %).…”

Staurosporine induces different cell death forms in cultured rat astrocytes

“…Cultures of rat cortical astrocytes were prepared from the brain of new-born rats in DMEM/F12 (1:1), 10% FBS, 1% Penicillin-Streptomycin culture medium as described previously 13. Cells were grown at 37°C in a humidified environment containing 10% CO2 until they became confluent.…”

“…The cells were stained simultaneously with Annexin V-FITC and 7-AAD dye according to the modified manufacturer’s instructions (for details see Šimenc&Lipnik-Štangelj13). Data acquisition was carried out using a flow cytometer.…”

“…Astroglial cell cultures have proven an appropriate in vitro model for studying cellular and molecular functions of astrocytes, including cell death 12. Recently we have reported, that staurosporine is able to induce necroptosis in cultured astrocytes 13. So far, the signalling pathways of staurosporine induced necroptosis have not been elucidated.…”