research article Staurosporine induces different cell death forms in cultured rat astrocytes Janez Simenc12, Metoda Lipnik-Stangelj1 1 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 2 University of Maribor, Faculty of Medicine, Maribor, Slovenia Radiol Oncol 2012 Received 22 March 2012 Accepted 18 May 2012 Correspondence to: Prof. Metoda Lipnik-Štangelj, PhD, University of Ljubljana, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology, Korytkova ulica 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 543 37 70; Fax: + 386 1 543 37 71; E-mail: metoda.lipnik-stangelj@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Materials and methods. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. Results. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10-7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10-6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Conclusion. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis. Key words: astrocytes; staurosporine; necroptosis; apoptosis; reactive oxygen species; flow cytometry Introduction In the central nerve system (CNS), astroglial cells are frequently involved in malignant transformation. It is believed that dysfunction of apoptosis underlies glioblastoma genesis, proliferation and resistance to chemotherapy and radiotherapy. Besides, necrosis seems to be related with gliob-lastoma proliferation, angiogenesis and invasion. Induction of apoptosis has not made sufficient achievements in the treatment of glioblastoma, mainly because the tumour cells are often resistant to apoptosis. Better result in this case may be achieved by modulating the necroptosis, thus circumvent the apoptosis resistance.1 The knowledge of molecular pathways, involved in different forms of cell death is therefore crucial. Apoptosis is a well-studied form of programmed cell death, with morphological characteristics such as cell shrinkage, fragmentation of cellular organelles and nucleus. In early apoptosis plasma membrane remains largely intact however it redistributes phosphatydilserine from the cytoplasmatic to the outer surface prior to any morphological changes. In late apoptosis, plasma membrane often becomes permeable, causing sec- ondary necrosis.23 Described changes are mostly due to the activated intracellular cysteine-pro-teases caspases.4 In contrast, primary necrosis is morphologically characterized by swelling of the cytoplasm and cell organelles, followed by early plasma membrane ruptures. The release of intra-cellular content outside of the cell may induce an inflammatory response and additional cell loss. Contrary to apoptosis, primary necrosis does not involve caspases.3 For long time, primary necrosis has only been considered as an unregulated, passive cell death caused by the devastating stress. However, at least a part of necrotic cells may die by necroptosis, a highly regulated form of primary necrosis.5 Apoptosis and necroptosis are rather interconnected and not entirely separated events. In different susceptible cell lines, Fas ligand or TNF-alpha, which normally activates apoptosis through the death receptors, can induce necroptosis when cas-pases are inhibited or deficient.6 While the mechanisms of apoptosis are well known, necroptosis is not fully understood. One critical molecular regulator, mediating different cellular responses upon activation through the death receptors, is a multifunctional receptor interacting protein (RIP) which is essential for the activation of necropto-sis, when caspases are inhibited.6 Recent reports revealed several details of necroptotic death signalling pathway and have confirmed a critical role of RIP1 kinase in necroptosis.78 In the central nerve system, astroglial cells in the brain may die by apoptosis or necroptosis in different pathologies.9-11 Distinction of the mechanisms of astrocytes apoptosis and necroptosis is necessary to improve our understanding of patho-physiology of neurological disorders, cancer or trauma and provide new therapy opportunities. 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. Therefore in the present study we clarified the molecular mechanisms of necroptosis, induced by staurosporine, and compared them with ap-optosis. We showed that necroptosis is caspases independent and occurs through the activation of RIP1 kinase. Furthermore, we demonstrated that the reactive oxygen species (ROS) production is increased through RIP1 kinase activity, while increased ROS is associated with necroptosis. Materials and methods Materials L-15 Leibowitz medium, foetal bovine serum (FBS), Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 (DMEM/F12), Penicillin (10,000 IU/ml), Streptomycin (10,000 mg/ml; P/S), Dulbecco's phosphate buffered saline (PBS) were purchased from Gibco BRL, Life Technologies, Scotland. Staurosporine, 2-7-dichlorodihydrofluo-rescin diacetate (DCFH-DA) probe, Necrostatin-1(nec-1), and Butylated hydroxyanisole (BHA) were obtained from Sigma Chemicals, USA. The pan-caspase inhibitor (z-vad-fmk) was purchased from R&D systems, USA. Petri plates were purchased from Nunc, Germany, and tissue culture flasks were obtained from TPP, Switzerland. Annexin V-fluorescin isothiocyanate (Annexin V-FITC) and 7-Aminoactinomycin D (7-AAD) staining kit for a flow cytometry was obtained from Beckman-Coulter, USA. All flow cytometry experiments were carried out on the Quanta SC MPL flow cytometer (Beckman Coulter, USA). Animals New-born Wistar rats (1-2 days old) were obtained from our own breeding colony. All animal studies were approved by the Veterinary Authority of the Republic of Slovenia (License number: 3440180/2008/4), and performed in accordance with the EU Directive 2010/63/EU and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123). Cell cultures 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. When they reached confluence, the cells were used for the treatment. Induction of cell death and production of ROS by staurosporine The astrocytes were treated with 10-7 M stau-rosporine for 6 hours to induce apoptosis. After the treatment, the cells were allowed to regenerate for 22 hours in a staurosporine free medium, or were analysed without regeneration. Similarly, the astrocytes were treated with 10-6 M staurosporine for 3 hours to induce the production of ROS and/or necroptosis. After the treatment, the cells were allowed to regenerate for 22 hours in a staurosporine free medium, or were analysed without regeneration. The cells were trypsinized and stained for an analysis with a flow cytometer. The control cells were not exposed to staurosporine. Attenuation of apoptosis The astrocytes were pre-treated with z-vad-fmk, an irreversible pan-caspase inhibitor, at 4 x 10-5 M concentration, for one hour. Then 10-7 M staurosporine was added into the culture medium, and the cells were incubated for an additional 6 hours. During 22 hours regeneration, the cells were incubated in z-vad-fmk containing medium without stau-rosporine. The cells were also exposed only to 4 x 10-5 M z-vad-fmk for 24 hours. Attenuation of necroptosis and ROS production The astrocytes were pre-treated with 10-4 M nec-1, a specific RIP1 kinase inhibitor, or with 4 x 10-5 M z-vad-fmk, one hour before 10-6 M staurosporine was added. Then the cells were incubated for an additional 3 hours. For the regeneration, the cells were incubated in the presence of nec-1 or z-vad-fmk for 22 hours. The cells were exposed only to 104 M nec-1 for 24 hours as well. For the attenuation of ROS production, the cells were pre-treated with 10-4 M nec-1, one hour before 10-6 M staurosporine was added. Then the cells were incubated for additional 3 hours. For the regeneration, the cells were incubated in the presence of nec-1 for 22 hours. Additionally, to attenuate the ROS associated necroptosis, the cells were treated one hour with 10-4 M BHA, a ROS scavenger, before 10-6 M staurosporine was added. Then the cells were incubated for an additional 3 hours. For the regeneration, the cells were incubated in the presence of BHA for 22 hours. The cells were exposed only to 10-4 M BHA for 24 hours as well. Flow cytometric analysis of a cell death and ROS production 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. The differentiation of early apoptotic, secondary necrotic, necroptotic and viable cells was made according to their phenotype: Annexin V+/7-AAD- were considered early apoptotic, Annexin V-/7-AAD+, necrop-totic, Annexin V+/7-AAD+ secondary necrotic, and Annexin V'/7-AAD' viable cells. In each sample, 10.000 cells were analysed. For the detection of ROS production, the cells were prepared according to the modified protocol.14 Statistical analyses Statistical analyses were made with SPSS 19 software (SPSS, Inc, USA). For each treatment and controls, ten samples from two independent groups of animals were analysed. For inhibitors toxicity (z-vad-fmk or nec-1), five samples were analysed. In the cell death experiments, data (means ± SEM) were expressed as the percentage of cell death. For statistical comparisons, only the proportions of early apoptotic and necroptotic cells were considered. For the ROS production, data (means ± SEM) were expressed as the percentage of DCF fluorescence. The differences between various groups were examined for a significance using the non-parametric Mann-Whitney U test. In all cases a p value of < 0.01 was considered statistically significant. Results Determination of apoptosis and necroptosis Apoptosis and necroptosis were detected by the flow cytometry dot plots (Figures 1, 2). The proportions of apoptotic and necroptotic cells were simultaneously detected with the binding of Annexin V-FITC and uptake of 7-AAD dye. The binding of Annexin V is considered independent of a cell death stimulus and specific marker of apoptosis. It precedes the loss of ability to exclude viability dyes, membrane ruptures, or occurrence of any morphological changes associated with apopto-sis.1516 On the other hand, the uptake of 7-AAD dye is a specific marker of necroptosis.17 However, it has been reported for the Jurkat cells with necrotic morphology, that the binding of Annexin V+ precedes the formation of membrane ruptures and necrosis.6 This observation suggests that secondary necrotic cells (Annexin V+/7-AAD+) may not necessarily die by apoptosis. Therefore, to avoid potential bias, secondary necrotic cells were omitted from the analyses, and only early apoptotic cells Secondary necrotic ® Annexin V-FITC 1D3 1D4 FIGURE 1. Examples of two-parameter flow cytometry dot plots showing simultaneous binding of Annexin V and 7-AAD uptake by cultured rat astrocytes after the induction of apoptosis. For each treatment, ten samples from two independent groups of animals were analysed. (A) Cells were not exposed to staurosporine (controls). (B) Cells were exposed to 10-7 M staurosporine for 6 hours, and regenerated for 22 hours in a staurosporine free medium. (C) Cells were exposed to 10-7 M staurosporine for 6 hours, and analysed without regeneration. 1П4 = 1D3-S O < < I 1D2-5 1 □1 ~= Necroptotic Secondary necrotic 1 D4 = Necroptotic 1D3-1 Secondary necrotic 1 D4 = Necroptotic 1D31 Secondary necrotic 1ПП 1D1 1D2-5 1D1"i 1QD-I CD Annexin V-FITC 1DJ-=. m ■ :Езг1у apoptotic ]—i 1111 m i—i i iiiiii|—i 11111111—i i Mini 1D2 1ПП 1D4 FIGURE 2. Examples of two-parameter flow cytometry dot plots, showing the simultaneous binding of Annexin V and 7-AAD uptake by cultured rat astrocytes after the induction of necroptosis. For each treatment, ten samples from two independent groups of animals were analysed. (A) Cells were not exposed to staurosporine (controls). (B) Cells were exposed to 10-6 M staurosporine for 3 hours, and regenerated for 22 hours in a staurosporine free medium. (C) Cells were exposed to 10-6 M staurosporine for 3 hours, and analysed without regeneration. (Annexin-V+ / 7-aad-) were considered apoptotic. Accordingly, only the cells with Annexin V-/7-AAD+ phenotype were considered as necroptotic cells, while secondary necrotic cells were omitted from the analyses. Induction and attenuation of apoptosis and necroptosis For the induction of apoptosis, the cells were exposed to 10-7 M of staurosporine for 6 hours, and regenerated for 22 hours in a staurosporine free medium. As shown in Figure 3, staurosporine increased early apoptosis approximately 5-fold in comparison to control cells, while necroptosis was not influenced. In order to confirm the induction of apoptosis, inhibition experiments with z-vad-fmk were carried out. The results showed that inhibition of caspases by z-vad-fmk strongly reduced early apoptosis, induced by staurosporine. On the contrary, the number of necroptotic cells was slightly increased, suggesting that the cells were switched from one form of a cell death (apoptosis) to another (necroptosis) (Figure 3). To induce necroptosis, cell cultures were exposed to 10-6 M staurosporine for 3 hours, and regenerated as described previously. The results showed that a higher concentration of staurosporine increased the extent of necroptosis (staurosporine 29.01 ± 1.03% vs control cells 5.76 ± 0.54%), whereas early apoptosis was not affected. To confirm the RIP1 ki-nase involvement in the transduction of the signal, FIGURE 3. Percentages of a cell death in cultured rat astrocytes after the exposure to 10-7 M staurosporine. Early apoptosis and necroptosis were determined by the binding of Annexin V-FITC and 7-AAD uptake, using a flow cytometry. Data are the means ± SEM of ten samples from two independent groups of animals. For the z-vad-fmk treatment, only five samples were analysed. (Control) Control cells were not exposed to staurosporine or z-vad-fmk. (STS) Cells were exposed to 1 0-7 M staurosporine for 6 hours, and regenerated for 22 hours in a staurosporine free medium. (STS+z-vad-fmk) Cells were exposed to staurosporine and 4 x 10-5 M z-vad-fmk for 6 hours, and regenerated for 22 hours in a staurosporine free medium with z-vad-fmk. (z-vad-fmk) Cells were exposed to 4 x 10-5 M z-vad-fmk for 24 hours. Data were analysed using the non-parametric Mann-Whitney U test; * p < 0.00 vs Control-early apoptosis, ** p < 0.00 vs STS-early apoptosis and *** p = 0.002 vs STS-necroptosis indicate significance. we inhibited necroptosis with nec-1. The results showed that nec-1 reduced staurosporine induced necroptosis to half, while early apoptosis remained unaffected. Furthermore, z-vad-fmk did not influence necroptosis which indicating that necroptosis, induced by staurosporine, was caspases independent (Figure 4). FIGURE 4. Percentages of a cell death in cultured rat astrocytes after the exposure to 10-6 M staurosporine and the regeneration period. Early apoptosis and necroptosis were determined by the binding of Annexin V-FITC and 7-AAD uptake, using a flow cytometry. Data are the means ± SEM of ten samples from two independent groups of animals. For the nec-1 treatment, only five saples were analysed. (Control) Control cells were not exposed to staurosporine or inhibitors. (STS) Cells were exposed to 10-6 M staurosporine for 3 hours, and regenerated for 22 hours in a staurosporine free medium. (STS+z-vad-fmk) Cells were exposed to staurosporine and 4 x 10-5 M z-vad-fmk for 3 hours, and regenerated for 22 hours in a staurosporine free medium with the z-vad-fmk. (STS+nec-1) Cells were exposed to staurosporine and 10-4 M nec-1 for 3 hours, and regenerated for 22 hours in a staurosporine free medium with nec-1. (nec-1) Cells were exposed to 10-6 M nec-1 for 24 hours. Data were analysed using the non-parametric Mann-Whitney U test; * p < 0.00 vs Control-necroptosis, ** p < 0.00 vs Control-necroptosis and *** p < 0.00 vs STS-necroptosis indicate significance. In order to show that increased ROS is associated with necroptosis, the cells were treated with 10-6 M staurosporine in the presence of 10-4 M BHA. As shown in Figure 7, BHA strongly reduced necroptosis, while early apoptosis was not influenced. Induction and attenuation of ROS production and ROS associated necroptosis In the cultures, exposed to 10-6 M staurosporine, the ROS accumulation was detected by DCF fluorescence. Linear amplification of the signal was observed in the staurosporine treated cells. Analyses were made by the logarithmically versus linear amplified DCF fluorescence cytometry dot plots (Figure 5). As shown in Figure 6, staurosporine increased the ROS production approx. 3-fold in comparison to control cells. Nec-1 reduced the ROS production, induced by staurosporine, to half. Induction of a cell death without regeneration To assess the impact of regeneration period on a cell death, the experiments without regeneration were carried out. As shown in Figure 8, in the cultures treated with 10-7 M staurosporine for 6 hours, early apoptosis was significantly decreased, while necroptosis was increased. Similarly, in the cultures, treated with 10-6 M staurosporine for 3 hours, early apoptosis was significantly decreased, while necroptosis was increased (Figure 8). These results indicated that regeneration of the cells is crucial for apoptosis, while it is not necessary for necroptosis. T——i—i—i—i—i—r \1D-3 1П-2 1D-1 1W 1D1 1D2 1D31D4 (0 ~~i—Г □ 1 1D2 1D31D4 r 100 CD o 80