NADPH oxidase inhibitor VAS2870 prevents staurosporine-induced cell death in rat astrocytes

Bovine serum albumin (BSA), Fetal bovine serum (FBS), L-15 Leibowitz medium, Dulbecco’s modified Eagle medium and Ham’s nutrient mixture F-12 (DMEM/F12), Penicillin (10,000 IU/ml), Streptomycin (10,000 mg/ml), Dulbecco’s phosphate buffered saline (PBS) were supplied from Gibco BRL, Life Technologies (Paisley, Scotland). Staurosporine, 3-benzyl-7-(2-ben-zoxazolyl) thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870), 3,3’-dihexyloxacarbo-cyanide iodide (DiOC6(3)), and 2-7-dichlorodihydrofluorescin di-acetate (DCFH-DA) were purchased from Sigma Chemicals (St. Louis, Missouri, USA). The pan-caspase inhibitor (z-vad-fmk) was purchased from R&D Systems (Minneapolis, Minnesota, USA). 7-Aminoactinomycin D (7-AAD) staining dye for flow cytometry was purchased from Beckman-Coulter, Inc. (Brea, California, USA). Bio-Rad protein assay kit was purchased from Bio-Rad Laboratories, München, Germany. 1,2-diaminocyclohexanetetraacetic acid (DCTA), dithiothreitol (DTT), Triton X-100, Tris-phosphate, and glycerol were supplied from Merck KGaA, Darmstadt, Germany. ATPlite 1-step assay system was supplied from Perkin Elmer, Boston, Massachusetts, USA. Caspase-Glo 3/7 Assay was purchased from Promega, Madison, Wisconsin, USA.

New-born Wistar rats (1–2 days old) were obtained from our own breeding colony. The animals were kept under constant environmental conditions with an ambient temperature of 22°C, relative humidity 55%, and a natural light-dark cycle. The breeding colony was kept in Ehret type-4 cages (Emmerdingen, Germany). The bedding material was Lignocel 3/4 (Altromin, Lage, Germany). The colony received a standard rodent diet (Altromin, Germany), and had free access to food and water. All animal studies were approved by the Veterinary Authority of the Republic of Slovenia (License number: 34401-7/2012/3), 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).

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.24 The cells were grown in tissue culture flasks at 37°C in a humidified environment containing 10% CO₂ until they became confluent (10 to 12 days). In order to reduce microglia contamination, confluent cultures were shaken at 150 rpm for a day. After shaking, the cells were trypsinized, reseeded onto 35 mm Petri dishes (approx. 1 x 10⁶ cells/ml) and grown for the next 5 to 7 days. When the cultures reached confluence, they were used for the treatment. The purity of the cell culture (> 98% of astrocytes) was checked using immuno-cytochemical staining for the glial fibrillary acidic protein, which is the major component of the astrocyte cytoskeleton.

After the cultures became confluent, culture medium was replaced with 1 ml of fresh serum-free medium. Then the cells were treated with 1 μM staurosporine for 3 hours, to induce cell death. After the treatment, the cells were allowed to regenerate for 22 hours in a staurosporine-free medium. The control cells were not exposed to staurosporine.

For the inhibition experiments, the cells were pre-treated with 5 and 10 μM VAS2870 or 20 μM z-vad-fmk, 1 hour before 1 μM staurosporine was added. Then the cells were incubated for an additional 3 hours. The cells were then regenerated in the staurosporine, VAS2870 and z-vad-fmk free medium for 22 hours. The cells were also exposed to 5 and 10 μM VAS2870 or to 20 μM z-vad-fmk only for 3 hours and allowed to regenerate in an inhibitor-free medium for 22 hours.

For the testing of viability, ROS and mitochondrial potential, the cells were trypsinized and stained for an analysis in a flow cytometer. For the detection of caspases-3/7 activity and level of cellular ATP, the cells were harvested and solubilized in a cell culture lysis buffer.

The cells from individual dishes (1-5 x 10⁶ cells/ml) were stained with 7-AAD viability dye, according to the manufacturer’s protocol. Briefly, the cells were harvested and washed in the ice cold PBS (500 μl). Then the cells were re-suspended in 100 ml PBS buffer with 1% BSA and 0.1% NaN₃. The cell suspension was stained with 20 ml of 7-AAD solution and incubated at 4°C for 15 minutes. After the incubation, an additional 400 μl PBS buffer with 1% BSA and 0.1% NaN₃ was added. Aliquots (200 μl) of cell suspensions were analysed on the Quanta SC MPL flow cytometer (Beckman Coulter, USA) with 488 nm argon laser. In each sample, approximately 5.000 cells were gated. Gating was done in a hierarchical fashion. First, total cell population was identified by plotting electronic volume (EV) versus side scatter (SSC). High EV and SSC population was gated. Next, viability (red fluorescence) scatter gate was used to identify dead cells. Three populations were observed: viable, dim and positive. The positive population (high red signal) was considered dead. The dead population was identified and gated by comparison of treated cells to untreated (negative) controls.

Harvested cells from individual dishes were solubilized in a 150 μl of cell culture lysis buffer (250 mM Tris-phosphate pH 7.8, 2 mM DCTA, 2 mM DTT, 1% Triton X-100, 10% glycerol). Aliquots (5 μl) of cell samples were used for determination of protein concentrations by Bio-Rad protein assay kit. Cellular ATP concentration was determined with the ATPlite 1-step assay system as described in the manufacturer’s manual. The chemiluminescent signals were measured by a Synergy HT microplate reader (BioTek USA). The amount of ATP in each sample, diluted to contain 1 mg protein/mL, was calculated using a generated standard ATP curve (1 pM - 1 mM). The same samples were used for detection of caspases-3/7 activity by Caspase-Glo 3/ 7 Assay (Promega, Madison, USA) as described in the manufacturer’s manual.

After the treatment, the cells (1-5 x 10⁶ cells/ml) were harvested and washed in the PBS (500 μl). For the detection of ROS production, the cells were resuspended in 500 ml of PBS at room temperature and stained with 2 μM DCFH-DA, as proposed by manufacturer’s protocol. Similar, for the detection of inner mitochondrial membrane potential, the cells were stained with 100 nM DiOC6(3), as described in the manufacturer’s manual. After the staining for either the detection of ROS or mitochondrial potential, the cell suspensions were incubated at 370C for 30 minutes. Aliquots (200 μl) of cell suspensions were analysed on the Quanta SC MPL flow cytometer (Beckman Coulter, USA) with 488 nm argon laser. Gating was done in a hierarchical fashion. First, total cell population (approximately 5.000 cells) was identified by plotting EV versus SSC. High EV and SSC population was gated. Then ROS (green fluorescence) scatter plot was used to identify the level of ROS and untreated (negative) controls were used to set the gate for high ROS population. Similar strategy was used for the identification of low mitochondrial membrane potential cell population. First, high EV and SSC population was identified. Next, green fluorescent histogram was plotted with marker showing low potential population. As previously, untreated (negative) cells were used to identify low mitochondrial membrane cell population.

All data are presented as a mean ± SEM of three to five determinations from five independent experiments. Data analysis was performed using Graph Pad Prism 5.0 (Graph Pad Software, Inc., USA). The statistical significance of differences between various groups was evaluated using one-way ANOVA followed by the Tukey multiple comparison test. A p value of < 0.05 was considered to be statistically significant.

3 h treatment with 1 μM staurosporine revealed a significant cytotoxic effect on cultured astrocytes causing 27.1 +/- 0.8% decrease of viability as detected by high red fluorescent signal in a flow cytometry (Figure 1). Pre-treatment with 5 μM VAS2870 reduced cell death by 1.3-fold, whereas 10 μM VAS2870 diminished staurosporine-triggered cell death even more, by 1.7-fold. In untreated control cells, on average 7.8 +/- 0.6% of dead cells was detected. The proportions of dead cells in the culture exposed to VAS2870 alone were low and did not differ significantly from the untreated controls (Figure 1).

Figure 1

1 μM staurosporine activated caspases-3/7 (Figure 2A), a key indicator of the execution phase of apoptotic cell death. While pre-incubation of cultured astrocytes with 20 μM z-vad-fmk lowered the activity of caspases-3/7 by 13.9-fold in comparison to the cells, treated by staurosporine alone, pre-treatment of the cells with 10 μM VAS2870 did not significantly inhibit caspases-3/7 activity. In the untreated controls, VAS2870 or z-vad-fmk caused no caspases-3/7 activation.

Figure 2

Furthermore, staurosporine critically reduced cellular energetic supply in astrocytes, decreasing the level of intracellular ATP by 15.9-fold compared to untreated controls, which represents 0.59 +/- 0.08 nM ATP/mg cell protein. While in the cells, pretreated with z-vad-fmk, the level of ATP remained low and did not differ significantly from the staurosporine treated cells (Figure 2B), pre-treatment of the cells with VAS2870 partially reversed the level of ATP, which represents 6.51-fold enhancement in comparison to the cells, treated by staurosporine. In the cells, exposed to either VAS2870 or z-vad-fmk only, the level of ATP remained high and did not differ significantly from the untreated cells (9.30 +/- 0.56 nM ATP/mg cell protein) (Figure 2B).

VAS2870 (5 and 10 μM) was applied to staurosporine-treated cells as a putative inhibitor of ROS production. Detection of ROS was performed with DCFH-DA probe by flow cytometry. In each experiment, fluorescence intensity of dichlorofluorescin (DCF), a product of DCFH-DA degradation and oxidation, was detected (Figure 3A-D). The results showed that 1 μM staurosporine induces ROS production; on average 81.6 +/- 1.8% of DCF fluorescent cells was detected (Figure 3E) and ROS production was increased by 10.2-fold. Pre-treatment with 5 μM VAS2870 reduced the levels of ROS by 1.6-fold, while 10 μM VAS2870 diminished ROS levels 3.0-fold compared to the cells, treated by staurosporine (Figure 3E). VAS2870 itself showed no influence on ROS production (Figure 3E).

Figure 3

The potential of inner mitochondrial membrane (Dpsi_m) was measured with DiOC6(3) potentiometric dye using flow cytometry. DiOC6(3) is taken up into the healthy cell and distributed across the inner mitochondrial membrane. The collapse of proton gradient across inner mitochondrial membrane of dying cells results in the loss of DiOC6(3) and hence loss of fluorescence intensity. In each experiment, green fluorescent signal of DiOC6(3) was analysed, identifying regions with low fluorescence intensity. As depicted in the representative flow cytometric experiment (Figure 4), 1 μM staurosporine potently reduced mitochondrial potential. The proportion of the cells with low mitochondrial potential was increased by 7.3-fold as compared to untreated controls (Figure 4B, E), while the untreated control cells and VAS2870 treated cells yielded a high fluorescent signal (Figure 4A, C). When astrocytes were pre-incubated with 5 μM VAS2870, the potential was partially restored (Figure 4E), whereas 10 μM VAS2870 restored the potential even more, as evidenced by 2.6-fold decrease of low potential cells compared to staurosporine only treated cells (Figure 4D, E).

Figure 4