If pre-incubation with α-T was performed for 18 h (n = 5) the rescue rates of 100 nM, 1 μM, 10 μM and 100 μM α-T against H₂O₂-induced cell death were found to be 48.3% ± 5.7%, 48.2% ± 7.8%, 47.1% ± 5.8% and 57.7% ± 4.2%, respectively (the difference between these values is not significant: p > 0.05 in all cases). Thus, the similar protection of PC12 cells against H₂O₂-induced death was achieved by long pre-incubation with α-T in the range from 100 nM to 100 μM. At a concentration of 10 nM, α-T still significantly inhibited the toxic effect of H₂O₂ by 29.6% ± 3.6% (p < 0.01), albeit to a lower extent than α-T at the higher concentrations tested (p < 0.02). The results of a typical experiment are shown in Figure 1.

If pre-incubation is performed for 3 or 6 h, the protective effect of 100 nM α-T is lower than the effect of 100 μM α-T (Figure 2A,B). However, no difference in the protective activity of α-T for these two concentrations is observed if pre-incubation takes place for 12 or 18 h (Figure 2C,D). The data obtained in four experiments were expressed as rescue rates of α-T at various concentrations at various time of pre-incubation. These results are shown in Table 1.

The data of Table 1 and Figure 2D provide evidence that the protective effect of α-T is concentration-dependent in the range 1–100 nM (1 nM < 10 nM < 100 nM) if pre-incubation is performed for 18 h. The data presented in Table 1 (like the previously discussed data of Figure 2) show that the protective effect of 100 nM α-T is lower than the effect of 100 μM α-T if pre-incubation with it is performed for 3 or 6 h, but no difference is revealed in the protective effect of α-T in these two concentrations at longer pre-incubation (12 or 18 h).

We were interested to find out if α-T at nanomolar concentration was protective following short pre-incubation (0.5 h) and a short (3 h) exposure of 1 mM H₂O₂. It is to be emphasized that we used the cell exposure to 1 mM H₂O₂ for 3 h only in a very limited number of experiments presented in Figure 3. In all other figures and tables presented, the results of experiments were made using cell exposure to 0.2 mM H₂O₂ for 24 h.

These data show that even the toxicity of a high concentration of H₂O₂ (1 mM) may be overcome by long pre-incubation with nanomolar α-T (Figure 3B). The rescue rates of 100 nM and 100 μM α-T calculated using the data of all five experiments made were found to be 53.6% ± 8.6% and 59.1% ± 17.8%, respectively, if pre-incubation was performed for 18 h (n = 5).

The anti-apoptotic effect of α-T was studied using cell staining with propidium iodide and flow cytometric analysis. The relative value of subG1 hypodiploid peak (left on the histograms) corresponds to the relative number of PC12 cells in late apoptosis. We compared the effect of short (0.5 h) and long (18 h) pre-incubation of PC12 cells with 100 nM and 50 μM α-T prior to their exposure to 0.2 mM hydrogen peroxide for 24 h (Figures 4 and 5, respectively).

It was found that 100 nM and 50 μM α-T possessed pronounced and similar anti-apoptotic activity on PC12 cells exposed to H₂O₂ if pre-incubation was performed for 18 h (Figure 5). However, α-T at nanomolar concentration was not effective if pre-incubation with it was performed for 0.5 h (Figure 4), while α-T in micromolar concentration had a pronounced anti-apoptotic effect under any conditions of the experiment. In each experiment, two parallel determinations were made; they produced the similar results.

The average percent of inhibition of the pro-apoptotic effect of H₂O₂ by α-T was calculated, taking into account the results of all experiments made. Pre-incubation of PC12 cells with 50 μM α-T for 0.5 h is highly effective, and decreases the number of PC12 cells in late apoptosis induced by H₂O₂ by 74.5% ± 8.2% (p < 0.01, n = 4). At the same time, pre-incubation with 100 nM α-T for 0.5 h does not produce any effect, as it inhibits the pro-apoptotic action of H₂O₂ by 9.2% ± 8.8% (p > 0.05, n = 4). When PC12 cells are pre-incubated with α-T for 18 h, α-T at concentrations 100 nM and 50 μM diminishes the pro-apoptotic effect of H₂O₂ by 63.8% ± 11.0% and 81.5% ± 9.0%, respectively (p < 0.01 in both cases, n = 5), the difference between the α-T effect at these two concentrations is not significant (p > 0.05).

The protective effect of pre-incubation of immature cortical neurons with 100 nM and 50 μM α-T for 18 h prior to their exposure to H₂O₂ was found to be pronounced and similar (Figure 6B). However, pre-incubation of cortical neurons for 0.5 h with 100 nM α-T is not effective, while pre-incubation for 0.5 h with 50 μM α-T increases the viability of immature neurons to a great extent (Figure 6A,B).

The rescue rates of α-T at concentrations 50 μM and 100 nM against H₂O₂-induced cell death of cortical neurons amounted in our study to 62.1% ± 9.8% and 59.0% ± 9.1%, respectively (n = 6), if pre-incubation with α-T was performed for 18 h. The difference between these values was not significant (p > 0.05). However, if cortical neurons were pre-incubated with α-T for 0.5 h, the rescue rates of 50 μM and 100 nM α-T constituted 54.2% ± 5.8% and 4.4% ± 4.6%, respectively. The difference between the effect of α-T at these two concentrations was significant (p < 0.001), but the effect of 100 nM α-T was absent (p > 0.05).

We used the inhibitor of MEK 1/2 (SL327) that inhibits ERK 1/2 phosphorylation and activation, the inhibitor of PI 3-kinase (LY294002) that inhibits phosphorylation and activation of downstream Akt (protein kinase B), and the inhibitor of PKC (GF109203X). The data presented in Table 2 provide evidence that in the presence of PKC inhibitor (1 μM GF109203X), the ability of 100 nM and 100 μM α-T to rescue PC12 cells from H₂O₂ -induced cell death is diminished and becomes insignificant. In the presence of 50 μM LY294002 or in the presence of 10 μM SL327, the protective effect of 100 nM and 100 μM α-T is significantly lower (p < 0.05) than in the absence of these inhibitors (Table 2). However, 10 μM LY294002 is ineffective, as this concentration does not appear to be sufficient to cause the total inhibition of PI 3-kinase activity [19]. One microliter SL327 or 0.1 μM GF109203X are also ineffective (data not shown).

One of the main aims of our study is to reveal how α-T modulates the activity of ERK 1/2 and Akt under conditions of oxidative stress induced by H₂O₂ by using immunoblotting. The results of such study will be given in the present section. We suggest that modulation of these protein kinase activities by α-T under conditions of oxidative stress makes a contribution to its protective effect against H₂O₂-induced toxicity.

We studied the effect of α-T on basal pERK 1/2 and pAkt levels in PC12 cells as well. It was difficult to make studies of the basal pERK 1/2 level as it was found to be very low in PC12 cells and diminished even more as a result of cell exposure to α-T.

If we used SuperSignal West Pico Chemiluminescent Substrate (Pierce, Thermo Scientific) as an enhancement solution (Figure 7B), the basal pERK activity was revealed on the blots only in controls, but after PC12 cell exposure to 100 nM and 100 μM α-T for 1, 3, 7 and 24 h, it was practically absent.

Thus, pERK 1/2 level was found to be low in PC12 cells, while the cell exposure to α-T was found to diminish even more the basal pERK 1/2 level at various times of cell exposure to α-T. No difference in the basal level of ERK 1/2, Akt or pAkt was revealed in PC12 cells as a result of cell exposure to 100 nM or 100 μM α-T for various time intervals.

It was of importance to study the effect of protein kinase inhibitors on the expression and activity of ERK 1/2 and Akt in PC12 cells exposed to hydrogen peroxide.

As Figure 8 shows, in the presence of 10 μM SL327, pERK 1/2 is not revealed in PC12 cells, while in the presence of 50 μM LY294002, pAkt is absent in these cells. The effect of other inhibitors used will be described later (after Figures 9 and 10).

The data presented in Figure 9A,B show that a well-pronounced and extended increase of pERK 1/2 levels in PC12 cells after their exposure to H₂O₂ is observed for 20–60 min, but no changes in total ERK 1/2 levels is revealed. These data provide evidence that ERK 1/2 is activated in PC12 cells by H₂O₂, but the level of its expression is not changed. The maximal level of pERK 1/2 is achieved in 20 min after PC12 cell exposure to hydrogen peroxide and then it is maintained at a high level for a rather long time, forming a plateau (Figure 9C,D). α-T does not change the initial increase in pERK 1/2 levels up to 20 min after cell exposure to H₂O₂. However, α-T at nanomolar and micromolar concentrations causes a significant and pronounced decrease of the pERK 1/2 level 30, 45 and 60 min after the exposure of PC12 cells to H₂O₂ (Figure 9A–D). In cells exposed both to α-T and H₂O₂ (as compared to cells exposed to H2O2 alone), a peak instead of a plateau is seen on the histograms (Figure 9C,D).

The studies of the effects of protein kinase inhibitors on pERK 1/2 levels were made during exposure to H₂O₂ for 20 min, as maximal activation of ERK 1/2 by H₂O₂ (maximal pERK 1/2 level) was already achieved after the same cell exposure time to H₂O₂. As Figure 8 shows, the basal pERK 1/2 level is very low, but H₂O₂ increases it to a great extent. As it was already mentioned, in the presence of MEK 1/2 inhibitor SL327, pERK l/2 was not practically revealed in cells exposed to H₂O₂, but total ERK 1/2 level was not changed. The inhibitors of PKC (1 μM GF109203X), of protein kinase A (1 μM H89) and of PI 3-kinase (50 μM LY LY294002) had no effect either on total ERK 1/2 or on pERK 1/2 level in PC12 cells exposed to hydrogen peroxide. It is of interest that the inhibitor of Trk receptor tyrosine kinase (1 μM K252a) was found to decrease significantly pERK 1/2 levels in PC12 cells exposed to H₂O₂. These data suggest that at early stages of ERK activation by H₂O₂, it may be activated downstream of Trk receptor tyrosine kinase. Trk receptors are neurotrophin receptors and activation of Trk receptor tyrosine kinase is usually protective.

The exposure of PC12 cells to H₂O₂ was found to result in a pronounced and prolonged increase of Akt activity (increase in pAkt levels) in the cells (Figure 10A,B), while the total Akt level did not change (data not shown). Pre-incubation of PC12 cells for 18 h with 100 μM or 100 nM α-T did not change pAkt level for the first 2–3 h after cell exposure to H₂O₂, but pAkt levels were significantly lower in cells exposed to α-T and H₂O₂ than in cell exposed to H₂O₂ alone 4, 5 and 24 h after the cell exposure to H₂O₂ (Figure 10A,B).

The studies of effects of protein kinase inhibitors on pAkt level were made using exposure to H₂O₂ for 20 min because maximal activation of Akt by H₂O₂ (maximal pAkt level) was achieved after the same period of cell exposure to H₂O₂ (Figure 10). The basal level of Akt activity is pronounced; exposure to H₂O₂ significantly increases it (Figure 8). As previously mentioned, in the presence of PI 3-kinase inhibitor 50 μM LY294002, pAkt was not revealed, but total Akt level was not changed. Neither did the inhibitors of PKC (1 μM GF109203X), of protein kinase A (10 μM H89) or of MEK 1/2 (10 μM SL327) have an effect on Akt expression, or on pAkt levels in PC12 cells exposed to H₂O₂. It is to be noted that the inhibitor of Trk receptor tyrosine kinase (1 μM K252a) was found to decrease significantly pAkt level in PC12 cells exposed to H₂O₂.

Thus, our immunoblotting data show that H₂O₂ activates both ERK 1/2 and Akt (increases pERK 1/2 and pAkt levels), but does not change the expression of these protein kinases. The pre-incubation with α-T prior to PC12 cell exposure to H₂O₂ diminishes the time of maximal activation of these protein kinases induced by H₂O₂.

Hydrogen peroxide, pyruvate, NADH, cytosine-arabinoside, α-T and propidium iodide were obtained from Sigma (USA), penicillin and streptomycin were from Serva (Germany), K-252a, SL327, LY294002 and GF109203X were from Calbiochem (USA). The incubation media, Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine, horse blood serum and fetal calf serum were purchased from the Biolot Company (Russia). The antibodies used for immunoblotting are listed in Immunoblotting section.

The experiments were performed on PC12 cells (ATCC) in CO₂ incubators in an atmosphere containing 5% CO₂ at 37 °C. DMEM containing 10% fetal calf serum, 5% horse blood serum, 50 U/mL of penicillin and 50 μg/mL of streptomycin was used as the complete growth medium and it was changed every 2–3 days. PC12 cells were seeded onto 24-well plates at a density of 2 × 10⁵ cells per well to determine the cell viability. The experiments started 24 h after the transfer of the cells to the plates, and were performed in the complete growth medium. PC12 cells were pre-incubated with α-T for 3, 6, 12, or 18 h prior to cell exposure to 0.2 mM H₂O₂ for 24 h. After pre-incubation with α-T for 0.5 h (Figure 3A), and in some experiments (Figure 3B) for 18 h, PC12 cells were exposed to 1 mM H₂O₂ for 3 h. In some experiments, PC12 cells were pre-incubated in the presence of protein kinase inhibitors (SL327, LY294002, GF109203X) for 0.5 h, before the exposure of the cells to α-T.

The viability of PC12 cells and cortical neurons was assessed by measuring the activity of LDH released from the damaged cells after their exposure to H₂O₂. The samples were centrifuged before the aliquots of supernatant were taken. The activity of LDH was determined in the samples by measuring the decrease in NADH levels. This reaction was performed in medium with the following composition: 80 mM tris-HCl (pH = 7.2), 200 mM NaCl, 1.6 mM pyruvate, 0.2 mM NADH. The decrease in optical density of the samples at 340 nm was registered at approximately 5–6 min as previously described [47], using the M40 spectrophotometer (Karl-Zeiss, Germany). The lysis of the cells was performed using 1% Triton X-100 at room temperature in order to determine total LDH activity in the samples. The percent of LDH activity in the incubation medium was estimated in comparison to the total LDH activity. The absence of viable cells corresponds to 100% of LDH activity in the incubation medium.

The difference in the amount of LDH released from the cells exposed to H₂O₂ in the absence and in the presence of α-T was determined. The ratio of this difference to the increase of LDH released from cells to the medium in the presence of H₂O₂ alone (taken for 100%) corresponds to the rescue rates of α-T against H₂O₂ toxicity (i.e., to the percent of inhibition of H₂O₂ toxic action by α-T). The formula is

Primary cultures of immature cortical neurons were prepared from embryonic day 16 Wistar rat fetuses by the modified method of Dichter [48], as previously described [49]. Cells were seeded on poly-d-lysine coated 24-well plates at a density of 3 × 10⁵ cells per well. After 24 h, cytosine-arabinoside (3 μM) was added to the culture for 24 h in order to minimize glial growth. Culture medium was replaced every 3 days. Treatments were performed on the 7th day in vitro. Pre-incubation of neurons with α-T was performed for 0.5 or 18 h prior to the exposure of cells to 0.2 mM H₂O₂ for 24 h.

After exposure to α-T and H₂O₂, PC12 cells were collected by centrifugation, rinsed in PBS, fixed with ice cold 70% ethanol and stored at −20 °C for 24 h–48 h. The cells were then incubated with a DNA extraction buffer as previously described [50], and washed with PBS. The cell pellets were resuspended in a DNA staining reagent (200 μg of RNase and 50 μg of propidium iodide in 1 mL PBS was used per 0.5–1 × 10⁶ cells) and incubated in the dark at 4 °C for 30 min. The amount of cells in hypodiploid (subG1) peaks and cell cycle distributions at different phases were analyzed using an EPICS XL flow cytometer (Beckman-Coulter, USA) and the WinMDI 2.1.4 software package. Between 40,000 and 80,000 cells were taken for the analysis of each sample. The relative content of DNA in the peaks was measured as the red propidium iodide fluorescence. The percent of cells in the subG1 peak corresponded to the relative number of cells undergoing late apoptosis in the sample.

The phosphorylated and non-phosphorylated forms of ERK1/2 and Akt were determined using Western blot analysis. After incubation with α-T and H₂O₂, PC12 cells were washed twice with ice-cold PBS and harvested in 60 μL of lysis buffer: 50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM β-glycerophosphate Na, 10 mM NaF, 1 mM Na₃VO₄, 1 mM phenyl methyl sulfonyl fluoride (PMSF), protease inhibitor cocktail (Roche). Cells were kept on ice for 1 h to complete lysis. Protein concentrations of the cell lysates were estimated in triplicate using the Lowry method with Folin & Ciocaltteu’s Phenol reagent and bovine serum as a standard. Equivalent amounts (35–40 μg) of protein-containing lysates were loaded into each lane and electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel, followed by transfer to Hybond-P PVDF membranes (Amersham, GE Healthcare). The non-specific binding sites of the membranes were blocked with PBS containing 5% (w/v) skimmed milk and 0.1% Tween 20. The blots were then probed overnight with antibodies for pERK1 (pThr202/pTyr204) and pERK2 (pThr185/pTyr187) (1:2000, Sigma), pAkt (Ser473) (1:1000, Cell Signaling), followed by three washes with 0.1% Tween 20 in PBS. After incubation with either an anti-mouse or anti-rabbit HRP-labeled secondary antibody (GE Healthcare) for 1 h at room temperature, blots were developed using enhanced chemiluminescence detection Western blotting reagents (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Thermo Scientific). To determine the level of basal pERK level, which was found to be low in PC12 cells, we used both long exposure of blots to the above-mentioned reagent and ECL PLUS (Amersham) in order to enhance chemiluminescent signals. For the normalization of data, membranes were incubated in buffer (65 mM Tris, pH 6.8, 2% SDS, w/v, β-mercaptoethanol) to strip the previous antibodies, and re-probed with antibodies for α-tubulin (1:2000, Sigma), total ERK (1:1000, Cell Signaling) or Akt(pan) (1:1000, Cell Signaling). The optical densities of the positive bands of the scanned films were quantified using NIH Image Analysis software version 1.43.

Data are presented as the means ± SEM. If three or more groups of data were compared, the statistical significance of differences was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. Student’s t test was used to characterize the significance of differences between two groups of data. The differences were considered significant at p < 0.05.