DMEM, heat inactivated FBS, Hank's balanced salt solution (HBSS) and PBS were purchased from Bio-Rad Laboratories, Inc. Penicillin/streptomycin sulfate, amphotericin B and L-glutamine were purchased from Mediatech, Inc. Cocaine hydrochloride (Ecgonine methyl ester benzoate; molecular weight, 339.8), trypan blue, sulfanilamide, LPS from Escherichia coli serotype 0111:B4, N-(1-naphthyl)-ethylenediamine (NED), phosphoric acid, and EDTA were supplied by Sigma-Aldrich; Merck KGaA. IFNγ protein was obtained from Novus Biologicals Ltd. All other routinely used agents were analytical grade.

C6 astrocyte-like cell line (CCL-107) was purchased from American Type Culture Collection and maintained as an adherent monolayer culture in complete DMEM in phenol red supplemented with 2 mM L-glutamine, 10% (v/v) FBS, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate and 0.25 µg/ml amphotericin B. Cells were grown in a humidified incubator at 37˚C with 5% CO₂, and sub-cultured twice a week. For cytotoxic studies, the culture was harvested by treating with 0.05% EDTA in PBS for ≤2 min, resulting in a single cell suspension. Cell count was assessed using 0.4% trypan blue dye exclusion assay (27˚C for 1 min) using a hemocytometer under a light microscope (magnification, x20).

Cells (5x10⁴/well) in 96-well plates were treated with cocaine (1-4 mM) for 24 h. Cocaine stock, working stocks and treatments were prepared as described previously (17). Cell viability was evaluated using a crystal violet dye uptake assay as described previously (17). At the end of treatment, 100 µl 0.25% glutaraldehyde was added per well and incubated for 30 min. After washing and air drying of plates, the dye was extracted with 100 µl 50 mM sodium phosphate mono basic solution, containing 50% ethyl alcohol. The plates were gently vortexed and the optical density (OD) measurements of incorporated dye in viable cells were measured at 540 nm using a microplate spectrophotometer (Bio-Tek Instruments Inc.).

Cells (5x10⁴/well) in 96-well plates in phenol red free media were treated with LPS (0.2 µg/ml), IFNγ (6 µg/ml) and cocaine (1-4 mM) for 24 h. Production of superoxide radicals from cells was detected using a cytochrome c reduction assay as described previously (21). Briefly, at the end of the 24 h treatment, the supernatant from samples was added to an equivalent volume of cytochrome c from horse heart (Sigma-Aldrich; Merck KGaA) prepared in PBS (final working concentration: 160 µM). Then the samples were incubated for 35 min at 37˚C with 5% CO₂. OD measurements of samples were obtained at 550 nm using a UV microplate spectrophotometer.

Cells (5x10⁴/well) in 96-well plates in phenol red free media were treated as described above, and H₂O₂ production was assessed using a previously described method with some modifications (22). After 24 h of treatment, sample supernatants were assessed for H₂O₂ production using a peroxidase linked continuous assay. The chromogenic solution contained (final working concentration) 1 mM vanillic acid, 500 µM 4-aminoantipyrine and horseradish peroxidase (4 U/ml) in PBS with 2 mM HEPES (pH 7.4). The chromogenic solution was added to each sample and incubated for 10 min at 37˚C. Samples were analyzed at 490 nm in a UV micro plate spectrophotometer (Bio-Tek Instruments Inc.). Controls and blanks were measured simultaneously, and subtracted from the final value to eliminate interference.

In order to determine the optimum concentrations of IFNγ (2.6, 3.3, 4.0, 4.6, 5.3, 6.0, 6.6 7.3, 8.0 and 8.6 µg/ml) or LPS (2.6, 3.3, 4.0, 4.6, 5.3, 6.0, 6.6 7.3, 8.0 and 8.6 µg/ml), a dose-response study was initially performed in 96-well plates. Then, the role of cocaine on NO generation was studied by treating the cells in phenol red free media as described above. At the end of treatments, Griess reagent (mixture of an equal volume of 1% sulfanilamide in 0.5 N HCl and 0.1% NED in deionized water) was added directly to the cells under reduced lighting at room temperature. The plates were read at 540 nm on a UV microplate spectrophotometer. Controls and blanks were measured simultaneously, and subtracted from the final value to eliminate interference. A standard curve was generated from a range of dilutions of sodium nitrite (1-100 µM) prepared in the plating medium.

For quantification of HIF-1α release in cell lysates, the cells (5x10⁴/well) were seeded in 96-well plates with DMEM. Supernatants from resting (unstimulated) and stimulated (with LPS and IFNγ) cells after 24 h cocaine exposure at different concentrations (1-4 mM) were discarded, and ice cold PBS was immediately added to each well, decanted and 100 µl cell lysis buffer (Abcam) supplemented with 1:500 µl protease inhibitor (cat. no. ab65621; Abcam) and N-methoxyoxoacetyl-glycine methyl ester (Sigma-Aldrich; Merck KGaA; cat. no. D3695, lot#:064M4728V) was added to each well. After three freeze/thaw cycles at -80˚C, the content of each well was centrifuged at 100 x g for 5 min at 4˚C. Then, 90 µl supernatant (centrifuged vial) was added to each ELISA well. ELISA (kit sensitivity, 0.78 pg/ml) was performed using a HIF-1α ELISA kit (CUSABIO; cat. no. E08540r) according to the manufacturer's protocol. Briefly, 90 µl supernatant from samples and standards were added to 96-well plates pre-coated with the capture antibody. After incubation for 2 h at 37˚C, the supernatant from each well was decanted and 100 µl of the prepared biotinylated primary antibody mixture was added. After 1 h of incubation at 37˚C, all wells were washed 3 times with 300 µl 1X wash buffer provided in the kit. The wash buffer was discarded and 100 µl prepared secondary antibody, HRP-avidin, was added to each well and incubated for 1 h at 37˚C. Subsequently, all wells were washed 5 times with 300 µl 1X wash buffer provided in the kit. After removal of the washing buffer, 90 µl of the color developing agent was added to each well and incubated at 37˚C for 25 min. Finally, 50 µl stop solution was added to each well and the absorbance was read at 450 nm using a UV microplate reader.

For quantification of VEGF release in cell lysates, the cells (5x10⁴/well) were seeded in 96-well plates with DMEM. After discarding the supernatant, the cell lysate was prepared as described for the HIF-1α ELISA. ELISA (kit sensitivity, 20 pg/ml) was performed using a VEGF ELISA kit (Boster Biological Technology; cat. no. EK0540) according to the manufacturer's protocol. Briefly, 100 µl of the samples and standards were added to 96-well plates pre-coated with the capture antibody. After incubation for 90 min at 37˚C, the supernatant from each well was decanted and 100 µl of the prepared biotinylated primary antibody mixture was added. After 1 h of incubation at 37˚C, all wells were washed 3 times with 300 µl 0.01 M PBS. The wash buffer was discarded and 100 µl of the prepared secondary antibody, Avidin-Biotin- Peroxidase Complex, was added to each well and incubated for 30 min at 37˚C. Subsequently, all wells were washed 5 times with 300 µl 0.01 M PBS. After removal of the washing buffer, 90 µl color developing agent was added to each well and incubated at 37˚C for 25 min. Finally, 100 µl stop solution was added to each well and the plate was read at 450 nm using an UV microplate reader.

Data are presented as the mean ± standard error of the mean. Differences between groups were compared using a one-way ANOVA followed by a post-hoc Dunnett's tests. Data analysis was performed using GraphPad Version 6 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Initially the effect of treatment with cocaine at concentrations ranging from 0.5 to 7 mM for 24 and 48 h on cell viability was assessed. The selection of cocaine concentrations was based on earlier reports (20,23), and the lag period and doubling time of these cells (17) were used as the criteria for treatment durations of 24 and 48 h. Compared with the corresponding controls, cocaine significantly decreased the cell viability at these time points (Fig. 1A and B; P<0.01 or P<0.001). The LC₅₀, where 50% of cells were killed by cocaine, was determined to be between 4.5 and 3.8 mM at 24 and 48 h, respectively.

Cells treated with cocaine for 48 h exhibited a higher rate of death compared with 24 h of treatment (Fig. 1A and B); in order to prevent severe loss of cells, 24 h of treatment was selected as the end point for subsequent experiments. The effect of cocaine on the production of O₂^- free radicals and H₂O₂ in cells with or without LPS and IFNγ stimulation was determined. It was found that there was no difference between the unstimulated or stimulated groups irrespective of cocaine treatment (P>0.05) for either of the products (Fig. 2A and B).

Next NO generation in cells was observed following stimulation and treatment. To determine the optimum concentrations of LPS and IFNγ, the cells were initially treated for 24 h with increasing concentrations of IFNγ against a fixed concentration (2.5 µg/ml) of LPS (Fig. 3A). Similarly, a dose-response experiment was performed for LPS was performed for 24 h against a fixed concentration (2.5 µg/ml) of IFNγ (Fig. 3B). It was found that 0.2 µg/ml LPS and 6 µg/ml IFNγ were ideal to stimulate NO/NO₂^- production without compromising the cell viability (data not shown), consistent with a previous study (24). Based on this, the cells were stimulated with LPS and IFNγ at these doses, and treated with cocaine for 24 h. Lack of LPS and IFNγ stimulation did not result in altered NO/NO₂^- production in the cocaine treated cells (Fig. 4); however, when stimulated with LPS alone, cocaine at different concentrations increased NO/NO₂^- production significantly in a dose-dependent manner compared with the unstimulated cocaine-treated cells (P<0.01), corroborating a previous report on LPS-induced NO/NO₂^- production (25). Conversely, IFNγ-stimulated cells did not produce NO/NO₂^- following cocaine treatment. However, LPS and IFNγ-stimulated cells treated with cocaine showed a significant increase in NO/NO₂^- production (40-50x; absorbance, 0.04 to 0.05, respectively; Fig. 4) compared with the unstimulated cells (absorbance, 0.001; Fig. 4; P<0.001).

In the absence of external challenge to the cells, there was no significant increase in HIF-1α with cocaine treatment at 3 and 4 mM (Fig. 5). However, when the cells were challenged with LPS and IFNγ, the HIF-1α levels in cells treated with 4 mM cocaine increased significantly by 5-fold (P<0.05). The average increase was 25.3±4.9 pg/ml of the control (5.1±2.1 pg/ml).

In the absence of external challenge, cells treated with cocaine at 3 and 4 mM did not increase VEGF levels (Fig. 6). However, when the cells were challenged with LPS and IFNγ, the VEGF levels in the cells treated with 3 and 4 mM cocaine increased significantly (P<0.05). Compared with the control (41.8±4.5), the average increase was 60.5±3.9 and 66.4±1.1 pg/ml, respectively at 3 and 4 mM cocaine.