Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities

Exclusion of trypan blue dye by viable U87MG cells was evaluated under a light microscope using a hemocytometer after all treatments. A pretreatment with DXM prevented decrease in cell viability (panel A, Fig. 1). Morphological features of apoptosis were detected following Wright staining (panel B, Fig. 1) and counted to determine the amount of apoptotic cell death (panel C, Fig. 1) based on characteristic morphological features such as condensation of the nucleus and cytoplasm, cytoplasmic blebbing, and the formation of apoptotic bodies. All treatment groups were examined under the light microscopy and cells were counted to determine the percentage of apoptotic cells (panel C, Fig. 1). Compared to control (CTL) cells, cells treated with 100 μM TMZ showed an increase in the percentage of apoptotic cells (P < 0.001). A pretreatment of cells with 40 μM DXM decreased TMZ induced apoptosis by three-fold, compared to treatment of cells with TMZ only.

Results obtained from Wright staining were further supported by the ApopTag assay (panel A, Fig. 2). Both CTL and DXM treated cells showed little or no brown color, confirming almost absence of ApopTag positive cells or apoptosis. The percentage of ApopTag positive cells was calculated (panel B, Fig. 2) and found to be highly significant (P < 0.001) in TMZ treated cells, compared to CTL cells. A pretreatment of cells with DXM considerably attenuated apoptotic DNA fragmentation in TMZ treated cells.

Using fura-2 assay, intracellular free [Ca²⁺] was determined in all treatment groups (Fig. 3). No significant difference (P = 0.928) was seen between CTL cells and cells treated with DXM alone. Cells treated with TMZ showed a significant increase (P = 0.007) in intracellular free [Ca²⁺], compared to CTL cells. This increase was attenuated almost 100% by a pretreatment of the cells with DXM. There was no significant difference (P = 0.999) between intracellular free [Ca²⁺] in CTL cells and those treated with DXM plus TMZ.

A commitment to apoptosis was measured by examining any increase in the ratio of Bax (pro-apoptotic protein) expression to Bcl-2 (anti-apoptotic protein) expression. The bax gene encodes different isoforms. The antibody we used in this investigation could recognize 21 kD Baxα and 24 kD Baxβ bands (panel A, Fig. 4). Here, we considered both bands in our estimation of total Bax expression. We also examined the level of Bcl-2 expression in all treatment groups (panel B, Fig. 4). Almost same level of β-actin expression in each treatment ensured that equal amount of protein was loaded in each lane (panel C, Fig. 4). Based on the Western blot experiments (panels A and B, Fig. 4), the Bax:Bcl-2 ratios were measured in all treatment groups (panel D, Fig. 4). There was no significant difference (P = 0.983) in Bax:Bcl-2 ratio between CTL and DXM treated cells (panel D, Fig. 4). Compared to CTL cells, a rise in Bax:Bcl-2 ratio (panel D, Fig. 4) in cells exposed to TMZ was influenced more by a change in Bax expression (panel A, Fig. 4) than a change in Bcl-2 expression (panel B, Fig. 4). Compared to CTL cells, cells treated with TMZ showed a significant increase (P = 0.007) in the Bax:Bcl-2 ratio (panel D, Fig. 4). There was a significant difference (P = 0.019) in Bax:Bcl-2 ratio between cells treated with TMZ alone and those treated with DXM plus TMZ, indicating a loss of commitment to apoptosis due to a pretreatment with DXM. There was no significant difference (P = 0.871) in Bax:Bcl-2 ratio between CTL cells and cells pretreated with DXM and then treated with TMZ.

Calpain and caspase-3 activities were assessed by Western blot analysis of the calpain-specific 145 kD SBDP and the caspase-3-specific 120 kD SBDP, respectively (panel A, Fig. 5). Level of β-actin expression, which was almost uniform in all treatments, was used as a loading control (panel B, Fig. 5). There was no significant difference (P = 0.911) between CTL cells and DXM treated cells in generation of 145 kD SBDP, indicating similar levels of calpain activity in these two cases (panel C, Fig. 5). The generation of 145 kD SBDP in cells treated with TMZ was about 1.5-fold more intense (P = 0.003) than CTL cells, indicating that the level of calpain activity was increased in cells due to treatment with TMZ. Cells pretreated with DXM and then treated with TMZ showed a significant decrease in the generation of 145 kD SBDP, indicating an inhibitory effect of DXM on TMZ mediated increase in calpain activity (panel C, Fig. 5).

Caspase-3 activity was also measured by Western blot analysis in the generation of caspase-3-specific 120 kD SBDP (panel D, Fig. 5). Compared to CTL cells, treatment of cells with DXM alone did not cause a significant change (P = 0.983) in caspase-3 activity. Caspase-3 activity in cells treated with TMZ was almost 1.5 times more (P = 0.001) than CTL cells (panel D, Fig. 5). Thus, pretreatment of cells with DXM prior to treatment with TMZ appeared to decrease the upregulation of caspase-3 activity. Furthermore, there was no significant difference (P = 0.785) between CTL cells and cells that were pretreated with DXM and then treated with TMZ (panel D, Fig. 5).

Caspase-3 activation was also measured by Western blot analysis of the production of active 20 kD caspase-3 fragment (panel A, Fig. 6). Again, almost uniform expression of β-actin in all treatments served as an internal standard and indicated equal amounts of protein loadings in all lanes (panel B, Fig. 6). The intensities of active 20 kD caspase-3 band were almost similar in CTL cells and cells treated with DXM alone (panel C, Fig. 6). Treatment of cells with DXM alone did not cause a significant change (P = 0.613) in caspase-3 activation over CTL cells. But there was a significant increase (P = 0.001) in production of active 20 kD caspase-3 fragment in cells treated with TMZ, compared to CTL cells. Treatment of cells with DXM prior to TMZ appeared to significantly decrease the activation of caspase-3 (panel C, Fig. 6), indicating an inhibitory effect of DXM on TMZ induced caspase-3 activation.

Human glioblastoma U87MG cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in 75-cm² flasks containing 10 ml of 1 × RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a fully-humidified incubator containing 5% CO₂ at 37°C. Prior to drug treatments, the cells were starved in 1 × RPMI 1640 supplemented with 0.5% FBS for 24 h. Dose-response studies were conducted to determine the suitable doses of the drugs for using in the experiments. Cells were pretreated with 40 μM DXM for 1 h. The DXM pretreated or untreated cells were subsequently treated with 100 μM TMZ for 6 h. Cells were washed with drug-free medium and allowed to grow for 48 h. Then, cells were collected for determination of viability, apoptosis, or Western blot analysis. DXM and TMZ were obtained from Sigma Chemical (St. Louis, MO) and Schering Corporation (Kenilworth, NJ), respectively. The drugs were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions, which were then stored at -20°C until used for treating cells.

Following all treatments the viability of attached and detached cell populations was evaluated by trypan blue dye exclusion test [46]. Viable cells maintained membrane integrity and did not take up trypan blue. Cells with compromised cell membranes took up trypan blue, and were counted as dead. At least 800 cells were counted in four different fields and the number of viable cells was calculated as percentage of the total cell population.

The cells from each treatment were washed with PBS, pH 7.4, and sedimented onto the microscopic slides using Cytobucket and Centra CL2 centrifuge (IEC) at 1200 rpm for 5 min. Cells were fixed in 95% (v/v) ethanol before examination of morphology with Wright staining [46]. The morphology of the apoptotic cells as detected by light microscopy included such characteristic features as chromatin condensation, cell-volume shrinkage, and membrane-bound apoptotic bodies. Four randomly selected fields were counted for at least 800 cells. The percentage of apoptotic cells was calculated from three separate experiments.

ApopTag Peroxidase kit (Intergen, Purchase, NY) was used to assess the extent of cell death following drug treatments. Briefly, cells for each treatment were grown on six-well cell culture plates (Corning Corporation., Corning, NY) and were treated as described above. Following treatments, cells were washed with PBS and then centrifuged to sediment onto the microscopic slides. Residual PBS was then removed and cells were fixed using 95% (v/v) ethanol and allowed to dry overnight. Slides were pretreated with a protein-digesting enzyme for 15 min and then washed with distilled water for 2 min. Cells were quenched with 3% (v/v) hydrogen peroxide for 5 min followed by washing with PBS. Terminal deoxynucleotidyl transferase (TdT) enzyme was added to the pre-equilibrated cells and incubated for 1 h at 37°C. Stop-buffer was added to the slide and agitated for 15 sec followed by 10 min incubation at room temperature. After washing three times with PBS for 1 min each, anti-digoxigenin peroxidase conjugate was added to the slides and incubated for 30 min. After slides were washed twice with PBS, freshly prepared peroxidase substrate 3,3'-diaminobenzidine was added to the slides and kept for 6 min and then slides were washed with water two times. Slides were counterstained with 0.5% (w/v) methyl green for 10 min followed by washing with water and then 100% n-butanol. After 10 min, cells were dehydrated in xylene for 2 min and then mounted with glass coverslip. Experiments were conducted in triplicates and the percentage of ApopTag-positive cells was determined by counting cells under light microscopy.

We recently reported this method [47], which was modified for determination of intracellular free [Ca²⁺] in U87MG cells. Briefly, cells were grown to 80% confluency in phenol red-free medium for 72 h, suspended in the culture medium, centrifuged at 2000 rpm for 5 min to obtain pellet, and washed twice in phosphate buffered saline (PBS, pH 7.4). Cells were resuspended in culture medium, and incubated at 37°C for 2 h with gentle shaking. Following incubation, cells wee washed twice in Ca²⁺-free Locke's buffer [48] and then counted on a hemocytometer. Cells (2 × 10⁷ cells/ml) were dispersed in Locke's buffer with 10% FBS. Fura-2 (Molecular Probes, Eugene, OR) was dissolved in DMSO and diluted in Ca²⁺-free Locke's buffer containing 10% FBS. Cells were mixed with 5 μM fura-2, incubated at 37°C for 30 min, washed twice and diluted to 1 × 10⁶ cells/ml in Ca²⁺-free Locke's buffer. The intracellular free [Ca²⁺] was calculated spectrofluorometrically using the equation [Ca²⁺] = K _d β(R-R _min )/(R _max -R), where β is the ratio of F³⁸⁰_max, fluorescence intensity exciting at 380 nM for zero free Ca²⁺ to F³⁸⁰_min, and fluorescence intensity at saturating free [Ca²⁺] as reported previously [49]. The determination of fluorescence ratio (R) was performed using an SLM 8000 fluorometer (Thermospectronic, Shelton, CT) at 340 and 380 nm wavelengths. The maximal (R _max ) and minimal (R _min ) ratios were determined using 200 μl of 250 μM digitonin (Sigma) and 500 mM EGTA (Sigma, St. Louis, MO), respectively. The value of K _d , a cell-specific constant, was determined experimentally to be 0.476 μM using standards of the Calcium Calibration Buffer Kit with Magnesium (Molecular Probes, Eugene, OR).

Monoclonal antibody against α-spectrin (Affiniti, Exeter, UK) was used to measure calpain activity as well as caspase-3 activity. Caspase-3 polyclonal antibody (MBL International, Woburn, MA) was used to determine caspase-3 activation. Bax and Bcl-2 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used to assess apoptotic threshold by determining the Bax:Bcl-2 ratio. Antibody to β-actin (monoclonal clone AC-15, Sigma) was used to standardize protein loading in Western blot experiments. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ICN Biomedicals, Aurora, OH), except in case of calpain and α-spectrin where HRP-conjugated goat anti-rabbit IgG was used (ICN Biomedicals).

Cells were washed in culture flasks using Hank's balanced salt solution without Ca²⁺ (GIBCO, Grand Island, NY). The cells were then washed twice in phosphate buffered saline (PBS, pH 7.4) and centrifuged in Eppendorf 5804R (Brinkmann Instruments, Westbury, NY) at 106 × g for 10 min. Cells were resuspended in a homogenizing buffer composed of 50 mM Tris-HCl (pH 7.4), 1 mM PMSF (Bethesda Research Laboratories, Gaithersburg, MD), and 5 mM EGTA (Sigma). A polypropylene pestle (Kontes Glass, Vineland, NJ) was placed inside the microcentrifuge tube (1.5 ml) containing cells and the tube was placed inside an ice bucket for 2 min to let the pestle cool to 4°C. Cells were completely homogenized for 30 sec (the homogenization was performed while holding the microcentrifuge tube in ice with one hand). Following homogenization, protein concentration was determined using Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) and spectrophotometric measurement at 595 nm (Spectronic, Rochester, NY). Samples were then diluted (1:1) in sample buffer (62.5 mM, Tris pH 6.8, 2% SDS, 5 mM β-mercaptoethanol, 10% glycerol) and boiled for 5 min. Samples were then loaded onto the 4–20% gradient gels for electrophoresis at 200 V for 30 min (Bio-Rad, Hercules, CA). For detection of α-spectrin bands, a 5% gel was used for electrophoresis at 100 V for 2 h. Following electrophoresis, gels with the resolved proteins were electroblotted to nylon membranes (Millipore, Bedford, MA) in an electroblotting Genie apparatus (Idea Scientific, Minneapolis, MN). The membranes were blocked for 1 h in blocking buffer (5% powdered non-fat milk, 20 mM Tris pH 7.6, 0.1% Tween 20 in saline). Primary antibody was diluted (1:100 for Bax, Bcl-2, caspase-3, and 1:500 for calpain, 1:2,000 for α-spectrin, and 1:15,000 for β-actin) in blocking solution and then added to the blots for 1 h. The blots were washed three times with a wash buffer (Tris/Tween solution) and covered with secondary antibody (goat anti-rabbit for calpain and α-spectrin and goat anti-mouse for all others) at a 1:2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia, Buckinghamshire, UK) and exposed to X-OMAT AR films (Eastman Kodak, Rochester, NY). The films were scanned on a UMAX PowerLook Scanner (UMAX Technologies, Fremont, CA) using Photoshop software (Adobe Systems, Seattle, WA), and optical density of each band was determined using Quantity One software (Bio-Rad, Hercules, CA). Estimation of the degradation products of α-spectrin indicated calpain and caspase-3 activities. The 145 kD spectrin breakdown product (SBDP) is specific for calpain activation [30], and the 120 kD SBDP is specific for caspase-3 activation [50]. Also, Western blot analysis was performed to examine the generation of active 20 kD caspase-3 fragment from 32 kD caspase-3, indicating activation of caspase-3.