Mechanism of mitomycin-induced apoptosis in cultured corneal endothelial cells

Materials

Cell culture materials including trypsin, minimal essential medium (MEM), glutamine, gentamicin, and fetal bovine serum were obtained from Gibco (Grand Island, NY). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was purchased from Sigma Chemical (St. Louis, MO). The general caspase inhibitor, Z-VAD-FMK; caspase-8 inhibitor, Z-IETD-FMK; caspase-9 inhibitor, Z-LEHD-FMK; and a TUNEL (terminal deoxyribonucleotidyl-transferase-(TdT)-mediated deoxyuridine-5′-triphosphate-digoxigenin (dUTP) nick-end labeling) apoptosis detection kit were purchased from Calbiochem (Bad Soden, Germany). A MitoLight mitochondrial apoptosis detection kit was purchased from Chemicon International Inc. (Temecula, CA). Rabbit antibodies to cytochrome c and p21 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Alexa Fluor 488-conjugated goat anti–rabbit antibody; the annexin V– FITC/propidium iodide double staining kit; and mouse anti–human antibodies to Bcl-2 and p53 were purchased from Invitrogen (Carlsbad, CA). Western Blot Chemiluminescence Reagent Plus was purchased from New England Nuclear (PerkinElmer, Boston, MA). Protein assay dye and agents used for electrophoresis were purchased from Bio-Rad (Richmond, CA). Horseradish peroxidase-conjugated sheep anti-mouse IgG and donkey anti–rabbit IgG were obtained from Amersham Pharmacia (Buckinghamshire, England). Mitomycin C (MMC) was purchased from Kyowa (Hakko Kogyo Co., Tokyo, Japan). All other chemicals were obtained from Merck (Darmstadt, Germany).

Culture of porcine corneal endothelial cells

Culture of porcine corneal endothelial cells was performed as published previously [11]. Briefly, porcine eyeballs were collected from a local slaughterhouse. Under sterile conditions, the corneal endothelial cells were separated using 0.25% trypsin for 30 min. The action of trypsin was stopped by adding minimal essential medium (MEM) containing 10% fetal bovine serum, 3.8 mM L-glutamine, and 50 μg/ml gentamicin. After centrifugation, the cells were resuspended in a culture flask. The culture was kept in a humidified chamber of 5% CO₂ at 37 °C, and the medium was changed every two to three days.

Assay of cell viability with MTT

Cell viability was measured with MTT dye following previously published procedures [14]. The MTT assay is based on the production of purple formazan from a methyl tetrazolium salt by the mitochondrial enzymes of viable cells. Cultured cells at a concentration of 4,000 cells/well were seeded in 96 well culture plates and allowed to form a monolayer for 24 h. The cells were then exposed to 150 μl of serum-free MEM medium containing various concentrations of MMC for 15 h and 24 h. For analysis of apoptotic caspase pathways, cells were pre-incubated with different caspase inhibitors including a general caspase inhibitor (Z-VAD-FMK), a caspase-8 inhibitor (Z-IETD-FMK), and a caspase-9 inhibitor (Z-LEHD-FMK) for 1 h before adding 0.001 mg/ml MMC. After exposure to the drug for 24 h, cells were washed twice with phosphate-buffered saline (PBS) and incubated with 150 μl MTT solution (0.833 mg/ml in PBS) for 4 h at 37 °C. At the end of the incubation period, the MTT solution was carefully aspirated, taking care not to disturb the crystal of purple formazan at the bottom of each well. The formazan reaction product was dissolved by the addition of 150 μl dimethyl sulfoxide (DMSO), and the optical density of the fluid in each well was read at 510 nm in a multi-well spectrophotometer (Titertek Multiscan, Flow Lab, Scotland, UK). Cytotoxicity was calculated based on a significant difference in the optical density between the MMC-treated and control groups.

Immunofluorescent staining of cytochrome c protein in corneal endothelial cells

Coverslips coated with a confluent monolayer of cells were treated with MMC following previously published procedures [15]. Cells were washed twice with PBS and rinsed with 0 °C acetone for 20 s. Following a thorough PBS washing, rabbit anti-cytochrome c antibody (1:50) was applied to cells in a humidified chamber at 37 °C for 1 h. Cells were again washed with PBS and then incubated with Alexa Fluor 488-conjugated goat anti–rabbit antibody (1:200) for 1 h at room temperature. The stained samples were then rinsed and mounted. All slides were examined and photographed with a fluorescence microscope (Leitz Ortholuxix II; Leitz, Wetzlar, Germany).

Flow cytometry assay of apoptotic changes in plasma membrane and mitochondrial membrane potential

Apoptosis in the plasma membrane was identified by flow cytometry with annexin V-FITC/propidium iodide (PI) staining. Mitochondrial membrane potential changes were assayed with MitoLight dye. In healthy cells, this dye accumulates in the mitochondria and yields a red fluorescence. In apoptotic cells where mitochondrial membrane potential has been depolarized, the dye aggregates in the cytoplasm and gives off a green fluorescence, allowing discrimination of apoptotic and non-apoptotic cells. In the absence or presence of various concentrations of MMC, cells were incubated with 1 μg/ml annexin V-FITC and PI for 10 min or with 50 μl of pre-diluted MitoLight solution (900 μl of water, 1 μl of MitoLight dye, and 100 μl of 10X incubation buffer) for 15 min following the manufacturer’s instructions. Cells were then analyzed by flow cytometry (Becton, Dickinson Inc., Franklin Lakes, NJ) for a cell count of 10000.

DNA apoptotic TUNEL staining

Immunohistochemical evidence for DNA strand breaks was obtained using the TUNEL assay. The cells were plated on coverslips for at least 24 h for attachment and then incubated with various concentrations of MMC at 37 °C. After exposure to MMC, the cells were washed twice with TBS (20 mM Tris-HCl, 140 mM NaCl, pH 7.6). According to the manufacturer’s instruction, the monolayer of cells was fixed for 20 min in 4% formaldehyde at room temperature and then washed three times in TBS. Cell membrane permeability was increased by treating the samples with 20 μg/ml proteinase K solution at room temperature for 5 min. Endogenous peroxidase activity was quenched by immersing the specimens in 2% H₂O₂ at room temperature for 5 min. The samples were rinsed by replacing the hydrogen peroxide solution with labeling buffer and reaction mixture solution. The specimens were then placed in a humidified incubator for 1 h at 37 °C. The labeling reaction was stopped by immersion in stop buffer at room temperature for 5 min. The specimens were subsequently washed in TBS. Then, 100 μl of streptavidin-horseradish peroxidase was applied to the cells for 20 min at room temperature. After washing with TBS, the specimens were immersed in diaminobenzidine (DAB) solution at room temperature for 5–10 min until a satisfactory color reaction was achieved. This assay was validated with the use of control slides, which had been ascertained to contain apoptotic (positive control) and non-apoptotic (negative control) cells.

Western blot assay of proteins involved in apoptosis

Bcl-2, p53, and p21 proteins were detected by sodium dodecyl sulfate (SDS)–PAGE following a previously published procedure [15]. Briefly, cells were treated with MMC and washed with 10 ml of buffer A (20 mM of N-2-hydroxyethyl-1-piperazine-N'-2-ethanesulfonic acid [HEPES], 1 mM of ethylene diamine tetraacetic acid [EDTA], 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 μg/ml pepstatin A, pH 7.4). Cells were then scraped into the ice-cold buffer A and immediately homogenized. Protein concentrations were determined with Bio-Rad protein assay dye. Protein (10 μg) from each sample was added to the SDS-PAGE sample buffer and heated in a boiling water bath for 5 min. An aliquot was then subjected to 10% SDS–PAGE. The proteins separated by SDS–PAGE were transferred in a Bio-Rad Trans-Blot cell onto nitrocellulose membranes. Transfer was performed at 100 V for 2 h in a buffer containing 25 mM Tris-HCl, 190 mM glycine, 0.01% SDS, and 20% methanol. The blots were blocked at room temperature for 1 h with buffer solution (20 mM Tris-HCI, 137 mM NaCl, pH 7.6) containing 5% nonfat milk and 0.1% Tween-20. Incubation was then performed for 1 h at 37 °C with monoclonal anti-Bcl-2 (1:50), anti-p53 (1:500), or rabbit anti-p21 (1:250) antibodies. The nitrocellulose membrane was washed three times with the same buffer used during the blocking phase and incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:2,000) or donkey anti–rabbit (1:10,000) as the secondary antibody at room temperature for 1 h. After washing, immunocomplexes were visualized by adding Western Blot Chemiluminescence Reagent Plus. The molecular size of the immunoreactive bands was determined by comparing them with a set of molecular weight marker proteins (Bio-Rad). Relative band intensity was then analyzed using the LabWorks software 4.6 from UVP Bioimaging systems (Upland, CA).

Statistical analysis

Data were analyzed by one way ANOVA followed by Dunnett’s post hoc analysis. The values were expressed as mean±standard deviation (SD). All data were significantly different at p<0.05.

Effects of mitomycin C on cell viability and apoptotic caspase pathways

The MTT assay is used as a marker for cell viability. In the present study, MMC produced toxic effects on corneal endothelial cells. After incubation with MMC for 15 h and 24 h, cell viability was significantly decreased in a time-dependent and dose-dependent manner at concentrations ranging from 0.1, 0.01, and 0.001 mg/ml (Figure 1A). To investigate the role of caspase in MMC-induced apoptosis, cells were pretreated with various caspase inhibitors for 1 h then incubated with 0.001 mg/ml MMC for 24 h. After application of caspase inhibitors, the cellular MTT values were significantly increased in comparison with the MMC only group. Figure 1B-D show that MTT values of cells were significantly significantly reduced when comparing the MMC only groups and the control groups. The addition of caspase-8 inhibitor, general caspase inhibitor, and caspase-9 inhibitors (Z-IETD-FMK, Z-VAD-FMK, and Z-LEHD-FMK) at 10^-5 and 10^-6 M reversed the MMC-induced cellular damage.

Assay of mitochondrial membrane potential change after mitomycin C exposure

Disruption of the mitochondrial transmembrane potential is one of the earliest intracellular changes induced by apoptosis. Following exposure to 0.00001 and 0.0001 mg/ml MMC for 24 h, the fluorescence of MitoLight dye in cells was obviously changed from the mitochondria to the cytoplasm as detected by the shift of the relative fluorescence intensity. Control cells showed red fluorescence, indicating a normal mitochondrial membrane potential, while cells treated with 0.00001, 0.0001 and 0.001 mg/ml MMC showed green fluorescence (Figure 2). The percentage of MitoLight accumulated in the mitochondria was decreased from 100%±3% of the control (red color) to 47%±4% at 0.00001 mg/ml (green color) and 27%±4% at 0.0001 mg/ml (black color). Exposure to 0.001 mg/ml MMC destroyed the mitochondrial membrane potential as shown by the blue color.

Cytochrome c released from the mitochondria into the cytoplasm is characteristic of cellular apoptosis. In control cells, the cytochrome c protein was found in the mitochondrial membrane (Figure 3A). After exposure to 0.001 and 0.01 mg/ml MMC for 24 h, typical apoptotic cells were recognized that were shrunken in shape and had lost contact with neighboring cells. Exposure to 0.001 mg/ml MMC caused the cytochrome c to slightly disperse in the cytoplasm (Figure 3B). Apoptotic changes throughout the cytosol were clearly visible following exposure to 0.01 mg/ml MMC for 24 h (Figure 3C).

Apoptotic TUNEL staining

Characterization of apoptosis was performed by TUNEL staining. In comparison to the negative staining observed in control cells (Figure 4A), cells exposed to 0.001 mg/ml MMC for 24 h were seen to contain cellular chromatin condensed and marginalized at the nuclear membrane (Figure 4B). After exposure to 0.01 mg/ml MMC for 15 h, almost all cells were seriously damaged and showed typical apoptotic bodies, which contained cytosol, condensed chromatin, and organelles (Figure 4C).

Annexin V-FITC/propidium iodide staining in cellular plasma membrane with flow cytometry

To identify apoptosis of the plasma membrane, annexin V-FITC/PI double staining in cells was performed by flow cytometry. In the nonapoptotic, viable control cells, the annexin V-FITC staining and PI negative staining were located in the bottom left quadrant of the dots (Figure 5A). After exposure of the cells to 0.001 mg/ml MMC for 24 h, a significant number of cells showed annexin V-FITC positive and PI negative staining, which increased the dot numbers in the bottom right quadrant from 7%±2% of control cells to 48%±3% (Figure 5B). The cells in this stage of apoptosis were still viable. Following the increase of MMC to 0.01 mg/ml, cells in advanced apoptosis stained positive with annexin V-FITC and PI (upper right quadrant) and were significantly augmented from 12%±3% of control cells to 34%±3% after 15 h (Figure 5C) and 49%±4% after 24 h incubation (Figure 5D). The population of cells progressed to advanced apoptosis, indicating that the cells were no longer viable.

Immunoblot analysis of proteins involved in apoptosis

To investigate the effect of MMC on proteins involved in apoptosis in cultured corneal endothelial cells, we examined the anti-apoptotic protein, Bcl-2, and two apoptotic proteins, p53 and p21, through western blot analysis. The results of three independent experiments demonstrated that MMC significantly decreased the content of Bcl-2 protein and increased the amount of p53 and p21 proteins in a dose-dependent manner (Figure 6). Densitometric analysis of Bcl-2 proteins bands showed that the optical density of proteins in control cells, 0.01 mg/ml MMC-treated cells, and 0.001 mg/ml MMC-treated cells was displayed as 336±8 (100%±4% of the control), 241±7 (64%±4% at 0.01 mg/ml MMC), and 292±9 (86%±5%), respectively. In contrast, the optical density for p53 proteins was 168±6 (control), 253±7 (0.01 mg/ml MMC) and 182±6 (0.001 mg/ml MMC). The protein only significantly increased at 0.01 mg/ml (139%±4% of the control). In the presence of 0.01 and 0.001 mg/ml MMC for 24 h, optical density of p21 proteins were 111±7 (control), 415±9 (0.01 mg/ml MMC), and 166±6 (0.001 mg/ml MMC), which corresponded to an increase of 373%±6% for 0.01 mg/ml and 149%±7% for 0.001 mg/ml over control values.