The human breast carcinoma cell line ZR-75-1 (American Type Culture Collection) were cultured in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 10% fetal bovine serum changing media every 3–4 days. Cells were incubated under a humidified atmosphere of 95% air/5% CO₂ at 37 °C and passed weekly by treatment with 0.25% trypsin/0.02% EDTA.

The primary polyclonal antibodies against human MnSOD and CuZnSOD were developed in our laboratory [25]. Human glutathione peroxidase (GPx1) and glutathione reductase (GR) primary antibodies were obtained from Lab Frontier (Seoul, Korea). Horseradish peroxidase conjugated to goat anti-rabbit IgG and blocking reagents were purchased from Boehringer Mannheim (Indianapolis, IN). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from HyClone (Logan, Utah). DCFH-DA and the oxidation-insensitive probe (C369) were bought from Molecular Probes (Eugene, OR). BCNU and Adriamycin were purchased from the clinical pharmacy at the University of Iowa Hospitals and Clinics. TNF-α and antimycin were purchased from Sigma Co. (Saint Louis, MO). Crystal violet and trypan blue were obtained from Fisher Scientific Co. (Pittsburgh, PA). Photofrin^TM (porfimer sodium) was a gift from QLT Phototherapeutics (Vancouver, British Columbia, Canada). A stock solution was made in 5% dextrose (pH 7.4), sterile filtered (0.22 μm), and stored at −20 °C.

The trypan blue dye exclusion was used to determine the cell viability. Twenty-four hours after treatment, cells were trypsinized and incubated with 0.2% trypan blue for 2 min at room temperature. The cells excluding the dye or stained were counted under a hemocytometer. The cell killing was indicated by the percentage of cells that were stained.

AdCMVMnSOD (AdMnSOD) was manufactured at the University of Iowa’s Vector Core Facility by inserting the MnSOD gene into the E1 region of an Ad5 E1/partial E3 deleted replication deficient adenoviral vector [24]. The cDNA was under the control of a CMV promoter. The MOI for all experiments were calculated from the plaque forming units (pfu), which was estimated as 1% of the total particles. ZR-75-1 cells were seeded in tissue culture plates and allowed to attach for 24 hours. Cells were then incubated with serum-free media containing adenovirus for 24 hours. AdLacZ was utilized as a vector control. Adenovirus-containing medium was replaced with complete medium for an additional 24 hours before cells were harvested or treated.

ZR-75-1 cells were seeded into tissue culture plates and exposed to 6 μg/mL Photofrin^TM in Hanks' buffer for 45 min. Cells were then irradiated with visible light (2.2 mW cm⁻²) for 2 min. After Photofrin^TM and light treatment, cells were allowed to recover for 6 h in full medium at 37 °C, 5% CO₂, and 95% air in the dark.

Cells were washed three times in phosphate-buffered saline (PBS, pH 7.0, KCl 2.7 mM, KH₂PO₄ 1.5 mM, NaHPO₄ 8 mM, and NaCl 136.9 mM), scraped from the dishes, and centrifuged at 82× g for 5 minutes. The supernatant was discarded and cells were resuspended in 50 mM phosphate buffer(pH 7.8). Cells were sonicated three times at maximum power, 30 seconds each time on ice using a Vibra cell cup horn sonicator (Sonics and Materials, Inc., Danbury, CT). Protein concentrations were estimated by the Bradford method and standardized with bovine serum albumin.

Briefly, total cellular proteins were electrophoresed, transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH), blocked (5% milk in TTBS: 0.01 M Tris / 0.15 M NaCl buffer, pH 8.0, and 0.1% Tween 20) at room temperature for 1 h, and incubated with primary antibody (1:1000, 1 h at room temperature. After washing, blots were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:1000, 1 h). Following a final wash, protein expression was visualized with chemiluminescence (ECL Plus) western blot detection solution (Amersham Pharmacia Biotech, Piscataway, NJ) exposed to X-ray film.

The SOD activity gel assay is based on the inhibition of the reduction of nitroblue tetrazolium (NBT) by SOD [26]. Equal amounts of protein from different samples were subjected to 12% native polyacrylamide gels by electrophoresis in nondenaturing running buffer (pH 8.3). For SOD band visualization after electrophoresis, the gel was incubated in 2.43 mM NBT, 28 μM riboflavin-5-phosphate, 28 mM tetramethylethylenediamine, and 0.75 mM NaCN in H₂O for 20 min under dark conditions. The gels were illuminated under a fluorescent light until achromatic SOD bands and a satisfactory blue background appeared.

The catalase activity gel assay was carried out according to the methods described by Sun et al. [27]. Equal amounts of protein (100 μg) from different samples were subjected to 8% native polyacrylamide gel electrophoresis in nondenaturing running buffer pH 8.3. For catalase band visualization, after electrophoresis, the gel was incubated in 0.003% hydrogen peroxide for 10 min and then stained with 2% ferric chloride and 2% potassium ferricyanide until achromatic catalase bands began to form.

Equal amounts of protein from different samples were subjected to 8% native polyacrylamide gels by electrophoresis in nondenaturing running buffer pH 8.3. For glutathione reductase band visualization, after electrophoresis, the gel was stained with 250 mM Tris (pH 8.0) containing 3.4 mM GSSG, 0.36 mM NADPH, 0.052 mM dichlorophenol-indophenol, and 1.1 mM 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide until blue precipitate GR bands began to form.

GR activity was measured according to the methods described by Mavis and Stellwagen [28]. The GR activity assay was based on the reduction of GSSG and oxidation of NADPH by the enzymatic action of GR. The disappearance of NADPH was monitored at 340 nm as the indicator of GR activity. Working buffer contained 0.65 mol of ddH₂O, 1.5 mL 100 mM potassium phosphate buffer with 3.4 mM EDTA (pH 7.6), 0.1 mL 30 mM GSSG, 0.35 mL 0.8 mM β-NADPH, and 0.3 mL 1.0% bovine serum albumin. At room temperature, the absorbance at 340 nm was monitored until constant then equal amount of cell extracts (500 μg/100 μL) were added in working buffer, and the decrease in absorbance in 340 nm for 3 minutes was immediately recorded. One unit of GR is defined as the amount of enzyme required to reduce 1.0 μmol of GSSG per minute at pH 7.6 at 25 °C.

ZR-75-1 cells were plated in 24-well microtiter plates at a density of 2 × 10⁴ cells/well for 24 hours. After different treatments, medium was removed, and the cells were washed with serum-free medium twice. A solution of either 10 μM DCFH-DA (1194) or 10 μM oxidation-insensitive DCFH-DA probe (C369) in serum free media was then added for 30 min. The cells were washed with serum-free media once. SDS (0.5%) was added to lyse the cells. Immediately after lysing the cells, fluorescence was measured using a SPECTRAFluor Plus spectrofluorimeter (excitation wavelength 485 nm, emission emission wavelength 530 nm for both probes).

DCFH staining with confocal microscopy was also used to measure intracellular ROS levels. ZR-75-1 cells were seeded at a density of 3 × 10⁵ cells/ well in slide-chambers. After different treatments, media was removed, either 10 μM DCFH-DA or 10 μM oxidation-insensitive DCFH-DA probe was added for 30 min in the dark, then washed with Hank’s buffer. Cells were fixed, and fluorescence was observed by confocal microscopy at 530/485 nm for both probes.

GR-siRNA were designed and manufactured by Ambion (Austin, TX). The target sequences for GR-siRNA were 5'-AAA GGG GTA AAT TCA ATT GGC GT-3' (forward), 5'-AAA AAC GCC AAT TGA ATT TAC CC-3' (reverse, NM 000637) respectively. SiNeg (siRNAs with sequences that do not target any gene product) was used to determine the transfection efficiency and to control for the effects of siRNA delivery. ZR-75-1 cells were transfected with siRNA (20 pmole/2 × 10⁵ cells by Lipofectamine 2000, Invitrogen) for 24 hours. The cells were incubated in full media for the indicated times prior to further experiments.

Total RNA was isolated from cells using the RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA synthesis was performed with a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Real time PCR was conducted using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems), and the PCR amplification was then detected with the SYBR Green I Nucleic Acid gel stain (Cambrex Bio Science Rockland, Inc., Rockland, ME). The housekeeping gene 18S was used as an endogenous control.

Mean values and standard errors were determined using the Microsoft Excel program. The ANOVA-Tukey test was used to compare the differences between two groups. In all cases, the statistical significance of differences between the two variants was determined at a level of P < 0.05. All the data presented are from the average of at least three independent experiments. All blots or activity gels were repeated at least twice to ensure reproducibility.

In cells infected with AdMnSOD, BCNU inhibited GR in both a dose- and time-dependent manner (Figure 1A). Treatment with BCNU caused a dose-dependent decrease in GR activity (5–30 μM) decreasing GR activity ≧90% at higher concentrations (Figure 1A). The inhibition of GR activity was also time-dependent as determined by activity gel analysis. Further characterization of an appropriate dosing regimen was determined over a 2 h time frame where inhibition of GR became significant after treating cells with BCNU (25 μM) for 60 minutes (Figure 1B). Moreover, the activity of GR increased gradually over time up to 48 hours after removal of BCNU whereupon the GR activity came back to control levels (Figure 1C). BCNU inhibited GR in a dose and time dependent manner. However, BCNU treatment did not affect the enzymatic activity of other major antioxidant proteins, including MnSOD, CuZnSOD and catalase (Figure 1D). Moreover, AdMnSOD plus BCNU did not greatly affect the levels of GPx immunoreactive protein (Figure 1E). Finally, MnSOD overexpression sensitized cells to BCNU-induced cytotoxicity. Following transduction with AdMnSOD, ZR-75-1 cells were treated with various concentrations of BCNU for 24 hours. Measuring cell killing by trypan blue dye exclusion assay, demonstrated that AdMnSOD plus BCNU increased cell killing when compared to AdMnSOD alone or BCNU alone (Figure 1F). Cell death was 6% with AdMnSOD (100 MOI) and increased to 16% with BCNU (50 μM). The combination of AdMnSOD with BCNU in ZR-75-1 cells increased cell killing to 28% (Means, P < 0.05 vs AdMnSOD alone, n = 3).

Besides inhibition of GR by a carbamoylating effect, BCNU also causes DNA or RNA crosslinking via an alkylating effect. To try to exclude the potential alkylating effect of BCNU from our results we designed small interfering RNA (siRNA) targeted to the GR sequence (siGR) as a specific means of inhibiting GR. The siGR oligos were transferred into ZR-75-1 cells. Figure 2 demonstrates that siGR inhibited GR at the mRNA, protein and enzymatic activity levels. GR mRNA was significantly lowered after infection for 72 and 96 hours (Figure 2A), GR protein was decreased by more than 50% (Figure 2B), and GR activity was also significantly decreased (Figure 2C). Cells treated with both siGR and AdMnSOD demonstrated both a decline in GR protein and an increase in MnSOD protein levels (Figure 2D). In addition, siGR and AdMnSOD significantly increased cell killing to 35% (Figure 2E). In contrast, siGR plus AdEmpty resulted in a 13% cell killing (Figure 2E). Furthermore, siGR plus AdMnSOD prolonged cell doubling time in ZR-75-1 cells from 2.8 ± 0.1 days to 3.8 ± 0.1 days (P < 0.05, Means ± SEM, n = 4) (Figure 2F).

Antimycin inhibits the electron transport chain by competitively binding to cytochrome b and displacing coenzyme Q resulting in electron leak at complex I and II. [30]. The combination of MnSOD, BCNU, and antimycin increased cell killing compared to any combinations of the other agents (Figure 3). For example, antimycin (20 μM) increased cell killing to 30% while the combination of AdMnSOD, BCNU and antimycin increased cell killing to >60%. Other combinations including AdLacZ plus antimycin, AdMnSOD plus antimycin without BCNU, or BCNU plus antimycin without MnSOD overexpression did not enhance the cytotoxicity of antimycin alone.