2.1. Chemicals and reagents
L-Buthionine sulfoximine (BSO), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), L-glutathione, glutathione reductase from bakers yeast, hydrogen peroxide solution (H2O2, 30%), pyruvate (sodium salt), phosphoric acid, meta-phosphoric acid, sodium phosphate (monobasic), Triton X-100, EDTA, NADH, NADPH, K2HPO4, KH2PO4, HEPES, D-mannitol, DMSO and DMF were from Sigma-Aldrich (St. Louis, MO). Tris-HCl, NaCl, 2-mercaptoethanol, perchloric acid and methanol were from Fisher (Pittsburgh, PA). SDS was from Bio-Rad Laboratories (Hercules, CA). MitoSOX was from Molecular Probes (Eugene, OR). Phosphate-Buffered Saline (PBS) was from Cellgro (Herndon, VA). Protease inhibitor cocktail tablets supplemented with EDTA were from Roche Diagnostics (Indianapolis, IN). Manganese(III) meso-tetrakis(N,N′-diethylimidazolium-2-yl)porphyrin (MnTDE-1,3-IP5+) was prepared as previously described in US patent #6,544,975B1 and was a kind gift from Aeolus Pharmaceuticals (Laguna Nigel, CA).
2.2. Synthesis of Cas IIgly
[Cu(4,7-dimethylphenanthroline)(glycinate)]NO3 (Casiopeína IIgly or Cas IIgly) was synthesized as previously described in US patent #5,576,326. Briefly, equimolar solution of copper(II) nitrate and the suitable substituted diimine were mixed together followed by addition of equimolar amount of the corresponding N–O donor (gly) previously deprotonated. Products were precipitated by partial evaporation of the solvent, recrystallized from water/ethanol at least two times, characterized by infrared spectroscopy (IR) and its purity was confirmed by elemental analysis. The ternary complexes exhibit IR absorptions typical of coordinated ligands: phen: 1625–1590 cm−1, 1524–1516 cm−1, 1430–1421 cm−1 (fused phenyl rings), 736–711 cm−1 and 896–811 cm−1 (δC–H out of plane); gly: 1634–1602 cm−1 (γas COO−), 648–597 cm−1 (δNH2), 3300–3240 cm−1 and 3449–3395 cm−1 (δNH2). The IR spectrum also showed an absorption band in 1384.7 cm−1 associated with the NO3− counter ion. Elemental analysis calculated for CuC16H16N4O5.2H2O (MW 443.90 g.mol−1): N (12.62), C (43.29), H (4.54); Found: N (12.96), C (43.00), H (4.42). The purity was estimated at 99%.
2.3. Cell lines and culture conditions
Human lung cancer A549 cells were purchased from ATCC (Manassas, VA) were grown in Ham’s F-12 medium with 2 mM L-glutamine (ATCC) supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep (10,000 unit, Cellgro), as commonly used for this cell line, at 37°C and 5% CO2 air atmosphere. Human lung cancer H157 cells were kindly provided by Dr. Daniel Chan (University of Colorado Denver, Aurora, CO) and were grown in RPMI-1640 medium (ATCC) supplemented with 10% FBS. It is worth noting that growing A549 cells in RPMI-1640 medium did not significantly alter the response of these cells to Cas IIgly treatment (data not shown). Cells grown in 24-well plates were used for polymerase chain reactions (PCR), flow cytometry studies, the assessment of intracellular GSH and GSSG levels and cytotoxicity. Cells grown in T-150 flasks were used for mitochondrial isolation. Cas IIgly and BSO were added from aqueous stock solutions (2.5 mM and 10 mM, respectively).
2.4. Intracellular levels of GSH and GSSG
Intracellular GSH levels were determined by HPLC with electrochemical detection (HPLC-EC) (Bode and Rose, 1999
). GSSG content was calculated as the difference between glutathione reductase (GR)-treated samples and GR-untreated samples. Cultured cells from 24-well plates were washed once with 1 ml of PBS, re-suspended in 0.5 ml of PBS and sonicated. Each sample was split into three samples (2 × 0.2 ml + 1 × 0.1 ml). The 0.2 ml samples were treated with 25 μl of GR reagent solution (11.25 U/ml GR, 0.675 mM NADPH, 25 mM Tris-HCl, 2.8 mM EDTA, pH 7.2) as described previously (Tietze, 1969
), and other 0.2 ml sample received 25 μl of PBS. Next, 10% meta-phosphoric acid (25 μl) were added to both GR-treated and untreated samples (1% final concentration), the samples centrifuged at 20,000 g
for 10 min, and the supernatants used for HPLC analysis. The HPLC column used was a Synergi 4u Hydro-RP 80A (150 × 4.6 mm) from Phenomenex (Torrance, CA) and the mobile phase was sodium phosphate buffer (125 mM sodium phosphate monobasic, pH adjusted to 3 with phosphoric acid) and 0.9% methanol. The flow rate was 0.5 ml.min−1
. The retention time for GSH under these conditions was 7.5 min. The HPLC instrument was from ESA, Inc. (Chelmsford, MA), equipped with an autosampler (model 540) and a Coul array detector (model 5600A). The potential applied was + 0.75 V vs. H/Pd electrode, and the injection volume was 5 μl. The remaining 0.1 ml sample was used to measure protein content as described above.
2.5. Assessment of cytotoxicity
The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to measure cancer cell survival, yet it has revealed artifacts when measuring the cytotoxicity of prooxidant agents (Bernhard et al., 2003
). Another simple method to evaluate drug-induced cytotoxicity is using membrane integrity as an index, which can be assessed by monitoring the release of cytosolic lactate dehydrogenase (LDH). LDH activity was measured in the culture medium and cell lysates (50 mM HEPES, Triton X-100 0.5%, pH 7) using a plate reader format as previously described (Day et al., 1995
). Briefly, 5 μl of cell culture supernatant and lysates were incubated with 0.24 mM NADH in a Tris/NaCl pH 7.2 buffer in 96-well plates for 5 min at 25o
C. The reaction was started by the addition of 9.8 mM pyruvate and the consumption of NADH was followed at 340 nm for 5 min at 30o
C. Percent LDH release was calculated by the following equation: supernatant LDH/(supernatant LDH + lysate LDH) × 100.
2.6. EPR studies
EPR spectra were obtained in 1 mm ID pyrex capillaries supported in a 4 mm OD quartz tube. Spectra were run on a Bruker EMX-Plus spectrometer with a Bruker rectangular resonator at the University of Denver. Instrument parameters were 9.42 GHz microwave frequency, 100 gauss scan width, 1 gauss modulation amplitude at 100 kHz, and microwave power 20 mW. Since signals are time dependent, data acquisition was started at 2 min. after mixing, for all samples shown in figures and reported in comparisons. Five scans with a time/scan of 42 sec were averaged. Due to the low solubility of Cas IIgly in phosphate buffers, experiments were carried out in water.
2.7. Flow cytometry
MitoSOX is an analog of hydroethidine and has been used to detect mitochondrial superoxide by flow cytometry (Julian et al., 2005
). The results obtained with this method remain qualitative rather than quantitative (Zhao et al., 2005
), yet flow cytometry allows the study of specific groups of cells. The oxidation products of MitoSOX were detected using the FL2 channel. Rhodamine 123 (Rh123) is a dye commonly used to assess the depolarization of the mitochondrial membrane, and therefore mitochondrial dysfunction. Such effect can be detected by a decrease in fluorescence using the FL1 channel (Follstad et al., 2000
). Briefly, control and drug-treated A549 cells (approximately 5 × 104
) were exposed to 5 μM MitoSOX or 2 μM Rh123 for 20 min. The supernatant was removed and the cells scraped in 0.5 ml ice-cold PBS, centrifuged at 2000 g
for 15 min, and re-suspended in 0.5 ml ice-cold PBS. Cells were analyzed within 30 min using a FACS Calibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). The total number of gated cell counted was 10,000.
2.8. Polymerase chain reaction of mitochondrial DNA
H157 and A549 cells were grown to near confluence and treated with 2.5 and 5 μM Cas IIgly, respectively. Following treatment, the media was removed and the cells were treated with 0.04% trypsin and collected by centrifugation at 500 g
. After two washes with PBS to remove residual media, the cells were resuspended in 250 μl of PBS, and total DNA was extracted using the DNeasy blood & tissue kit from Qiagen (Valencia, CA). The resulting DNA recovery was measured by UV absorption (260 nm) using a ND-100 UV/vis spectrophotometer from Nanodrop Technologies (Wilmington, DE). Although the DNA extracted from cells grown in one well of a 24-well plate was sufficient for one PCR reaction, more reliable readings of DNA extracts were obtained by combining the cells from 2 wells (total number of approximately 100,000 cells, leading to DNA concentrations of ~ 40 ng/μl). As described previously, the L2 (5′-GCC CGT ATT TAC CCT ATA GC-3′) and H3 (5′-GTC TAG GGC TGT TAG AAG TC-3′) primers, obtained from Integrated DNA Technologies (Coralville, IA), were used to amplify a 5595 bp product of the human mitochondrial genome (Velsor et al., 2004
). The amount of extracted DNA used for each PCR was 5 ng in a 25-μl reaction cocktail. Final concentrations were 300 nM of each primer, 2 mM MgSO4
, 200 μM of each dNTP and 0.5 U of Platinum Taq Polymerase High Fidelity in the corresponding buffer (Invitrogen, Carlsbad, CA). The total number of cycles was 20. The purity of the amplification product was checked by electrophoresis using FlashGel DNA cassettes (1.2% agarose) from Lonza (Rockland, ME). The amount of DNA resulting from the reaction was measured by fluorescence using the Quant-Ti™
Picogreen dsDNA reagent from (Invitrogen) and a multi-well fluorescence plate reader (Cytofluor 4000 from PerSeptive Biosystems, Framingham, MA).
2.9. Mitochondria isolation
Isolation of mitochondria was achieved through differential centrifugation as previously described (Velsor et al., 2004
). Briefly, cells were trypsinized (0.04% trypsin in Puck’s EDTA), pelleted by centrifugation (2,000 g
for 10 min at 4°C), re-suspended in PBS and spun down (2,000 g
for 10 min at 4°C) to yield a final cell pellet. The pellet was re-suspended in 550 μL of ice-cold hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2
, 10 mM Tris-HCl, pH 7.5). The cell suspension was homogenized (Kontes glass homogenizer, Fisher-Scientific, Fair Lawn, NJ) and, immediately after homogenization, 400 μL of 2.5-X mannitol-sucrose buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5) was added. Cellular debris was pelleted by centrifugation at 1,300 g
for 10 min at 4°C. Centrifugation was repeated twice and mitochondria from the supernatant were isolated by centrifugation at 17,000 g
for 15 min at 4°C. The mitochondrial pellet was washed with a mannitol-sucrose buffer and centrifuged again at 17,000 g
for 15 min at 4°C to limit cytosolic contamination. Mitochondrial enrichment was determined by the relative activity of a cytosolic enzyme marker (LDH) and a mitochondrial enzyme marker (glutamate dehydrogenase, GDH) in the fractions as previously described (Velsor et al., 2004
). The purity, according to LDH contamination, was 95%.
2.10. Immunoblotting of mitochondrial respiratory complexes I and II
Mitochondria pellets were lysed in a ground glass homogenizer with 50 μl of 50 mM HEPES, 0.5% Triton-X-100, pH 7.0 lysis solution. After homogenization, total volume was brought to 10 μl with protease inhibitor cocktail in H2O. Total protein concentration was measured at 595 nm on Spectra Max 340PC micro plate reader (Molecular Devices Corp., Sunnyvale, CA) using Coomassie Plus kit (Pierce, Rockford, IL). PAGEr® Gold Precast Polyacrylamide 4–20% Tris-Glycine (Cambrex Bio Science, Rockland, ME) were loaded with the mitochondrial fractions obtained as described above. Samples were run at 150 volts for 60 min and transferred to PVDF-plus membrane (Osmonics Inc., Westborough, MA) at 100 volts for one hour. Blocking, washing, and stripping solutions were prepared as suggested by manufacturer for optimal results with the ECL Plus Western Blotting Detection Reagents Kit (Amersham Biosciences, Buckinghamshire, UK). All wash steps were performed in triplicate for 10 min in Tris-buffered-saline-Tween (TBS-T). Membranes were blocked for one hour at room temperature in TBS-T and plus 10% horse serum. Complex I primary antibody (0.25 μg/ml of monoclonal 39 kDa antibody #A-21344, Molecular Probes, Eugene, OR) was applied for 2.5 h. Secondary antibody (peroxidase-conjugated AffiniPure goat anti-mouse IgG, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was diluted 1:40,000 in TBS-T and applied for 30 min. ECL Plus Western Blotting Detection Reagents were used to detect proteins. Following complex I detection membranes were submerged in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and incubated at 55°C for 45 min with occasional agitation. Membranes were re-probed for complex II (0.125 μg/ml of monoclonal 70 kDa antibody #A-11142, Molecular Probes). The secondary antibody (same as used above) was diluted 1:40,000 in TBS-T and applied for 30 min.
2.11. Immunoblotting of HO-1
Cells were grown in 24-well plates and, after treatment, washed with PBS. Cells were then sonicated in 200 μl of water. Cell debris was spun down and the supernatant volume reduced to approximately 30 μl by evaporation using a speed vacuum system. The resulting cytosolic proteins were run in a gradient gel and blotted as described above. The membrane was probed using heme hoxygenase 1 (HO-1) antibody (2.0 μg/ml of monoclonal 34.6 kDa antibody ab13248, Abcam, Cambridge, MA) applied over-night. The secondary antibody (same as used above) was diluted 1:40,000 in TBS-T and applied for 30 min. The membrane was re-probed using glyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibody applied for 2.5 h (0.2 μg/ml of monoclonal 40.2 kDa antibody ab9484, Abcam, Cambridge, MA). The secondary antibody was the same as used above.
2.12. Statistical analysis
Data are presented as means ± standard error. For percentage of LDH relase and GSH levels measurements, results were reported from experimental groups consisting of three to four wells and experiments replicated once. Data were subsequently analyzed for significant differences using ANOVA analysis coupled with a Tukey’s range test where significance was preset at P < 0.05 (Prizm v.4, GraphPad, San Diego, CA).