Tumor cells and culture conditions
Murine B16F10-Nex2 melanoma cells were cloned at the Experimental Oncology Unit, Federal University of São Paulo, UNIFESP, as described elsewhere [16]. Human melanoma cell line SKmel25 was obtained from the Memorial Sloan Kettering Cancer Center, New York, and all other human cell lines were obtained from the Ludwig Institute for Cancer Research (São Paulo). Tumor cells were cultivated in complete RPMI-1640 medium, pH 7.2, supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 24 mM sodium bicarbonate, 40 mg/ml gentamycin, 100 U/mL penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS), all from Invitrogen (CA, USA). Cells were maintained in culture flasks at 37°C in humidified atmosphere with 5% CO2, and were collected using PBS-EDTA (1 mM) solution.
Animals
C57Bl/6 male mice (8 weeks old) were purchased from CEDEME (Centro de Desenvolvimento de Modelos Experimentais, UNIFESP), and maintained in sterilized environment, with food and water ad libitum, in 12 h cycles of light/dark. All animal experiments were approved by the Animal Experimentation Ethics Committee of UNIFESP, under protocol No. 1507/09.
Cyclopalladated compound
The cyclopalladated complex C7a was synthesized from N, N-dimethyl-1-phenethylamide (dmpa), complexed to 1, 2 ethanebis (diphenylphosphine, dppe) ligant, as previously described in Rodrigues et al. [14] and its chemical formula is shown in Figure 1. The compound is diluted to a final concentration of 10 mM in DMSO (cell culture tested, Sigma Aldrich), and for in vivo and in vitro assays diluted to the final concentration in complete RPMI-1640 medium.
In vitro Cell Viability Assay
Tumor cells were seeded at 104 cells/well into 96 well-plates (Corning Costar Co, NY, USA), and 12 h later, they were incubated with serially diluted C7a to a final volume of 200 μL in complete RPMI medium. After 24 h incubation with C7a, the cytotoxic activity was determined by measuring cell viability by two different methods, Trypan Blue exclusion and the Cell Proliferation kit (MTT, Roche Diagnostics Comp., Indianopolis, IN), following the manufacturer's instructions. A 50% inhibition of cell growth was taken as a comparative index of cytotoxicity (IC50). To verify the inhibitory effect of DTT on C7a cytotoxic effect, cells were pre-incubated with 2 mM dithiothreitol (DTT, Sigma Aldrich, MO) for 10 minutes, and then with a high dose of C7a (10 μM) for 1 or 2 h. Alternatively, cells were incubated for 2 h with DTT, carefully washed with serum-free medium and then incubated with 1 μM C7a for 1 and 2 h. Viable cells were counted by Trypan Blue exclusion, and each IC50 value was calculated using at least 3 separate experiments.
Isolation of rat liver mitochondria (RLM)
Mitochondria were isolated by conventional differential centrifugation [17] from the liver of adult rats. Male Wistar rats weighing approximately 180 g were sacrificed by cervical dislocation and the liver was immediately removed and homogenized in 250 mM sucrose, 1 mM EGTA, and 10 mM HEPES-KOH buffer (pH 7.2) in a Potter-Elvehjem homogenizer. Homogenates were centrifuged at 770 g for 5 min and the resulting supernatant was further centrifuged at 9800 g for 10 min. Pellets were suspended in the same medium containing 0.3 mM EGTA and centrifuged at 4500 g for 15 min. The final pellet was resuspended in 250 mM sucrose and 10 mM HEPES-KOH buffer (pH 7.2) to a final protein concentration of 80-100 mg/ml. All studies with mitochondria were performed within 3 h and mitochondrial protein content was determined by the Biuret reaction [18].
Mitochondrial swelling
Rat liver mitochondria (0.25 mg protein/ml) were suspended in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH, pH 7.4, at 30°C plus 5 mM potassium succinate, 2.5 μM rotenone and 10 μM CaCl2. Complex 7a was added 10 seconds after the start of data recording. The mitochondrial swelling was estimated from the decrease in the relative absorbance at 540 nm in a Hitachi U-2000 Spectrophotometer (Tokyo, Japan).
Measurement of mitochondrial membrane potential (ΔΨ) in isolated mitochondria
Mitochondrial ΔΨ was estimated under the same experimental conditions of the swelling assay. Changes of 0.4 μM rhodamine 123 fluorescence were recorded on a Hitachi F-2500 Spectrofluorometer (Tokyo, Japan) operating at 505/525 nm with a slit width of 5/5 nm, excitation/emission, respectively. The results are expressed as percentage of dissipation in relation to uncoupled mitochondria (carbonyl cyanide trifluoro-methoxyphenylhydrazone, FCCP, Sigma Chemical, MO, 1.0 μM).
Measurement of extracellular acidification rate
HCT-8, SiHa and SKmel25 human tumor cells (3 × 105) were seeded on 3 μm pore transwells (Corning Costar), and grown in complete culture medium containing 10% FCS in 12-well culture plates for 12 h before the experiment. The extracellular acidification rate of C7a-treated and untreated cells was determined using a Cytosensor Microphysiometer (Molecular Devices, Gräfelfing, Germany). Capsules containing the adherent cells were transferred to sensor chambers and kept in a low buffered RPMI containing 1% BSA at 37°C for 20 min, until extracellular acidification rate stabilization, producing a basal line. The perfusion medium was then pumped through each sensor chamber at 50 μl/min, and pumping cycles consisted of a flow period of 90 sec, followed by a flow-off period of 30 sec. During these periods, protons released from the cells accumulated in the sensor chamber, and the slope of the H+ profile was quantified every 2 minutes. Cells were perfused with low-buffered medium containing 1% BSA (control), 10 μM C7a or 200 μM Cisplatin, both compounds diluted in the same medium. Compounds were maintained until the end of the experiment.
ΔΨm (mitochondrial transmembrane potential) measurement in intact tumor cells
Mitochondrial membrane potential measurements were carried out as described previously [19]. Briefly, 5 × 104 B16F10-Nex2 cells were seeded on 25 mm2 poly-lysine (1 mg/ml) pre-treated coverslips. After cell attachment, coverslips were placed in Leiden coverslip chambers, and cells were incubated with 50 nM of TMRE (tetramethylrhodamine ethyl ester, Molecular Probes, OR, USA) for 15 minutes at room temperature. The apparatus was transferred to a thermostatically regulated microscope chamber (37°C, Harvard Instruments, MA, USA) and 1 μM C7a, or alternatively 10 μM C7a in the presence of 2 mM DTT, was added. TMRE fluorescence (548 nm excitation and 585 nm emission) was acquired immediately after C7a addition at 1 frame/6 seconds using a TE300 Nikon inverted microscope (Nikon Osaka, Japan) and a 16 bit cooled CCD camera CoolSnap (Roper Sci, Princeton Instruments, USA) controlled by imaging software (BioIP, Wilmington, DA). Because of the high resolution, individual mitochondria were localized, especially at the borders of the cells, and the regions of interest (ROI) were drawn surrounding each mitochondrion. For calibration, at the end of each experiment (after 90-100 images captured) 5 μM FCCP (Sigma Chemical, MO, USA), a protonophore uncoupler that collapses ΔΨm, was added. Fluorescence intensity was measured in arbitrary units. At least 100 mitochondria/coverslip and nine coverslips/treatment were analyzed. In addition, cells showing depolarization (decrease), hyperpolarization (increase) or no change in the mitochondrial fluorescence were visually counted. The assay was done in triplicate, and at least 200 cells were counted in each slide.
Bax translocation
B16F10-Nex2 cells (5 × 104) were seeded on 25 mm2 poly-lysine (1 mg/ml) pre-treated coverslips. After attachment, cells were transfected with a GFP-Bax plasmid as described previously [20]. Briefly, 0.5 μg of GFP-Bax plasmid and 4 μl of LipofectAmine (Life Technologies, Gaithersburg, MD, USA) were used per coverslip. Cells were incubated for 5 h in the transfection mixture at 37°C, placed in Leiden coverslip chambers and adapted to the microscope, where the temperature of the specimen was maintained at 35-37°C. The complex C7a (1 μM) was then added and GFP-Bax translocation from cytosol to intracellular compartments was observed using an inverted confocal microscope LSM510 (Carl Zeiss, Heidelberg, Germany) equipped with ArKr 488/568, HeNe 543 lasers and 40 × Apochromat objective. Alternatively, intracellular calcium was chelated with 20 μM 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM, Sigma Chemical, MO, USA) for 20 minutes before C7a addition. For mitochondrial colocalization of Bax, transfected cells were loaded with 20 nM tetramethylrhodamine ethyl ester perchlorate (TMRE) for 10 minutes previously to the addition of C7a complex. Time course analyses of treated cells were carried out at 30 min intervals to monitor changes in GFP-Bax and TMRE localization
Calcium measurements assay
B16F10-Nex2 cells (5 × 104) were seeded on 25 mm2 poly-lysine (1 mg/ml) pre-treated coverslips. After cell attachment, coverslips were incubated with 2 μM Fura-2-AM (Molecular Probes, OR, USA) plus 20% Pluronic F127 (Sigma Chemical, MO, USA) for 30 minutes at room temperature. The coverslips were then washed and placed in Leiden coverslip chambers, adapted to the microscope, where the temperature of the specimen was maintained in 35-37°C. Cytosolic concentration of calcium on untreated cells was normalized to the zero value of Fluorescence Ratio 340/380 nm. Complex C7a (1 μM) was then added (Image number Zero) and images were collected at 3 sec intervals by using a TE300 Nikon inverted microscope (Nikon Osaka, Japan) coupled to 16 bit cooled CCD camera CoolSnap (Roper Sci, Princeton Instruments, USA) controlled by imaging software (BioIP, Wilmington, DA). Fura-2, a ratiometric calcium dye, was excited at 340 and 380 nm with emission acquired at 505 nm. Readings at 340 and 380 nm were used to calculate fluorescence ratios, which represented the variations in cytosolic calcium under these circumstances. Single cells were then analyzed using the ROI tool, fluorescence intensities obtained were normalized and plotted using the BioIP software and Kaleida Graph Synergy software. The effect of C7a was evaluated in the presence or absence of external calcium, and the maximum effects evoked by C7a under these conditions were plotted in a histogram. Seven cells/coverslip from at least nine different experiments were analyzed.
Quantification of cytosolic ATP
Cytosolic ATP concentration was measured using a bioluminescence assay kit (Adenosine 5'-triphosphate bioluminescent somatic cell assay kit, Sigma-Aldrich). Briefly, 2 × 104 plated cells were treated with 1 μM C7a (100 μl) for 15, 30, 45 or 60 minutes. Cells (or medium, as control) were lysed in 100 μl of ATP-releasing reagent and 50 μl of this suspension was added to 50 μl of ATP assay mix solution into each well of white 96-well plates. Light emission was measured at 570 nm in a SpectraMaxL luminometer (Molecular Devices, CA, USA). A standard curve obtained with diluted ATP solutions was used to calculate ATP concentrations in samples.
Identification of activated caspases
The ApoTarget™ Caspase Colorimetric Protease Assay kit (Invitrogen, CA, USA) was used for measurement of caspase-2, caspase-3, caspase-6, caspase-8 and caspase-9 activities in cell lysates. Briefly, B16F10-Nex2 cells (5 × 106) were treated in vitro with 1 μM C7a for 5 minutes. As a positive control, B16F10-Nex2 cells were exposed to ultraviolet (UV) light for 7 minutes. Cell lysates were obtained following manufacturer's instructions and caspase activity was measured at 400-405 nm as free p-nitroaniline released from p-nitroaniline-labeled specific substrates. Alternatively, caspase-3 activity was measured by flow citometry. B16F10-Nex2 cells (1 × 106) were treated for 12 h with 1 μM C7a or 10 μM Actinomycin D, as a positive control. The cells were collected and incubated with Anti-ACTIVE® Caspase-3 policlonal antibody (Promega, WI, USA) diluted 1:1000 in PBS/BSA 1% for 1 h on ice and maintained in the dark. Samples were evaluated in a FACScalibur Flow Cytometer (BD Biosciences, CA, USA), using CellQuest® software.
Analysis of nuclear alterations
B16F10-Nex2 cells (5 × 104) were grown for 12 h on sterile 25 mm2 coverslips in 6-well tissue culture plates. Cells were treated with 1 μM C7a and coverslips collected at different times were washed with PBS and stained with Hoechst 33342 (Sigma-Aldrich, MO, USA) for 15 minutes. Coverslips were inverted on slides and analyzed in an Olympus BX61 microscope (magnification 400 ×) at 360 nm. The images were acquired using Cell^M Software. Nuclear alterations in at least 200 cells for each time point were visually observed and counted, and the frequency of cells showing alterations was calculated. The assay was repeated three times.
Morphological analyses
For analysis of morphological changes in tumor cells by light microscopy, B16F10-Nex2 cells (1 × 104) were grown for 12 h on sterile 13 mm2 coverslips inserted into 12-well tissue culture plates (Corning Costar). Cells were treated with 1 μM C7a and coverslips collected at different times were washed in PBS, inverted on glass slides and analyzed in an Olympus BX61 microscope (magnification 1000 ×). The images were acquired using Cell^M Software. For transmission electron microscopy (TEM) analysis, B16F10-Nex2 cells (5 × 104) were grown for 12 h on sterile 13 mm2 coverslips in 12-well tissue culture plates. Cells were treated with 1 μM C7a for 15 min and fixed in glutaraldehyde 2.5% in sodium cacodylate buffer 0.1 M, pH 7.4. The post-fixation was performed using 1% osmium tetroxide, 0.08% potassium ferricyanide, 5 mM calcium chloride in the same buffer for 60 minutes in the dark. Dehydration was made in series of acetone and infiltration in polybed epoxy resin (Polysciences, PA, USA). Ultrathin sections obtained by ultramicrotomy were collected in grids (300 mesh) and contrasted in uranyl acetate and lead citrate. The ultrastructural analysis was done in a transmission electron microscope Zeiss EM-900.
Treatment of Experimental Tumor Metastasis
C57Bl/6 mice were injected intravenously into the tail vein with 5 × 105 B16F10-Nex2 viable cells in 100 μL of RPMI medium. Intraperitoneal injections of C7a (200 ng·kg-1) or PBS (control group) started 24 h after tumor inoculation, and treatment was repeated 3 times a week for 13 days. Animals were killed by cervical dislocation on day 15th, lungs were collected and pulmonary nodules were counted using an inverted microscope.
Statistical analysis
Statistical analysis was performed using Student's t Test from Microsoft Excel (Microsoft Office Software). Values (p) equal to or less than 0.05 were considered significant.