Tumor Cytotoxicity by Endothelial Cells

IMPAIRMENT OF THE MITOCHONDRIAL SYSTEM FOR GLUTATHIONE UPTAKE IN MOUSE B16 MELANOMA CELLS THAT SURVIVE AFTER IN VITRO INTERACTION WITH THE HEPATIC SINUSOIDAL ENDOTHELIUM*

Angel L. OrtegaDagger §, Julian CarreteroDagger , Elena ObradorDagger , Juan GambiniDagger , Miguel AsensiDagger , Vicente Rodilla||, and José M. EstrelaDagger **

From the Dagger  Departamento de Fisiología, Universidad de Valencia, 46010 Valencia, Spain and the || Departamento de Fisiología, Farmacología y Toxicología, Universidad Cardenal Herrera CEU, 46113 Moncada, Spain

Received for publication, July 17, 2002, and in revised form, January 23, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H2O2 caused GSH depletion and a decrease in gamma -glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma -glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H2O2 damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O2 consumption, lower ATP levels, higher O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and H2O2 production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha -induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The interaction of cancer and endothelial cells is a critical process in the initiation of metastasis (1, 2). Direct in vitro lysis of metastatic tumor cells by cytokine-activated murine vascular endothelial cells has been shown (3). The liver is a common site for secondary metastatic growth, and it was reported that arrest of B16 melanoma (B16M)1 cells in the liver microvasculature induces endogenous NO and H2O2 release leading to intrasinusoidal tumor cell killing (4-6). Recently, we studied in vitro the tumoricidal mechanism of endothelial cells and found that H2O2 was not cytotoxic in the absence of NO (6). However, NO-induced tumor cytotoxicity was increased by H2O2 because of the formation of potent oxidants, likely ·OH and -ONOO radicals, via a trace metal-dependent process (6). A high percentage of B16M cells with high GSH (gamma -glutamyl-cysteinyl-glycine) content survived the combined nitrosative and oxidative attack and may represent the main task force in metastatic invasion (6). In agreement with this concept, previous in vivo video microscopic studies on the viability of intraportally injected B16M cells left untreated and or treated with L-buthionine (S,R)-sulfoximine (BSO) (a specific inhibitor of GSH synthesis) (7), arrested in mouse liver microvasculature, showed that the rate of tumor cell death increased dramatically in GSH-depleted cells (8). Therefore, because GSH content and metastatic growth appear to be directly related (9, 10), maintenance of high intracellular levels of GSH may be critical for the extravascular growth of those metastatic cells that survive after interaction with the endothelium. Thus, we investigated GSH homeostasis in this metastatic cell subset and found that invasive melanoma (iB16M) cells show an impairment in the mitochondrial system for GSH uptake. This is important because mitochondrial GSH (mtGSH) depletion may facilitate mitochondrial membrane permeabilization, permeability transition pore opening, and the release of apoptosis-inducing molecular signals (11). Our results reveal that NO and H2O2 damage the high and the low affinity components of this system. This fact can challenge iB16M cells to maintain their physiological mtGSH levels under conditions of low cytosolic GSH (cytGSH) levels (<1 mM). We propose that maintenance of mtGSH homeostasis may be a limiting factor for the survival of metastatic cells in the immediate period following intrasinusoidal arrest and interaction with activated vascular endothelial cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Culture of B16 Melanoma Cells-- B16M derived from B16M-F10 subline cells were cultured (8) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), pH 7.4, supplemented with 10% fetal calf serum (Invitrogen), 10 mM HEPES, 40 mM NaHCO3, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Isolation and Culture of Hepatic Sinusoidal Endothelium-- Male C57BL/6J mice (10-12 weeks old) were from IFFA Credo (L'Arbreole, France) and received care according to the criteria outlined by the National Institutes of Health. Hepatic sinusoidal endothelium (HSE) was separated and identified as previously described (12). Sinusoidal cells were separated in a 17.5% (w/v) metrizamide gradient. Cultures of HSE were established and maintained in pyrogen-free DMEM supplemented as described above for the B16M cells. Differential adhesion of endothelial cells to the collagen matrix and washing allows a complete elimination of other sinusoidal cell types (Kupffer, stellate, and lymphocytes) from the culture flasks.

B16 Melanoma-Endothelial Cell Adhesion and Cytotoxicity Assays-- B16M cells were loaded with 2',7'-bis-(2-carboxyethyl)-5-and-6-carboxyfluorescein acetoxymethylester (BCECF-AM; Molecular Probes, Eugene, OR) (106 cells were incubated in 1 ml of HEPES-buffered DMEM, containing 50 µg of BCECF-AM and 5 µl of Me2SO, for 20 min at 37 °C). After washing, BCECF-AM-containing cells were resuspended in HEPES-buffered DMEM without phenol red at a concentration of 2.5 × 106 cells/ml and added (0.2 ml/well) to endothelial cells (plated 24 h before) and also to plastic or collagen precoated control wells. The plates were then incubated at 37 °C, and 20 min later the wells were washed three times with fresh medium and read for fluorescence using a Fluoroskan Ascent FL (Labsystems, Manchester, UK). The number of adhering tumor cells was quantified by arbitrary fluorescence units based on the percentage of the initial number of B16M cells added to the HSE culture (4). Damage to B16M cells during their in vitro adhesion to the HSE was measured, as previously described (8), using tumor cells loaded with calcein-AM (Molecular Probes, Eugene, OR). The integrity of B16M cells cultured alone was assessed by trypan blue exclusion and by measuring lactate dehydrogenase activity released to the extracellular medium (9). The other reagents used in the experiments of tumor cytotoxicity were from Sigma.

Cytokines-- Recombinant murine TNF-alpha (2 × 107 units/mg protein) and recombinant murine interferon-gamma (IFN-gamma ; 105 units/mg protein) were obtained from Sigma. Stock solutions (5 × 105 units of TNF-alpha /ml and 25 × 104 units of IFN-gamma /ml) were diluted in sterile physiological saline solution (0.9% NaCl), adjusted to pH 7.0, and stored at 4 °C.

eNOS-deficient Mice-- Generation of eNOS-deficient mice was carried out as previously described (6, 13). We interbred heterozygous (+/-) eNOS-deficient mice to generate eNOS+/+ and eNOS-/- mice. We used eNOS+/+ and wild-type C57BL/6J mice as controls. Genotyping of the animals was performed by Southern blotting DNA from tail biopsies. The identification of eNOS+/+ and eNOS-/- mice was essentially as previously described (13).

Isolation and Compartmentation of Invasive B16M Cells-- Anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting were used to isolate viable B16M cells after co-culture with HSE cells. Anti-Met-72 monoclonal antibodies, which react with a 72-kDa cell surface protein (Met-72) expressed at high density on B16M clones of high metastatic activity, were produced as previously described (14) through syngenic immunizations of C57BL/6J mice with clones of B16M-F10. B16M and HSE cell dispersion was carried out by trypsinization (in Mg2+- and Ca2+-free PBS supplemented with 0.2% trypsin, 0.5 mM EDTA, and 5 mM glucose for 3 min at 37 °C). Then cells were washed three times in PBS and resuspended in DMEM, and an aliquot containing 2 × 106 of B16M cells was incubated with a predetermined excess of anti-Met-72 monoclonal antibody for 1 h on ice. After three washings with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated sheep anti-mouse IgF(ab)2 (Cappel Laboratories, Westchester, PA) for 1 h on ice. After another three washing steps with PBS at 4 °C, the cell pellets were resuspended in 1 ml of ice-cold PBS, filtered through a 44-µm pore mesh and analyzed using an EPICS ELITE (Coulter Electronics, Hieleah, FL). Fluorescent B16M cells were separately gated for cell sorting and collected into individual tissue culture chambered slides (Nalge Nunc International Corp., Naperville, IL). Then the sorted iB16M cells were harvested and plated in 25-cm2 polystyrene flasks (Falcon Labware) as above.

To separate cellular compartments, cultured B16M cells were harvested by exposure to 0.02% EDTA (5 min at 37 °C), then washed twice, and resuspended in ice-cold Krebs-Henseleit bicarbonate medium, pH 7.4. To separate cytosolic and mitochondrial compartments, we modified and combined the original methods by Zuurendonk and Tager (15) and Reinhart et al. (16). An aliquot of the B16M cell suspension (1 ml containing 25 × 106 cells) was mixed at 4 °C with a digitonin solution (4 ml containing 2 mM digitonin, 250 mM sucrose, 20 mM MOPS, and 3 mM EDTA, pH 7.0) for 30 s. The mixture (5 ml) was then taken to a previously prepared propylene tube (containing two layers: 2.5 ml of 200 mM mannitol, 50 mM sucrose, 1 mM EGTA, 5 mM MOPS, 5 mM KH2PO4, and 0.1% fatty acid-free bovine serum albumin, adjusted to pH 7.4 with KOH, at the bottom; and 2.5 ml of AR200 silicone oil from Serva, Heidelberg, Germany, at the top), placed on the silicone oil layer, and centrifuged immediately (10,000 × g for 30 s at 4 °C). Up to 98% of the total lactate dehydrogenase activity was found in the supernatant (cytosolic fraction). The cytosolic compartment contained maximally 2.5 and 0% of the total amount of glutamate dehydrogenase and cytochrome c oxidase, respectively. The high percentage of the total lactate dehydrogenase found in the supernatant of centrifugation shows that a complete rupture of the plasma membrane occurs. On the other side, digitonin fractionation did not damage the mitochondria, as shown by the very low percentages of glutamate dehydrogenase and cytochrome c oxidase found in the supernatant. Contamination of the lower phase with supernatant from above the oil was estimated to be 1.38 ± 0.05 µl/mg dry weight (n = 6) in parallel fractionations where B16M cells were preincubated for 10 min in the presence of 0.5 µCi of 3H2O/ml and 0.2 µCi of [U-14C]inuline/ml (Amersham Biosciences). The inuline-impermeable space was found to be 0.12 ± 0.02 µl/mg cell dry weight, a value that represents 13.6 ± 2.7% (n = 7) of cell volume. The samples (2 ml) were removed from the lower phase (mitochondria-enriched fraction) by inserting a syringe through the oil and rapidly layered on to an ice-cold Percoll gradient prepared in 13.5-ml polycarbonate tubes. Previously, the Percoll (Sigma) was passed through a Chelex 100 (Bio-Rad) chelating ion exchange resin to remove contaminating cations. The step gradient corresponded to approximately 60, 50, 40, and 30% Percoll (v/v; 2.5 ml was the volume for each phase) in medium containing 200 mM mannitol, 50 mM sucrose, 10 mM KCl, 10 mM sodium succinate, 1 mM ADP, and 0.5 mM dithiothreitol in 10 mM MOPS/KOH buffer, pH 7.4) and centrifuged at 32,000 × g for 45 s in a Centrikon T1075 refrigerated centrifuge (Kontron Instruments, Zürich, Switzerland) fitted with a fixed angle TFT 60.13 rotor. The gradients were fractionated into 10 fractions, and the distribution of organelles was analyzed with cytochrome c oxidase, glucose 6-phosphatase, 5'-nucleotidase, acid phosphatase, and catalase as marker enzymes for mitochondria, endoplasmic reticulum, plasma membrane, lysosomes, and peroxisomes, respectively. Because only mitochondria were required, the gradient was aspirated to the 40-50% interface, and the mitochondrial fraction was removed by automatic pipette, and resuspended to a final protein concentration of 20 mg/ml (n = 10). All of the operations were performed at 4 °C. Enzyme activities recovered in the purified mitochondrial fraction (expressed as a percentage of each total activity measured in B16M cells) were 74 ± 9% of cytochrome c oxidase, 1 ± 0.5% of glucose 6-phosphatase, 2 ± 1% of 5'-nucleotidase, 2 ± 0.5% of acid phosphatase, and 3 ± 1% of catalase (n = 12). Cytochrome c oxidase was measured as described below; glutamate dehydrogenase, glucose 6-phosphatase, 5'-nucleotidase, and acid phosphatase were measured as described by Bergmeyer (17); and catalase was measured as described by Aebi (18). Electron microscopy (see below for technical details) applied to mitochondria isolated from LD B16M or iB16M cells showed normal mitochondrial shapes without swelling or damage of the mitochondrial crests or other structures (not shown). For functional characterization, mitochondrial respiration in the absence and in the presence of ADP (0.2 mM) was measured using a Clark-type oxygen electrode in an incubation medium containing 100 mM sucrose, 50 mM KCl, 10 mM KH2PO4, 50 mM MOPS, 0.2 mM MgCl2, 1 mM EGTA, and 5 mM sodium succinate, pH 7.4. Oxygen uptake in states 3 and 4 was 121 ± 27 and 23 ± 8 nmol of O2/min/mg of protein, respectively, in mitochondria isolated from LD B16M cells (12 h cultured) and 53 ± 7 and 11 ± 4 nmol of O2/min/mg of protein, respectively, in mitochondria isolated from iB16M cells (18 h cultured) (n = 12 in both cases).

GSH-related Enzyme Activities-- B16M cells were detached (see above), washed twice at 4 °C in Krebs-Henseleit bicarbonate medium (without Ca2+ or Mg2+) containing 0.5 mM EGTA, pH 7.4, resuspended, and homogenized in 0.1 M phosphate buffer, pH 7.2, at 4 °C. gamma -Glutamylcysteine synthetase (gamma -GCS) and GSH synthetase activities were measured as described elsewhere (10). GSH transferase activity was determined as previously described (9).

gamma -Glutamylcysteine Synthetase Expression Analysis-- Total RNA was isolated by the acid phenol-guanidine method (19). cDNA was obtained using a random hexamer primer and a MultiScribe Reverse Transcriptase kit as described by the manufacturer (TaqMan Reverse Transcription Reagents, Applied Biosystems, Foster City, CA). A PCR master mix containing the specific primers (gamma -GCS heavy subunit (gamma -GCS-HS): forward, 5'-ATC CTC CAG TTC CTG CAC ATC TAC, and reverse, 5'-GAT CGA AGG ACA CCA ACA TGT ACTC; gamma -GCS light subunit (gamma -GCS-LS): forward, TGG AGT TGC ACA GCT GGA CTC T, and reverse, 5'-CCA GTA AGG CTG TAA ATG CTC CAA; glyceraldehyde-3-phosphate dehydrogenase: forward, 5'-CCT GGA GAA ACC TGC CAA GTA TG, and reverse, 5'-GGT CCT CAG TGT AGC CCA AGA TG) and AmpliTaq Gold DNA polymerase (Applied Biosystems) were then added. Real time quantitation of gamma -GCS-HS and gamma -GCS-LS mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA was performed with a SYBR Green I assay and evaluated using a iCycler detection system (Bio-Rad). Target cDNAs were amplified in separate tubes using the following procedure: 10 min at 95 °C, then 40 cycles of amplification (denaturation at 95 °C for 30 s, and annealing and extension at 60 °C for 1 min/cycle). The increase in fluorescence was measured in real time during the extension step. The threshold cycle (CT) was determined, and then the relative gene expression was expressed as follows: fold change = 2-Delta (Delta CT), where Delta CT = CT target - CT glyceraldehyde-3-phosphate dehydrogenase, and Delta (Delta CT) = Delta CT treated - Delta CT control.

Mitochondrial GSH Transport-- Isolated mitochondria were resuspended in incubation medium and used immediately after B16M cell fractionation (see above) to avoid any loss of their initial GSH content. Using a variation of the technique described by Martensson et al. (20), the net rates of GSH uptake into the matrix were measured at 25 °C in mixtures (volume, 500 µl) containing incubation medium and 0.1-10 mM (1 µCi/assay) [35S]GSH (see the legend to Fig. 2). Uptake was initiated by adding 20 µl of the mitochondrial suspension (final concentration, 1 mg of protein/ml). Protein was determined by the Bradford method (21). The incubations were terminated by centrifugation of the mitochondria through Ficoll at 10,000 × g for 3 min at 4 °C. The mitochondrial pellet was washed twice at 4 °C with a medium containing 0.3 M sucrose, 1 mM EGTA, 5 mM MOPS, 5 mM KH2PO4, and 0.1% bovine serum albumin (fatty acid free), adjusted to pH 7.4 with KOH, to remove GSH from the intermembranous space and then suspended in 50 µl of 5% sulfosalicylic acid and centrifuged at 2,000 × g for 3 min. The 35S was measured in the supernatant, whereas the residual pellet was treated as previously described (20) to measure matrix protein-bound GSH, which was 8-10% of the total mtGSH in all of our experimental conditions.

The net rates of GSH uptake into mitochondria were estimated in intact B16M cells by using a variation of the technique described by Lash (22) for renal cells. B16M cells (approximately 5 mg of dry weight/ml) were suspended at 37 °C in Krebs-Henseleit bicarbonate medium, pH 7.4, and the incubations were performed in 25-ml Erlenmeyer flasks in a Clifton shaking water bath (60 cycles/min). Glucose (5 mM) was always present. The gas atmosphere was O2/CO2 (19:1). Cell membrane integrity was determined as previously described (23). To prevent possible GSH degradation, B16M cells were pretreated with acivicin (L-(alpha S,5S)-alpha -amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; an irreversible inhibitor of gamma -glutamyltranspeptidase (24)) (Sigma) (0.1 mM) for 15 min prior to the addition of GSH. After this period, GSH (2 mM, containing 2 µCi of [35S]GSH from PerkinElmer Life Sciences) and bathophenanthroline disulfonate (0.1 mM; to minimize metal ion-catalyzed GSH oxidation) were added to the incubation mixture, which was then taken to an electroporation unit for eukaryotic cells (Bio-Rad) to permeabilize cell membranes and allow uptake of intact GSH molecules into the cytosol. The field strength applied to each sample was of 1.0 kV/cm with a time constant of 50 ms. The cells were washed twice in ice-cold Krebs-Henseleit bicarbonate medium, pH 7.4, to remove extracellular GSH and then were incubated again, as described above. Kinetics of mtGSH transport was calculated after B16M cell fractionation was applied as explained above.

Preparation of GSH Ester-- GSH monoisopropyl(glycyl) ester was prepared as previously described (25).

Measurement of Redox Activities of the Respiratory Enzymes-- All of the enzyme activities were measured spectrophotometrically with a double-beam, dual wavelength spectrophotometer (Cecil Instruments, Cambridge, UK) by suspending 0.1 mg of protein of mitochondrial particles prepared by freezing and thawing (three times) of isolated mitochondria in a medium containing 50 mM potassium phosphate buffer, pH 7.4, and 25 mM EDTA (final volume, 1.5 ml) at 25 °C.

NADH-CoQ oxidoreductase (complex I) was assayed by following the rotenone-sensitive initial rate of NADH oxidation at 360-374 nm (Delta epsilon  = 2.3 mM-1·cm-1). The basic reaction medium also contained 30 mM NADH, 1.5 mM antimycin A, and 1.5 mM KCN. The reaction was initiated by adding 50 mM decyl-ubiquinone.

Succinate-CoQ oxidoreductase (complex II) was assayed by following the reduction of 2,6-dichlorophenolindophenol at 600 nm (Delta epsilon  = 19.1 mM-1·cm-1). The basic reaction medium also contained 1 mg/ml rotenone, 1.5 mM antimycin A, 1.5 mM KCN, 50 mM 2,6-dichlorophenolindophenol, and 10 mM succinate. The reaction was initiated by adding 50 mM decyl-ubiquinone.

Ubiquinol-cytochrome c oxidoreductase (complex III) was assayed by following at 540-550 nm the initial rate of antimycin-sensitive cytochrome c reduction upon the addition of 10 mM reduced decyl-ubiquinone. The reduced quinine was obtained from the oxidized form as previously described (26). The reaction medium also contained 1 mg/ml rotenone, 1.5 mM KCN, and 10 mM ferricytochrome c.

Cytochrome c oxidase (complex IV) activity was estimated from the initial rate of ferrocytochrome c oxidation at 540-550 nm. The reaction mixture was supplemented with 10 mM ferrocytochrome c and 1.5 mM antimycin A. The reaction was started by the addition of mitochondrial particles.

Measurement of H2O2, Nitrite, and Nitrate-- The assay of H2O2 production was based, as previously reported (6) on the H2O2-dependent oxidation of the homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) to a highly fluorescent dimer (2,2'-dihydroxydiphenyl-5,5'-diacetic acid) that is mediated by horse-radish peroxidase (27). Nitrite and nitrate determinations were performed as previously described (6) and based on the methodology of Braman and Hendrix (28). Total NOx (NO<UP><SUB>2</SUB><SUP>−</SUP></UP> plus NO<UP><SUB>3</SUB><SUP>−</SUP></UP>) determinations were made by monitoring NO evolution (chemiluminiscence detection) from a measured sample placed into a boiling VCl3/HCl solution (which will reduce both NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> to NO). Quantitation was accomplished using a standard curve made up of known amounts of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

Flow Cytometry-- Cellular suspensions were diluted to ~250,000 cells/ml. Analyses were performed with an EPICS PROFILE II (Coulter Electronics) as previously described (29). Samples were acquired for 10,000 individual cells. Fluorochromes were from Molecular Probes (Poortgebouw, Leiden, The Netherlands), excepting dihydroethidium, which was from Sigma. Cell viability was determined by the fluorescent dye propidium iodide (final concentration, 10 µM). The rest of the studies were limited to viable cells. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation was determined using dihydroethidium (2 µg/ml) (30). Size of isolated tumor mitochondria was estimated using the forward angle light scatter that represents the mitochondrial size that contributes to the refraction of light as it travels through the particle (31).

Quantitative Determination of Plasma and Mitochondrial Membrane Potentials-- Plasma membrane potential was measured using a standard technique (32). B16M cells were incubated as described above, seeded at 5 × 104 cells/cm2 in glass Petri dishes, and cultured for 24 h to about 50% confluency. The culture dishes were mounted on a tube-focusing microscope (Nikon, Tokyo, Japan). Intracellular measurements were performed at 25 °C with glass micropipettes filled with 3 M KCl and with a 20 MOmega DC resistance. The membrane potentials were measured with a WP Instruments M4-A electrometer amplifier (Sarasota, FL), and the output was displayed using a MacLab System (Castle Hill, Australia). Measurements were made only in cells (from at least three different preparations) that gave a stable membrane potential within 10 s of penetration, which indicates a good seal of the plasma membrane with the recording electrode.

The measurements of the mitochondrial membrane potential (MMP) were performed by the uptake of the radiolabeled lipophilic cation methyl-triphenylphosphonium (TPMP), which enables small changes in potential to be determined (28). Briefly, B16M cells (2 × 106) were incubated at 37 °C for 60 min in 1 ml of DMEM supplemented as mentioned above but including 1 µM TPMP, 250 nCi of [3H]TPMP (Amersham Biosciences), and 1 µM sodium tetraphenylboron. After incubation the cells were pelleted by centrifugation (1,000 × g for 5 min), 100 µl of supernatant was removed, the pellet was resuspended in 100 µl of 10% Triton X-100, and the radioactivity in the supernatant and pellet was measured using an LKB Wallace 8100 LSC liquid scintillation counter with quench corrections. Nonspecific TPMP binding was corrected as previously described (28). Energization-dependent TPMP uptake was expressed as an accumulation ratio in units of (TPMP/mg protein)/(TPMP/µl supernatant) (33).

Metabolites-- GSH was measured by using the glutathione S-transferase reaction (34). GSSG was determined by high performance liquid chromatography as previously described (35). Formation of GSH-bimane conjugates in B16M cells incubated with monochlorobimane (MCB, Molecular Probes) was followed as previously described (36) using a Leica TCS-SP2 confocal laser scanning microscopy system. Bimane fluorescence in subcellular fractions was measured by using a Perkin-Elmer LS-50B fluorometer operating at 385-nm (excitation) and 470-nm (emission) wavelengths (37).

For amino acid analysis, the intracellular fractions (see above) were treated for protein precipitation with 5% (w/v) sulfosalicylic acid in 0.3 M lithium citrate buffer, pH 2.8, as previously described (23). Fifty µl of the supernatant were collected and injected into an LKB 4151 Alpha Plus amino acid analyzer (LKB Biochrom, Cambridge, UK).

ATP, alpha -ketoglutarate, oxaloacetate, malate, citrate, succinate, phosphoenolpyruvate, pyruvate, and lactate levels in cell compartments were measured spectrophotometrically or fluorimetrically following standard enzymatic methods (17). Malate and alpha -ketoglutarate uptake by isolated tumor mitochondria were performed as previously described (see Ref. 39 and references therein).

Electron Microscopy-- Mitochondria were fixed in phosphate-buffered (0.1 M, pH 7.2) glutaraldehyde solution for 2 h and then postfixed in phosphate-buffered (0.1 M, pH 7.2) 2% osmium tetroxide solution for a further 2 h. After embedding the mitochondria blocks in Epon, ultrathin sections were cut with an LKB ultramicrotome ULTRATOME III (LKB Ultratome, Uppsala, Sweden) and then contrasted with uranyl acetate and lead citrate for transmission electron microscopy. Electron micrographs were taken with a JEOL JEM 100 B (Tokyo, Japan).

Statistical Analysis-- The data were analyzed by Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HSE-induced Changes in the Mitochondrial Glutathione Status of iB16M Cells-- LD B16M cells show a high GSH content in vitro and a high metastatic activity in vivo (9). Most LD B16M cells survive the NO- and H2O2-mediated tumoricidal activity of endothelial cells (6) (Table I). However, it is unknown whether these survivors suffer transient or permanent alterations in their intracellular glutathione redox status that could challenge their growth capacity or their resistance against a subsequent stress. To investigate this possibility, in the first set of experiments we measured NO- and H2O2-dependent damage in LD B16M cells attached to cytokine-activated HSE (Table I). In these experiments, the combination of TNF-alpha and IFN-gamma was used as a potent activator of NO and H2O2 generation by the HSE (6) and also by the B16M cells (6, 17). We used HSE cells isolated from eNOS-deficient (eNOS-/-) mice to abolish eNOS-dependent NO production and L-NAME to inhibit both inducible NOS and eNOS activities, whereas the addition of exogenous catalase was used to eliminate H2O2 released to the extracellular medium (6). As shown in Table I, eNOS+/+ HSE-induced tumor cytotoxicity is low (6) (approximately 15%). A similar value was obtained by directly treating LD B16M cells with NO and H2O2 (Table I). Abrogation of eNOS-mediated NO generation or elimination of H2O2 accumulation decreased HSE-induced LD B16M cytotoxicity to extremely low values (3-5%) (Table I).


                              
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Table I
GSH and GSSG contents and GSH synthesis-related enzyme activities in B16M cells that survive after interaction with the HSE in vitro
24-h cultured HSE cells (~2.5 × 105cells/well) isolated from wild-type, eNOS+/+, or eNOS-/- mice were co-cultured with LD B16M cells (~5.0 × 105 cells/well; precultured for 12 h to low cellular density). Twenty min after B16M addition to the HSE, the plates were washed as described under "Experimental Procedures." The ratio of tumor cells adhering to the HSE was ~1:1. TNF-alpha (100 units/ml) and IFN-gamma (50 units/ml) or vehicle (physiological saline) were added to the co-cultures when all tumor cells present were attached to the HSE. During the 6-h period where B16M and endothelial cells were co-cultured, the percentage of HSE cell viability was 99-100% in all cases. NO (10 µM) and H2O2 (100 µM) were added to B16M cells cultured alone. L-NAME and catalase, when present, were incubated at 1 mM and 103 units/ml, respectively. Tumor cytotoxicity (expressed as the percentage of B16M cells that lost viability within the 6-h period of co-culture) was determined after 6 h of incubation. GSH and GSSG contents and GSH-related enzyme activities were measured in viable cancer cells isolated after their interaction with the HSE (see under "Experimental Procedures"). cyt, cytosolic; mt, mitochondrial. The data are expressed as the means ± S.D. of six or seven different experiments.

Intracellular GSH depletion and a decrease in catalase activity was recently reported in NO- and H2O2-treated B16M cells, whereas the GSH peroxidase/GSH reductase system or the total superoxide dismutase activity remained unaffected (6). LD B16M cells surviving the combined nitrosative/oxidative attack induced by the HSE, named here iB16M cells, showed a decrease in cytGSH and mtGSH contents and a parallel increase in GSSG in both cell compartments (Table I). This decrease in glutathione redox status (GSH/GSSG) associated with a decrease in gamma -GCS activity (Table I), the rate-limiting enzyme in GSH synthesis (7). Direct exposure of LD B16M cells to H2O2 did not affect gamma -GCS activity (not shown); however, NO decreased this activity to approximately 59% of control values (Table I) (H2O2 and NO were incubated at concentrations that reflected those found in co-cultures of HSE and B16M cells (6) (see the Introduction).) These results are in agreement with a previous report showing inhibition of gamma -GCS activity by nitric oxide donors (40). On the other hand, among other pro-oxidants, both NO and H2O2 have been postulated as potential inducers of mammalian gamma -GCS subunit genes (see Ref. 41 for a recent review). We found that NO-mediated partial inactivation of this enzyme in LD B16M cells (Table I) is followed by overexpression of gamma -GCS-HS and gamma -GCS-LS in the immediate period (maximum values were found at 3 h) following interaction with the HSE (Table II). Expression of gamma -GCS subunits in iB16M cells isolated after interaction of LD B16M and endothelial cells was similar if the HSE was isolated from eNOS+/+ (Table II) or from eNOS-/- (not shown) mice, which suggests that NO is not responsible for this effect. However, when exogenous catalase was present in the LD B16M + eNOS+/+ HSE co-cultures (as in Table I), overexpression in iB16M cells was abolished (results were not significantly different from those displayed in Table II for LD B16M cells; not shown), which indicates that H2O2 promotes gamma -GCS expression in iB16M cells. As shown in Fig. 1, gamma -GCS overexpression is associated with a rapid increase in cytGSH in iB16M cells within the 6-18-h period of growth in vitro (gamma -GCS activity 12 h after plating was of 217 ± 34, n = 5; a value not significantly different from that found in control LD B16M cells, see Table I). Nevertheless, mtGSH values did not recover, and even decreased, during the period of cytGSH increase (Fig. 1). Only after 18 h of plating, when cell number began to increase, did mtGSH levels increase (Fig. 1). Removal of the fetal calf serum from the culture medium did not allow B16M cell growth and mtGSH increase (not shown). Therefore, it is possible that (a) interaction between B16M cells and the HSE impairs the mitochondrial system for GSH uptake in the tumor cells and (b) repairing this damage is associated with cell division.


                              
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Table II
gamma -GCS overexpression in iB16M cells
B16M cells (~5.0 × 105 cells/well) were precultured for 12 h to low cellular density. HSE isolated from eNOS+/+ mice and B16M cells were co-cultured for 6 h as described in Table I, and then iB16M cells were isolated as described under "Experimental Procedures." gamma -GCS-HS and gamma -GCS-LS expression in LD B16M cells was determined 3 and 6 h after the initial 12-h period of preculture, whereas expression in iB16M cells was determined in viable cancer cells during their co-culture with endothelial cells. The figures, expressing fold induction, show the mean values ± S.D. from five different experiments. No significant differences were found when results obtained using iB16M cells co-cultured with wild-type HSE were compared with those displayed in the Table (LD B16M cells and HSE isolated from eNOS+/+ mice).


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Fig. 1.   Cytosolic and mitochondrial GSH contents in growing iB16M cells. The data are the means ± S.D. for five or six independent experiments. a, p < 0.01 comparing all data versus controls (0 h); b, p < 0.05 comparing all data versus controls (0 h).

HSE-induced Impairment of GSH Transport into iB16M Mitochondria-- We measured GSH uptake by mitochondria in intact B16M cells and found that LD B16M cells (12 h after plating) transport GSH into mitochondria at a rate of 0.77 ± 0.12 nmol/mg of mitochondrial protein/min (n = 7), whereas mitochondria in iB16M cells take up GSH at a rate of 0.18 ± 0.05 (n = 7; p < 0.01) 12 h after plating and of 0.68 ± 0.14 nmol/mg of mitochondrial protein/min (n = 7; not significantly different as compared with LD B16M cells) 48 h after plating. As shown in Fig. 2, in agreement with previous observations in liver mitochondria (20), isolated LD B16M or iB16M cell mitochondria showed maximal GSH uptake rates during the first minute. Equilibrium was reached in 1-2 min (Fig. 2) at external GSH levels ranging from 0.5 to 10 mM (see the legend to Fig. 2). As previously suggested for liver mitochondria (20), GSH uptake into B16M mitochondria reflects function of a multicomponent system. Measurement of GSH uptake at different external GSH levels (Fig. 2) revealed at very low external GSH levels (0.1-1 mM; which can only be expected in malignant cells under conditions of severe, e.g. drug-induced, intracellular GSH depletion) a high affinity component with an apparent Km of 88 ± 17 µM (n = 7). At higher external GSH levels (2-9 mM), a low affinity component transports GSH with an apparent Km of 4.5 ± 0.66 mM (n = 7). From the [35S]GSH transported into the matrix, we calculated that 90 ± 5% of the 35S associated with free GSH, and the remainder associated with protein-bound GSH (n = 6).


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Fig. 2.   Impairment of GSH transport into isolated mitochondria of growing iB16M cells. Tumor mitochondria were isolated from LD B16M cells (12 h after plating, ) and iB16M cells (12 h, black-triangle; and 48 h after plating, open circle ). Incubations were performed as described under "Experimental Procedures" in the presence of 10 mM external GSH (which is approximately the highest cytGSH found in cultured LD B16M cells) (9) (panel A) or 0.5 mM external GSH (panel B), and 1 mM external ATP (which fully preserves the ATP dependence of the mitochondrial GSH transport system) (20). No significant differences were found when the data in panel A were compared with those found in the presence of 2 mM external GSH (which corresponds approximately to the lowest cytGSH found in HD B16M cells) (9) (not shown). No GSH uptake by tumor mitochondria, isolated from iB16M cells 12 h after plating, was detected in the presence of 0.1 or 1 mM external GSH (not shown). The data are the means ± S.D. (n = 6-7). a, p < 0.01 comparing uptake rates obtained in mitochondria from LD B16M cells with those measured in mitochondria isolated from iB16M cells 12 or 48 h after plating.

Tumor mitochondria isolated from iB16M cells (12 h after plating) showed a marked decrease in GSH uptake (to approximately 26% of control values; see above) in the presence of 2-10 mM external GSH; however, they could not transport the tripeptide when external GSH was of 1 mM or lower (Fig. 2). These facts suggest that vascular endothelium-induced tumor cell toxicity partially impairs transport through the low affinity component of the mitochondrial GSH transport system but completely abrogates transport through the high affinity component.

To investigate the possibility that the low mitochondrial GSH levels cause decreased exchange transport with label, thereby artifactually leading to the conclusion that the transporter is functionally altered, we induced mtGSH depletion by incubating LD B16M cells in the presence of diethylmaleate (DEM) and BSO (Table III). DEM- and BSO-induced GSH depletion did not affect B16M cell viability in vitro (>99%), although mtGSH decreased to 43% of control values (a similar value to that found when LD B16M cells are co-cultured with eNOS+/+HSE; Table I). HSE induced a marked decrease in the rate of GSH uptake by mitochondria isolated from B16M cells (Fig. 2); however, mtGSH depletion by itself did not alter the transport of GSH into the organelles (Table III).


                              
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Table III
Effect of DEM- and BSO-induced GSH depletion on GSH transport into LD B16 mitochondria
B16M cells were cultured for 12 h to low density. DEM (50 µM) was added where indicated and remained present for 15 min. Then the culture medium was replaced by fresh medium. BSO (0.2 mM) was added to cells pretreated with DEM. All flasks were incubated for 6 h more before performing the measurements. GSH uptake by isolated mitochondria was determined as described under "Experimental Procedures" in the presence of 10 mM external GSH (see also the legend to Fig. 2). No significant differences were found when data in this figure were compared with those found in the presence of 2 mM external GSH (not shown). The data are the means ± S.D. (n = 7).

Because GSH is negatively charged, hypothetically it could be also co-transported with a cation or exchanged with another anion. Anions and amino acids require specific carrier systems to be transported into the mitochondria. However, we found that different metabolic intermediaries at their cytosolic concentrations in 12/48-h cultured iB16M cells (3.60 ± 0.47/2.15 ± 0.34 mM glutamate, 0.55 ± 0.13/0.36 ± 0.09 mM alpha -ketoglutarate, 0.02 ± 0.01/0.01 ± 0.005 mM oxaloacetate, 0.16 ± 0.04/0.33 ± 0.12 mM aspartate, 0.26 ± 0.07/0.17 ± 0.05 mM pyruvate, 9.39 ± 1.22/5.44 ± 0.78 mM lactate, 0.04 ± 0.02/0.10 ± 0.03 mM phosphoenolpyruvate, 7.82 ± 0.97/5.06 ± 0.88 mM alanine, 0.30 ± 0.10/0.19 ± 0.06 citrate, 0.33 ± 0.08/0.15 ± 0.04 malate, and 0.02 ± 0.005/0.07 ± 0.02 mM succinate; n = 5-6 different determinations) or a complete mixture of amino acids (42) did not alter rates of GSH uptake (similar to those reported in Fig. 2; not shown) by mitochondria isolated from 12- or 48-h cultured iB16M cells. Furthermore, an inhibitor of the dicarboxylate carrier (butylmaloneate, 1 mM), which decreases malate transport into isolated mitochondria (from 48-h cultured iB16M cells) by 77.4 ± 10.6% (n = 5; p < 0.01), or an inhibitor of the alpha -ketoglutarate carrier (phenylsuccinate, 1 mM), which decreases alpha -ketoglutarate transport into isolated mitochondria (from 48-h cultured iB16M cells) by 80.6 ± 7.9% (n = 5; p < 0.01), did not significantly change the rate of GSH uptake by iB16M mitochondria (not shown).

Nitrogen- and Oxygen-derived Reactive Species Damage the Mitochondrial GSH Transport-- Tumor cytotoxicity of reactive nitrogen and oxygen species (RNS and ROS) was recently reported in B16M cells directly exposed to NO and H2O2 (6). To investigate the possible involvement of RNS and/or ROS in damaging the system of GSH transport into mitochondria, in the first set of experiments we observed that a high rate of NOx and H2O2 accumulation, as it occurs when LD B16M and eNOS+/+ HSE cells are co-cultured in the presence of cytokines, associated with a marked decrease (approximately 28% of control values at 30 s) in the rate of GSH uptake by tumor mitochondria (Table IV). Co-culture of LD B16M cells with eNOS-/- HSE or with eNOS+/+HSE in the presence of L-NAME, which decreased/abolished NOx accumulation without affecting H2O2 generation, also associated with a decrease in GSH uptake by mitochondria but to a lesser extent (66-44% of control values) (Table IV). On the contrary, catalase, which practically abolished H2O2 accumulation without altering NOx levels, slightly decreased mtGSH transport (Table IV). However, when NOx accumulation was abolished and H2O2 levels were limited to less than 20% of control values by incubating LD B16M and eNOS+/+ HSE cells in the presence of L-NAME and catalase, rates of GSH uptake were not significantly different as compared with controls (Table IV). Therefore, although under TNF-alpha and IFN-gamma stimulation HSE cells may also release other molecular effectors in addition to NO and H2O2, our results suggest that ROS and RNS are the main signals that cause impairment of the mitochondrial GSH transport system.


                              
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Table IV
NO- and H2O2-dependent damage of the mitochondrial GSH transport in LD B16M cells attached to the HSE
HSE cells were co-cultured as described in Table I. TNF-alpha and IFN-gamma (concentrations were identical to those used in Table I) or vehicle (physiological saline) were added, and determination of NOx and H2O2 accumulation was restricted to co-cultures in which all tumor cells were attached to HSE cells. L-NAME and/or catalase were incubated as in Table I. The data represent the total amount of NOx and H2O2 that accumulated in the culture medium during the first 3 h of incubation (where tumor cell viability was >95% in all cases; data not shown). GSH uptake was measured in mitochondria isolated from B16M viable cells after 6 h of incubation. During the 6-h period of incubation, the percentage of HSE cell viability was 99% in all cases. The data are the means ± S.D. for six or seven different experiments. mt., mitochondrial; ND, not detectable.

In addition, GSH uptake by mitochondria was measured in viable LD B16M cells directly exposed to NO and/or H2O2. As shown in Table V, NO caused a decrease in the rate of GSH uptake to approximately 63% of control values. H2O2 did not affect significantly GSH transport, but NO in the presence of H2O2 further decreased GSH uptake to only 31% of controls (Table V). Less available cytGSH in the presence of NO and/or H2O2 cannot be argued to explain the decrease rate of GSH uptake by mitochondria if one takes into account cytGSH contents (Table V) and the Km values of the transport system (see above). -OONO decreased GSH uptake to approximately 41% of controls (Table V). Incubation of B16M cells in the presence of NO and superoxide dismutase, which removes O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> anions and increases H2O2 levels, decreased GSH uptake by mitochondria more than NO alone (Table V). EGTA-mediated removal of metal ions prevented NO- and H2O2-induced damage to the mitochondrial GSH transport system, whereas addition of NO, H2O2, and FeCl3 to EGTA-pretreated LD B16M cells did not alter the rate of GSH uptake as compared with NO- and H2O2-treated LD B16M cells (Table V). These results indicate that NO and H2O2 promote formation of potent oxidants (e.g. ·OH or -OONO) that have tumoricidal activity (6) and also appear to be responsible for the damage to the components of the mitochondrial GSH transport system in those cells that survive the interaction with the vascular endothelium (Tables IV and V).


                              
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Table V
Nitrogen and/or oxygen-derived reactive species damage the mitochondrial GSH transport system in intact LD B16M cells
B16M cells were cultured for 12 h to low cellular density. NO (10 µM), H2O2 (100 µM), superoxide dismutase (SOD; 100 units/ml), -OONO (10 µM, prepared as in Ref. 6), or EGTA (0.5 mM) were added at 12 h of culture. FeCl3 (1 mM) was added 5 min after EGTA addition. Uptake measurements were performed 1 h later in viable cells. The data are the means ± S.D. of five or six different experiments.

Mitochondrial GSH Depletion Increases TNF-alpha -induced Oxidative Stress and iB16M Cell Death-- TNF is a macrophage/monocyte-derived cytokine with cytostatic and cytotoxic anti-tumor effect (43). TNF-alpha interferes with electron flow in the mitochondria (44) and increases ROS production (45). Therefore we tested its effect in growing iB16M cells in vitro. The cytokine was added 12 h after plating, when mtGSH content is decreasing to low values. As shown in Table VI, 12 h after cytokine addition when mtGSH levels are increasing in controls, rmTNF-alpha increased ROS production, kept mtGSH levels at very low levels, and caused a dramatic decrease in cell number and viability. We also tested whether GSH replenishment under conditions of mtGSH depletion could prevent TNF-alpha -induced B16M cytotoxicity. Previous studies showed that GSH esters, but not GSH itself, are effectively transported into cells and converted into GSH (46) and that administration of GSH monoesters to mice leads to increased mtGSH levels in various tissues (47). As shown in Table VI, GSH ester increased the mitochondrial pool, decreased TNF-alpha -induced ROS generation, and completely abolished the deleterious effect of TNF-alpha by increasing cell number and viability. However, because GSH ester boosts both GSH pools, the cytosolic and the mitochondrial (Table VI), it was necessary to answer whether the pool relevant for cell survival is the mitochondrial one. iB16M cells were incubated in the presence of BSO and MCB, a substrate of the GSH transferases originally developed for fluorimetric determination of GSH (36). In agreement with previous findings in rat hepatocytes (37), GSH transferase activity in iB16M mitochondria is very low as compared with the cytosolic activity (iB16M cells contain 60 ± 9 milliunits of GSH transferase activity/106 cells, but only 0.9 ± 0.3% of this activity is localized within isolated mitochondria; n = 10). Thus, in iB16M cells preloaded with GSH ester, the addition of MCB caused a rapid decrease in free cytosolic GSH (to approximately 32% of controls), whereas the mitochondrial pool remained close to control values (Table VI). Under these experimental conditions, TNF decreased cytGSH and mtGSH to approximately 19 and 66%, respectively, of control values; however, cell viability remained high and close to control values (Table VI). These results prove that mtGSH levels directly regulate TNF-alpha -induced B16M cytotoxicity.


                              
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Table VI
Response of iB16M cells to treatment with rmTNF-alpha in vitro
rmTNF-alpha (100 units/ml) was added to the culture medium 12 h after plating. GSH ester (0.5 mM) was added to the culture medium 6 h before cytokine addition. MCB (70 µM) was added 3 min before cytokine addition. To remove all extracellular MCB, before cytokine addition cells were washed twice with PBS, and fresh culture medium was added. BSO (50 µM) was added together with the rmTNF-alpha . All of the parameters were measured 12 h after cytokine addition. The data are the means ± S.D. for five or six independent experiments.

To improve its therapeutic efficacy, nontoxic TNF-alpha doses could be combined with other cytokines (e.g. IFN-gamma ) (43), thiol-depleting agents such as BSO or DEM (7, 48), or combined with a glutamine-enriched diet that may facilitate a glutamate-induced inhibition of GSH transport into tumor mitochondria (11). Thus, the present results may have applications in the therapy of metastatic tumors.

Mitochondrial Function and Morphology in iB16M Cells-- Obviously, interaction with the HSE may also alter mitochondrial functions and/or morphology in surviving B16M cells. To investigate this question different mitochondrial function-related parameters were analyzed in growing iB16M cells (Table VII). Interaction with endothelial cells causes a decrease of activity of respiratory complexes II (succinate dehydrogenase), III (cytochrome c reductase), and IV (cytochrome c oxidase) and, as a consequence, a decrease in the rate of O2 consumption (measured in intact cells in vitro at 37 °C with a Clark-type electrode in the presence of 5 mM glucose and 5 mM L-glutamine) and in cytosolic and mitochondrial ATP levels (Table VII). Our results appear partially in agreement with previous data identifying respiratory complexes I and III as mitochondrial sites of damage in hematopoietic progenitor cells exposed to ionizing radiation and NO (49) or demonstrating how astrocytic NO/-ONOO causes damage to the activities of complexes II, III, and IV of neighboring neurons (50). Inhibition of respiratory enzyme activities, as previously postulated, may cause an increase in oxygen free radical formation from mitochondrial complex I (51). Indeed, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and H2O2 generation increases significantly in iB16M cells as compared with LD B16M cells (Table VII). These effects associated with a decrease in the MMP in intact iB16M cells (Table VII). Changes in cell volume and the plasma membrane potential can affect TPMP accumulation by whole cells (28). However, we did not find significant changes in the cell volume when LD B16M and iB16M cells were compared (e.g. 4.0 ± 0.3 µl/mg dry weight in LD B16M cells, n = 4). Moreover, plasma membrane potential measured in growing iB16M cells (0, 18, or 48 h after plating) were similar (e.g. 47.5 ± 2.2 mV at 48h, n = 5). Therefore changes in TPMP accumulation are due to mitochondria alone. Furthermore, electron microscopy of both types of B16M cells showed normal mitochondrial shapes without swelling or damage of the mitochondrial crests or other structures (not shown). Despite the transient fall in different mitochondrial function-related parameters (reaching maximum values ~18 h after plating) (Table VII), growing iB16M cells maintained their viability in vitro above 98% (not shown), and 48 h after plating showed no significant differences (as compared with LD B16M cells) in all of the parameters studied (Table VII). These results indicate that iB16M cells were able to repair the HSE-induced mitochondrial damages.


                              
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Table VII
Mitochondrial function-related parameters in growing iB16M cells
MMP, mitochondrial volume, O2 consumption, H2O2 and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation, and ATP contents were determined in intact cells, whereas specific activity of respiratory enzymes was measured using isolated mitochondria as described under "Experimental Procedures." The data are the means ± S.D. for four or five independent experiments. cyt, cytosolic; mt, mitochondrial.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GSH protects highly metastatic B16M cells against nitrosative and oxidative stress in the murine hepatic microvasculature (6, 8). Indeed, metastatic growth can be implemented in these cells by directly increasing their GSH content with GSH ester (10). Endothelial cell-derived RNS and ROS caused cytGSH and mtGSH depletion and a decrease in gamma -GCS activity in B16M cells that survived after interacting with the HSE (Table I). Direct exposure of LD B16M cells to NO, but not to H2O2, decreased gamma -GCS activity (see under "Results"). This appears to be in agreement with previous observations showing that rat gamma -GCS is inactivated by S-nitrosylated metabolites such as S-nitroso-L-cysteine and S-nitroso-L-cysteineglycine, although not by S-nitroso-GSH (40). In contrast, an NO-dependent induction of GSH synthesis through increased expression of gamma -GCS has also been reported (52); however, this effect required nanomolar concentrations of NO (1-3 nM/s), which can only be expected under physiological conditions but not in the metastatic microenvironment (6). This is not surprising because, as recently discussed (6), experiments in isolated mitochondria showed that NO reversibly inhibited permeability transition pore opening with an IC50 of 11 nM, although at higher concentrations (>2 µM) NO accelerated pore opening; similarly, low levels of H2O2 (3-5 µM) may cause a mitogenic response, whereas higher concentrations (>100 µM) may cause growth arrest and cell damage.

On the other hand, the surviving cell subset (iB16M cells), when cultured, showed a rapid gamma -GCS overexpression-associated (Table II) increase of cytGSH levels (Fig. 1). Interestingly, our findings suggest that H2O2 promotes gamma -GCS overexpression (see under "Results"). This could be especially relevant because signaling through NF-kappa B is involved in the oxidative stress-mediated regulation of gamma -GCS-HS expression (53) and because NF-kappa B activity correlates with growth and metastasis of human melanoma cells (54). In addition, mtGSH further decreased before recovering during the initial culture period (approximately 18 h) (Fig. 2). In fact, HSE-derived NO and H2O2 can damage the ATP-dependent mitochondrial system for cytGSH uptake (Fig. 2 and Tables IV and V), and the following experimental facts support directly and/or indirectly this conclusion: (a) cytosolic and mitochondrial GSH contents are similar in LD B16M cells co-cultured with HSE cells or directly exposed to H2O2 and NO (Table I); (b) after interaction with HSE cells during the first 18 h, cytGSH increases, whereas mtGSH decreases within LD B16M cells (Fig. 1); (c) there was a decrease in GSH uptake by iB16M mitochondria as compared with LD B16M mitochondria (Fig. 2); (d) rates of GSH uptake by tumor mitochondria are similar under conditions where cytGSH values are different: high in controls and low in BSO- and DEM-treated cells (approximately 20% of controls) (Table III); (e) mitochondrial GSH transport in LD B16M cells attached to the HSE is similar to controls in the presence of L-NAME and catalase (a fact that correlates with NOx and H2O2 values) (Table IV); and (f) direct exposure of LD B16M cells to H2O2 and NO (Table V) decreases cytGSH levels to values (approximately 6 mM) that cannot limit per se GSH transport into mitochondria (see also Km values above).

Mitochondrial dysfunction is a common event in the mechanisms leading to cell death (55), and recently it has been found to be an essential step for killing non-small cell lung carcinomas resistant to conventional treatments (56). Mitochondrial permeability transition is critical in the process leading to apoptosis, and it is linked to the opening of a permeability transition pore complex (57). This molecular gate is regulated by many endogenous factors, including divalent cations (e.g. Ca2+ and Mg2+), protons, the MMP, the concentration of adenine nucleotides, the thiol (controlled by GSH) and the pyrimidine redox state, the rate of ROS and NO generation, the concentration of lipoids (e.g. ceramide), the concentration of certain peptides targeting proteins for mitochondrial import, and the function of different pro- and anti-apoptotic proteins (57). GSH, which is not synthesized by mitochondria but taken up from the cytosol through a multicomponent transport system (20), is the only defense against peroxides generated from the electron transport chain (58) and may be an important regulator of the mitochondrial permeability transition and permeability transition pore opening (57, 59, 60). Thus, impairment of GSH uptake by mitochondria (Fig. 2) may be important to sensitize invasive cells to molecular effectors (e.g. oxidative stress inducers) capable of activating the mitochondrion-based death mechanism.

Direct evidence for TNF-alpha -induced mitochondrial ROS and their involvement in DNA damage (61) and cytotoxicity (45) was obtained using the murine fibrosarcoma cell line L929. In addition, we observed in Ehrlich ascites tumor cells that mtGSH depletion facilitates activation of the apoptotic cascade upon TNF-alpha stimulation (11). In agreement with these findings, when TNF-alpha was added to growing iB16M during the immediate period following their interaction with the HSE, ROS production was increased, mtGSH was further depleted, and the cell number and viability decreased (Table VI). Moreover, prevention of TNF-alpha -induced B16M cytotoxicity by GSH ester (Table VI) proved that the tripeptide is directly involved in regulating mitochondrion-based death mechanisms.

The prevalent mode of iB16M cell death under different conditions is an open question now being studied in our laboratory, although it is important to remark that mtGSH or ATP depletion to very low levels may cause a bioenergetic catastrophe (57) changing the mode of cell death from apoptosis to necrosis. Meanwhile, our present results indicate that approaches capable of maintaining low mtGSH levels in invasive cells may be important to challenge their survival during macrophage attack or when cancer therapy is applied. Interestingly, it has been shown in hepatoma cells that Kupffer cells have anti-tumor activity through NO-mediated mitochondrial damage to tumor cells (62).

GSH, in addition to regulating mitochondrial pore opening, could also regulate invasive cell growth and survival at other steps. Disulfide formation activates OxyR transcription factor, whereas inactivation occurs via reduction by glutaredoxin 1 (a transcriptional target of OxyR) (63). In addition, other transcription factors (including NF-kappa B, AP-1, and p53) are also sensitive to redox changes affecting their DNA-binding domains (64), which suggests that GSH may be an important transcriptional regulator. Moreover, a recent report, in which the role of GSH in the growth of HepG2 cells was studied, showed that changes in cell growth and DNA synthesis paralleled changes in GSH levels, suggesting a causal relationship between the two (65). Hypothetically, GSH could also regulate (a) DNA synthesis by providing reducing equivalents to glutaredoxin and/or thioredoxin, both necessary for ribonucleotide reductase, or (b) mechanisms of genomic surveillance, e.g. cell cycle checkpoints systems (66). Furthermore, GSH levels in invasive cells could also be affected by microenvironment conditions, e.g. cysteine availability (10), heavy metals, heat shock, high glucose, NO, or oxidants (67); and intercellular signals, e.g. GSH synthesis in hepatocytes is down-regulated in response to stress-related hormones (68) through a Ca2+-dependent, protein kinases A and C pathway that causes gamma -GCS inhibition by direct phosphorylation of this enzyme (69). On the other hand, invasive cells may benefit of oxidative stress-promoting metastatic mechanisms, e.g. increasing cell adhesion molecule expression (70), activation of early growth response-1 transcription factor gene (71), activation of metalloproteinases (72), or increasing resistance to oxidative stress (73, 74). In fact in mammalian cells at least 40 different gene products are involved in adaptive responses to oxidative stress (74). Also, a growing body of evidence suggests that many cellular responses to oxidative and nitrosative stress are regulated at the transcriptional level (75). Nitrosylation or oxidation of critical cysteine residues in the DNA-binding domains or at allosteric sites may regulate transcription of target genes (75), although up to now the molecular mechanisms underlying redox control of mammalian gene expression have not been elucidated in any well defined cellular system. Therefore, a complex balance between pro- and anti-metastatic ROS and RNS effects may underlie the progression of invasive cells within a tissue. Nevertheless, availability of microarray technology makes feasible large scale multigene expression analysis, which might represent a powerful approach to elucidate potential RNS- and ROS-induced adaptive responses in selected invasive cells, and is likely to explain the mechanisms by which they resist anti-cancer treatments. Indeed, gene expression patterns in apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma reveal that the multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential (38). On the other hand, cells resistant to apoptosis down-regulate these biochemical pathways while elevating their GSH content (38). In fact, these properties may likely apply in our model because, as we have shown here, surviving iB16M cells increase their GSH content after interacting with the HSE (Fig. 1). In conclusion, the biochemical mechanisms described in this report indicate that mitochondria and modulation of mtGSH in particular might be novel targets in anticancer therapy.

    FOOTNOTES

* This work was supported by Comisión Interministerial de Ciencia y Tecnología Grants SAF99-112 and 1FD97-548 and Generalitat Valenciana Grant GV01-140 (Spain).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a fellowship from the Ministerio de Ciencia y Tecnología (Spain).

Supported by a fellowship from the Fundación Científica de la Asociación Española contra el Cáncer.

** To whom correspondence should de addressed: Dept. de Fisiología, Fac. de Medicina y Odontología, Av. Blasco Ibáñez 17, 46010 Valencia, Spain. Tel.: 96-3864646; Fax: 96-3864642; E-mail: jose.m.estrela@uv.es.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M207140200

    ABBREVIATIONS

The abbreviations used are: B16M, B16 melanoma; LD cells, cells cultured to low density; HSE, hepatic sinusoidal endothelium; iB16M, invasive B16 melanoma; gamma -GCS, gamma -glutamylcysteine synthetase; cytGSH, cytosolic GSH; mtGSH, mitochondrial GSH; gamma -GCS-HS, gamma -GCS heavy subunit; gamma -GCS-LS, gamma -GCS light subunit; BSO, L-buthionine (S,R)-sulphoximine; DMEM, Dulbecco's modified Eagle's medium; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-and-6-carboxyfluorescein acetoxymethylester; MMP, mitochondrial membrane potential; TPMP, methyl-triphenylphosphonium; MCB, monochlorobimane; ROS, reactive oxygen species; RNS, reactive nitrogen species; DEM, diethylmaleate; TNF, tumor necrosis factor; IFN, interferon; PBS, phosphate-buffered saline; eNOS, endothelial nitric-oxide synthase; MOPS, 4-morpholinepropanesulfonic acid; L-NAME, Nw-nitro-L-arginine methyl ester; rmTNF, recombinant murine TNF.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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