From the 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
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ABSTRACT |
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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 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 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- eNOS-deficient Mice--
Generation of eNOS-deficient mice was
carried out as previously described (6, 13). We interbred heterozygous
(+/ 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.
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-( 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
(
Succinate-CoQ oxidoreductase (complex II) was assayed by following the
reduction of 2,6-dichlorophenolindophenol at 600 nm (
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 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 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 M
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, 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.
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-
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 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).
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).
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
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
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). Mitochondrial GSH Depletion Increases TNF-
To improve its therapeutic efficacy, nontoxic TNF- 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/ 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 On the other hand, the surviving cell subset (iB16M cells), when
cultured, showed a rapid 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- 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--glutamylcysteine synthetase activity in iB16M cells.
Overexpression of
-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
-induced oxidative stress and death.
Therefore depletion of mtGSH levels may represent a critical target to
challenge survival of invasive cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ONOO radicals, via a
trace metal-dependent process (6). A high percentage of
B16M cells with high GSH (
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2 × 107 units/mg protein) and recombinant murine interferon-
(IFN-
; 105 units/mg protein) were obtained from Sigma.
Stock solutions (5 × 105 units of TNF-
/ml and
25 × 104 units of IFN-
/ml) were diluted in sterile
physiological saline solution (0.9% NaCl), adjusted to pH 7.0, and
stored at 4 °C.
) 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).
-Glutamylcysteine synthetase (
-GCS) and GSH synthetase activities
were measured as described elsewhere (10). GSH transferase activity was
determined as previously described (9).
-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 (
-GCS heavy subunit
(
-GCS-HS): forward, 5'-ATC CTC CAG TTC CTG CAC ATC TAC, and reverse,
5'-GAT CGA AGG ACA CCA ACA TGT ACTC;
-GCS light subunit
(
-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
-GCS-HS and
-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
(
CT), where
CT = CT target
CT glyceraldehyde-3-phosphate dehydrogenase, and
(
CT) =
CT
treated
CT control.
S,5S)-
-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; an irreversible inhibitor of
-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.
= 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.
= 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.
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.
-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
-ketoglutarate uptake by isolated tumor mitochondria were performed as previously described (see Ref. 39 and references therein).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IFN-
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).
GSH and GSSG contents and GSH synthesis-related enzyme activities in
B16M cells that survive after interaction with the HSE in vitro
/
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-
(100 units/ml) and IFN-
(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.
-GCS activity (Table I), the rate-limiting enzyme in GSH
synthesis (7). Direct exposure of LD B16M cells to
H2O2 did not affect
-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
-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
-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
-GCS-HS and
-GCS-LS in the immediate period
(maximum values were found at 3 h) following interaction with the
HSE (Table II). Expression of
-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
-GCS expression
in iB16M cells. As shown in Fig. 1,
-GCS overexpression is associated with a rapid increase in cytGSH in
iB16M cells within the 6-18-h period of growth in vitro
(
-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.
-GCS overexpression in iB16M cells
-GCS-HS and
-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).
View larger version (18K):
[in a new window]
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).
View larger version (20K):
[in a new window]
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,
; and 48 h after plating,
). 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.
Effect of DEM- and BSO-induced GSH depletion on GSH transport into LD
B16 mitochondria
-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
-ketoglutarate carrier (phenylsuccinate, 1 mM),
which decreases
-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).
/
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-
and IFN-
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.
NO- and H2O2-dependent damage of the
mitochondrial GSH transport in LD B16M cells attached to the HSE
and IFN-
(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.
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
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).
Nitrogen and/or oxygen-derived reactive species damage the
mitochondrial GSH transport system in intact LD B16M cells
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.
-induced Oxidative
Stress and iB16M Cell Death--
TNF is a macrophage/monocyte-derived
cytokine with cytostatic and cytotoxic anti-tumor effect (43). TNF-
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-
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-
-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-
-induced ROS generation, and completely abolished the
deleterious effect of TNF-
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-
-induced B16M cytotoxicity.
Response of iB16M cells to treatment with rmTNF- in vitro
(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-
. All of the parameters were measured 12 h after
cytokine addition. The data are the means ± S.D. for five or six
independent experiments.
doses could be
combined with other cytokines (e.g. IFN-
) (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.
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
Mitochondrial function-related parameters in growing iB16M cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
-GCS activity
(see under "Results"). This appears to be in agreement with
previous observations showing that rat
-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
-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.
-GCS overexpression-associated (Table II)
increase of cytGSH levels (Fig. 1). Interestingly, our findings suggest
that H2O2 promotes
-GCS overexpression (see under "Results"). This could be especially relevant because
signaling through NF-
B is involved in the oxidative stress-mediated
regulation of
-GCS-HS expression (53) and because NF-
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).
-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-
stimulation (11). In agreement
with these findings, when TNF-
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-
-induced B16M cytotoxicity by GSH ester (Table VI) proved that
the tripeptide is directly involved in regulating mitochondrion-based
death mechanisms.
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
-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.
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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
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ABBREVIATIONS |
---|
The abbreviations used are:
B16M, B16 melanoma;
LD cells, cells cultured to low density;
HSE, hepatic sinusoidal
endothelium;
iB16M, invasive B16 melanoma;
-GCS,
-glutamylcysteine synthetase;
cytGSH, cytosolic GSH;
mtGSH, mitochondrial GSH;
-GCS-HS,
-GCS heavy subunit;
-GCS-LS,
-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.
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REFERENCES |
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