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J. Biol. Chem., Vol. 276, Issue 28, 25775-25782, July 13, 2001

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Tumoricidal Activity of Endothelial Cells

INHIBITION OF ENDOTHELIAL NITRIC OXIDE PRODUCTION ABROGATES TUMOR CYTOTOXICITY INDUCED BY HEPATIC SINUSOIDAL ENDOTHELIUM IN RESPONSE TO B16 MELANOMA ADHESION IN VITRO^*

Julian Carretero^§, Elena Obrador^¶, Juan M. Esteve, Angel Ortega, José A. Pellicer, Francisco Vera Sempere^**, and José M. Estrela

From the  Departamento de Fisiología, Universidad de Valencia, and the ^** Servicio de Anatomía Patológica, Hospital Universitario La Fe, Valencia, Spain

Received for publication, February 6, 2001, and in revised form, April 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanism of NO- and H₂O₂-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N^G-nitro-L-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H₂O₂ was removed by exogenous catalase. H₂O₂ was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H₂O₂ due to the formation of potent oxidants, likely ^·OH and OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H₂O₂ than B16M cells cultured to high density (HD cells; with ~25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H₂O₂ were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H₂O₂. NO and H₂O₂ caused an ~75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H₂O₂. In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H₂O₂.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interaction of cancer and endothelial cells in capillary beds is a critical step in the initiation of metastasis (1). Early studies on the organ distribution of B16 melanoma (B16M)¹ cells showed that <0.1% of circulating cells survive and may promote secondary metastatic growth (2). The liver is a common site for metastasis development, and it has been shown that, under experimental conditions, a high percentage of circulating cancer cells are mechanically trapped in the liver microvasculature (3). Interaction of metastatic cancer cells with the hepatic sinusoidal endothelium (HSE) and Kupffer cells activates local release of pro-inflammatory cytokines, which can promote cancer cell adhesion, invasion, and proliferation (4-6). Indeed, interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) induce binding of very late antigen-4-expressing mouse B16M cells by up-regulating vascular adhesion molecule-1 on endothelial cells (6, 7) and thereby facilitate metastatic growth (8, 9). In addition, endothelial cells also release harmful reactive oxygen species in response to endotoxins and cytokines or in response to cancer cell contact (10-12). However, at low micromolar levels, these reactive species appear to act as intra- and intercellular messengers capable of promoting growth responses (13). In fact, vascular adhesion molecule-1 gene transcription and expression in vascular endothelial cells are coupled to an oxidative stress-dependent mechanism (14). Therefore, a balanced process between pro- and anti-metastatic reactive oxygen species effects may underlie the interaction of metastatic cells with the vascular endothelium.

According to this idea, it has been shown that IL-1-dependent HSE production of H₂O₂ contributes to melanoma cell adhesion enhancement, which would compensate for its direct cytotoxic effects on adherent, vulnerable melanoma cells and lead to the metastatic progression of H₂O₂-resistant melanoma cells (6). The involvement of H₂O₂ in cancer cell cytotoxicity is supported by experiments in which the hepatic metastasizing ability of B16M cells, following intrasplenic inoculation of IL-1-treated mice, was reduced by administration of exogenous catalase or enhanced by exogenous superoxide dismutase (6). Nevertheless, to date, data reported on H₂O₂ released by the vascular endothelium during the process of cancer cell adhesion regard to relative values. Quantitative determination of H₂O₂ levels is necessary to assess whether H₂O₂ induces tumor cytotoxicity by itself or requires some additional factor(s).

Elegant studies by Fidler and co-workers (15) showed that NO, the formation of which is catalyzed by constitutive NO synthetase or inducible NO synthetases expressed in many cells, promotes lysis of tumor cells by cytokine-activated endothelial cells. Interestingly, an inverse correlation between expression of inducible NO synthase activity and production of metastasis in murine melanoma cells was found (16). In addition, recent results have identified the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation (17). Although the biochemical mechanisms by which NO elicits its cytotoxic action are not well understood, it has been proposed that, for example, NO inactivates iron-containing prosthetic groups, a mechanism that leads to inhibition of the mitochondrial respiratory chain and DNA synthesis (18), and that NO reacts very rapidly with O₂, which results in formation of the highly oxidant peroxynitrite (19). Moreover previous reports have shown cooperative cytotoxic action of nitric oxide and H₂O₂ in, for example, Escherichia coli (20) and Fu5 rat hepatoma cells (21). Therefore, it is plausible that endothelial NO and H₂O₂ or their derived reactive species may have synergistic cytotoxic effects on cancer cells. Interestingly, reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion have been recently involved in the killing of weakly (but not highly) metastatic colorectal cancer cells (22). In addition, we have shown that GSH, which is involved in cell defense against nitrosative and oxidative stress (23), protects circulating B16M cells against HSE-induced cytotoxicity (24). In the present work, we have used endothelial nitric-oxide synthetase (eNOS) gene disruption and N^G-nitro-L-arginine methyl ester (L-NAME)-induced inhibition of NO production to study the role of endothelium-derived NO in the killing of B16M cells. In these studies, we have compared B16M cells cultured to low (LD) and high (HD) densities, which have different GSH contents and different metastatic activities (25). Our results show that H₂O₂ released by the HSE was not cytotoxic; however, NO was particularly tumoricidal in the presence of the peroxide. A high percentage of tumor cells with high GSH content survived the combined nitrosative and oxidative attacks and may represent the main task force in metastatic invasion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Culture of B16M Cells-- B16M cells derived from B16-F10 subline cells were cultured (24) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), pH 7.4, supplemented with 10% fetal calf serum (Life Technologies, Inc.), 10 mM HEPES, 40 mM NaHCO₃, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Isolation, Identification, and Culture of the Hepatic Sinusoidal Endothelium-- Male C57BL/6J mice (10-12 weeks old) were from the Institut Français pour la Fiévre Aphtose Credo (L'Arbreole, France). All animals received care according to the criteria outlined by the National Institutes of Health (Rockville, MD). The HSE was separated and identified as previously described (26). Tissue digestion was carried out by sequential perfusion of type E Pronase and collagenase A (Roche Molecular Biochemicals, Mannheim, Germany) plus Pronase solutions. The liver was then minced and stirred in another solution containing Pronase, collagenase, and DNase (type I deoxyribonuclease, Sigma). Sinusoidal cells were separated in a 17.5% (w/v) metrizamide gradient. Cultures of the HSE were established and maintained in pyrogen-free Dulbecco's modified Eagle's medium supplemented as described above for the B16M cells. Differential adhesion of endothelial cells to the collagen matrix and washing allow complete elimination of other sinusoidal cell types (Kupffer, stellate, and lymphocytes) from the culture flasks.

B16 Melanoma-Endothelial Cell Adhesion Assay-- This assay was based on a previously published methodology (6). Briefly, B16M cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Inc., Eugene OR) (10⁶ cells were incubated in 1 ml of HEPES-buffered Dulbecco's modified Eagle's medium 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 Dulbecco's modified Eagle's medium 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. This was measured using a Fluoroskan Ascent FL (Labsystems, Manchester, United Kingdom). 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.

In Vitro Cytotoxicity Assays-- Damage to B16M cells during their in vitro adhesion to the HSE was measured, as previously described (24), using tumor cells loaded with calcein-AM (Molecular Probes, Inc.). Integrity of B16M cells cultured alone was assessed by trypan blue exclusion and by measuring lactate dehydrogenase activity (27) released to the extracellular medium (24). Other reagents used in experiments of tumor cytotoxicity were from Sigma (L-NAME, catalase, and superoxide dismutase).

Cytokines-- Recombinant murine TNF- (2 × 10⁷ units/mg of protein), recombinant murine IL-1 (20 µg/mg of protein), and recombinant murine interferon- (IFN-; 10⁵ units/mg of protein) were obtained from Sigma. Stock solutions (5 × 10⁵ units/ml TNF-, 100 ng/ml IL-1, and 25 × 10⁴ units/ml IFN-) were diluted in sterile physiological saline solution (0.9% NaCl), adjusted to pH 7.0, and stored at 4 °C.

Measurement of H₂O₂, Nitrite, and Nitrate-- The assay of H₂O₂ production was based on the H₂O₂-dependent oxidation of homovanillic acid (4-hydroxy-3-methoxyphenylacetic acid) to a highly fluorescent dimer (2,2'-dihydroxydiphenyl-5,5'-diacetic acid), which is mediated by horseradish peroxidase (28). For this purpose, cells were cultured in the presence of 100 µM homovanillic acid and 1 unit/ml horseradish peroxidase. A linear relationship between fluorescence (_ex = 312 nm and _em = 420 nm) and amount of H₂O₂ was found in the range of 0.1-12 nmol/2-ml assay.

Nitrite and nitrate determinations were performed using the methodology of Braman and Hendrix (29). Briefly, measurement of NO levels was made by monitoring NO evolution via chemiluminescence detection from a measured sample placed into a refluxing solution of iodide/acetic acid (which will reduce only NO, but not NO, to NO). Total NO plus NO (NO_x) determinations were made by monitoring NO evolution from a measured sample placed into a boiling VCl₃/HCl solution (which will reduce both NO and NO to NO). The determination of NO levels was made by subtracting the NO value from the NO_x value. Quantitation was accomplished using a standard curve made up of known amounts of NO and NO.

Measurement of GSH, GSSG, and S-Nitrosothiols-- For GSH determination, cells were treated at 4 °C with ice-cold perchloric acid (2%). Samples were centrifuged at 15,000 × g for 5 min at 4 °C and GSH was measured in the supernatants using the GSH S-transferase reaction as previously described (30).

For GSSG analysis, cells were treated at 4 °C with ice-cold perchloric acid (12%) containing 40 mM N-ethylmaleimide (Sigma) and 2 mM bathophenanthrolinedisulfonic acid (Sigma). Samples were centrifuged at 15,000 × g for 5 min at 4 °C and the acidic supernatants were derivatized and analyzed by high performance liquid chromatography as previously reported (31).

The acid-soluble fractions (200 µl) obtained for GSH determination (see above) were added to 25 µl of 2.0% ammonium sulfamate. To the mixtures were added 200 µl of 0.5 N HCl containing 0.25% HgCl₂ and 5% sulfanilamide. Then, 250 µl of a 0.5 N HCl solution containing 0.25% N-(1-naphthyl)ethylenediamide dihydrochloride were added to the mixtures. After incubation for 30 min at 25 °C, the amount of S-nitrosothiols (RSNO) was determined spectrophotometrically at 550 nm using S-nitrosoglutathione (GSNO) as a standard (32). Fresh GSNO solution was obtained, before each experiment, by incubating equimolar GSH and sodium nitrite in acidified water at 0 °C as previously described (33). GSNO concentration was determined spectrophotometrically at 335 nm ( = 992 dm³ × mol¹ × cm¹) and at 545 nm ( = 15.9 dm³ × mol¹ × cm¹). No significant decomposition of GSNO occurred during the assay procedures.

Preparation of GSH Ester-- GSH monoisopropyl(glycyl) ester was obtained as previously described (34).

Preparation of NO Solution-- Thionitrobenzoic acid was used to characterize the stability of a NO solution obtained by bubbling a 50 mM HEPES-NaOH buffer (pH 7.4) with NO gas as previously described (35). Levels of NO were measured as previously reported (36) using a Sievers NOA 280A chemiluminescence analyzer. Briefly, samples (100 µl) were injected into a nitrogen purge vessel containing a 1% solution of sodium iodide in glacial acetic acid to liberate gaseous NO from dissolved NO and nitrite. The sample gas was then exposed to ozone to form activated nitrogen dioxide (NO₂*), which luminescences in the red and far-red spectrum and is detected by a red-sensitive photomultiplier tube. For calculations, we used a calibration curve obtained by measuring a series of sodium nitrite standards. The saturated NO solution (2 mM) was stored at 4 °C and used for experiments within 15 min. During this time period, NO concentration in stock solutions remained unchanged.

Preparation of Peroxynitrite Solution-- Peroxynitrite was prepared from amyl nitrite and H₂O₂ following the methodology described by Uppu and Pryor (37). Amyl nitrite (20 ml) was added to 80 ml of 2 M H₂O₂, 2 M NaOH, and 2 mM DTPA, and the solution was stirred for 2 h at 4 °C. The lower aqueous layer was removed and washed three times with ice-cold hexane. Residual H₂O₂ was removed by gradually adding 10 g of manganese dioxide to the OONO solution while it was being stirred on ice for 1 h. The solution was filtered and passed through a 10 × 1-cm MnO₂ column equilibrated with 0.1 M NaOH to remove traces of H₂O₂. The OONO was diluted with 0.1 N NaOH to ~50 mM immediately before use. The concentration of OONO was measured spectrophotometrically (extinction coefficient of 1670 M¹ × cm¹ at 302 nm). Nitrite contamination of the OONO solution was ~20% of the OONO concentration (less than that reported in the original method (37)). Addition of catalase to decomposed peroxynitrite at pH 7.0 resulted in no oxygen evolution (measured at 37 °C with a Clark-type electrode), which implies no H₂O₂ contamination in the solution.

Measurement of Antioxidant Enzyme Activities-- B16M cells were detached (see above) and washed twice at 4 °C in Krebs-Henseleit bicarbonate medium (pH 7.4) without Ca²⁺ or Mg²⁺ added and containing 0.5 mM EGTA and then resuspended in ice-cold lysis buffer (0.1 M phosphate buffer (pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin A (Sigma)) at a density of ~15 × 10⁶ cells/ml. Cells were disrupted by sonication (Braun Labsonic; 50% output, three times for 10-s each), and the suspension was centrifuged at 12,000 × g for 15 min at 4 °C. GSH reductase activity (30), GSH peroxidase (selenium-dependent) activity (using H₂O₂ as a substrate) (38), superoxide dismutase activity (39), and catalase activity (40) were measured in the supernatants as described previously.

eNOS-deficient Mice-- Generation of eNOS-deficient mice was carried out as previously described (41). 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 as previously described (41). Briefly, 20-µg samples were digested with BamHI, separated on 1.0% agarose gels, and then transferred to nylon-supported nitrocellulose. The blots were hybridized using a random primer-labeled 1.4-kilobase pair eNOS cDNA probe (41). A 5.3-kilobase pair fragment was diagnostic of the endogenous eNOS locus, and a 6.4-kilobase pair fragment was diagnostic of the targeted allele.

Immunohistochemical Localization of eNOS-- Livers were rapidly removed and embedded in Miles Tissue-Tek OCT compound (Sciscope, Iowa City, IA) for preparing 5-µm frozen sections. Sections were fixed with acetone (20 °C at the start) for 10 min at 4 °C and washed twice with PBS containing 0.1 g of bovine serum albumin/100 ml at room temperature. Sections were then incubated with 0.25% H₂O₂ in methanol for 30 min at room temperature to block endogenous peroxidase activity and washed again with PBS containing 0.1% bovine serum albumin. Nonspecific antibody binding was blocked by incubation for 3 h with PBS containing 5% bovine serum albumin. Sections were then washed twice with PBS containing 0.1% bovine serum albumin and incubated for 1 h with a mouse anti-eNOS antibody (diluted 1:100; kit 482731, Calbiochem). After sections were washed for 15 min with PBS, biotinylated goat anti-mouse immunoglobulin G (kit 4002, Vector Labs, Inc., Burlingame, CA) was applied for 30 min. Sections were washed and developed with the Vectastain ABC system (Vector Labs, Inc.) using tetrahydrochloride. Sections were counterstained with hematoxylin and examined by light microscopy for staining of eNOS (dark-purple color), which was positive in wild-type and eNOS^+/+ mice and completely negative in eNOS^/ mice (data not shown).

Statistical Analyses-- Data were analyzed by Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In Vitro NO and H₂O₂ Generation by Cytokine-stimulated HSE-- The incubation of endothelial cells with cytokines can induce the production of different mediators, among which are NO (15) and H₂O₂ (12). Rolling and early adhesion of B16M cells to the HSE, release of H₂O₂ by the HSE, and late adhesion of surviving melanoma cells are sequential steps during B16M cell attachment to the HSE that occur in a short period (3-6 h) (6). In the first set of experiments, to determine the rates of NO and H₂O₂ generated by the HSE, freshly isolated sinusoidal cells were treated with different concentrations of TNF-, IL-1, and/or IFN- (Table I). All these cytokines have been shown to be capable of activating endothelial cells (12, 15). In preliminary experiments, we identified the optimal concentrations of the cytokines to be 100 units/ml TNF-, 1 ng/ml IL-1, and 50 units/ml IFN- (data not shown). We found that NO_x and H₂O₂ accumulated in the culture medium in a time-dependent fashion and that NO_x and H₂O₂ production was maximal in the presence of TNF- and IFN- (Table I).

NO and H₂O₂ Levels during B16M Adhesion to the HSE in Vitro-- In the second set of experiments, we used an in vitro B16 melanoma-endothelial cell adhesion assay to investigate NO and H₂O₂ production during the process of tumor cell attachment to the HSE. In these experiments, the combination of TNF- and IFN- was used as a potent activator of NO and H₂O₂ generation by the HSE (Table I) and also by the melanoma cells (16). For comparison, IL-1 was also used. B16M cells obtained from cultures with low cellular density (LD cells, which have high GSH content and high metastatic activity in vivo) or from cultures with high cellular density (HD cells, which have lower GSH content and metastatic activity) (25) were independently co-cultured with HSE cells, and the results were compared (Table II). In these experiments, before addition to the HSE, GSH content in the B16M cells was 37 ± 6 nmol/10⁶ LD cells and 10 ± 3 nmol/10⁶ HD cells (n = 5-6 in both cases). The percentage of tumor cell adhesion to the HSE was not significantly different when both types of B16M cell subsets were compared (e.g. 47.6 ± 6.5% in the case of HD B16M cells, n = 7). To calculate the percentage of NO_x produced by the HSE during the adhesion process, in parallel experiments, we used HSE cells pre-cultured for 12 h in the presence of 1 mM L-NAME, which blocks NO synthesis activity (42, 43) (under these conditions, after washing with medium and culturing in the absence of tumor cells, HSE cells do not produce detectable amounts of NO or NO in the following 4 h). Similar results were obtained when NO_x produced by the HSE was calculated by subtracting NO_x produced by co-cultured HSE and B16M cells from NO_x produced by cultured B16M cells alone (data not shown). The percentage of H₂O₂ generated by the HSE was calculated by subtracting H₂O₂ produced by co-cultured HSE and B16M cells from H₂O₂ produced by cultured B16M cells alone (data not shown). Our data show that, during the adhesion process, most of the NO and H₂O₂ were generated by the HSE (Table II). Pre-culture of endothelial cells in the presence of L-NAME did not alter the percentage of B16M cell adhesion (data not shown) as compared with controls (see above).

HSE-induced Tumor Cytotoxicity-- The specific NO- and/or H₂O₂-induced tumor cytotoxicity during the adhesion process was studied in co-cultures of HSE and B16M cells (Table III). We used HSE cells isolated from eNOS-deficient (eNOS^/) mice to abolish eNOS-dependent NO production and L-NAME to inhibit both inducible and endothelial NO synthase activities. Addition of exogenous catalase was used to eliminate H₂O₂ released to the extracellular medium. LD and HD B16M cell subsets where compared in the absence or presence of TNF- and IFN-. As shown in Table III, HSE-induced tumor cytotoxicity was significantly higher in B16M cells pre-cultured to high density, a fact that is in agreement with our previous observations (24, 25). In addition, we observed that a high rate of NO_x and H₂O₂ accumulation (as occurs in the presence of inducing cytokines) correlated with an increasing percentage of nonviable tumor cells (Table III). Tumor cytotoxicity decreased significantly when B16M cells were co-cultured with eNOS^/ HSE cells, where only the rate of NO_x accumulation decreased (Table III). Moreover, L-NAME treatment of eNOS^+/+ HSE cells completely abolished NO_x accumulation and tumor cell death without altering H₂O₂ accumulation (Table III). These facts indicate that H₂O₂ is not cytotoxic by itself or that it requires additional factors, likely NO, to promote irreversible tumor cell damage. When B16M cells where co-cultured with eNOS^+/+ HSE cells in the presence of exogenous catalase, which decreases H₂O₂ accumulation to very low levels (10% or less of control values), only in the case of HD B16M cells was a significant decrease in the percentage of tumor cytotoxicity observed (Table III).

View this table: [in this window] [in a new window]   Table III NO- and H2O2-dependent cytotoxicity in B16M cells attached to the HSE eNOS-deficient mice were generated as described under "Experimental Procedures." HSE cells isolated from wild-type, eNOS+/+, or eNOS/ mice were co-cultured with B16M cells as described in legend to Table II. TNF- and IFN- (concentrations were identical to those used in Table I) or vehicle (physiological saline) was added, and determination of NOx and H2O2 accumulation was restricted to co-cultures in which all tumor cells were attached to HSE cells. In these experiments, 1 mM L-NAME and 103 units/ml catalase were used (see "Results"). 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). Tumor cytotoxicity (expressed as the % of tumor cells that lost viability within the 4-6-h period of incubation, see "Experimental Procedures") was determined after 6 h of incubation. During the 6-h period of incubation, the percentage of HSE cell viability was 99-100% in all cases. Data are means ± S.D. of five to six different experiments. ND, not detectable.

To investigate whether these effects are tumor GSH content-related, we used, in parallel experiments, LD B16M cells pre-cultured in the presence of L-buthionine (RS)-sulfoximine (BSO; a nontoxic and selective inhibitor of -glutamylcysteine synthetase, the rate-limiting step in GSH synthesis) (44) and HD B16M cells pre-cultured in the presence of GSH ester (which rapidly enters the cell and delivers free GSH) (25). GSH content in BSO-treated LD B16M cells was not significantly different from that found in control HD B16M cells, whereas GSH content in GSH ester-treated HD B16M cells was not significantly different from that found in control LD B16M cells (Table IV). HSE-induced tumor cytotoxicity in BSO-treated LD B16M cells was similar to that found in HD B16M cells, whereas HSE-induced tumor cytotoxicity in GSH ester-treated HD B16M cells decreased to values similar to those found in control LD B16M cells (Table IV). These facts indicate that GSH is directly involved in the protection of B16M cells against HSE-induced cytotoxicity. Nevertheless, which molecular mechanisms relevant to metastatic activity are regulated by GSH, e.g. enzyme activities or signaling molecules, is an important question that needs further study.

View this table: [in this window] [in a new window]   Table IV Role of GSH in preventing HSE-induced B16M cytotoxicity in vitro HSE and B16M cells were cultured as described in the legend to Table II. TNF- and IFN- (concentrations were identical to those used in Table I) were present under all conditions and were added to co-cultures as described in the legend to Table III. B16M cells were pre-cultured in the absence or presence of BSO or GSH ester. GSH values in LD B16M cells represent those found, in the absence or presence of 100 µM BSO (added to the culture at 0 h), 12 h after plating. GSH values in HD B16M cells represent those found, in the absence or presence of 1 mM GSH ester (added to the culture at 71 h), 72 h after plating. Tumor cytotoxicity is expressed as the % of tumor cells that lost viability within a 6-h period of incubation. Data are means ± S.D. of five to six different experiments.

Tumor Cytotoxicity of Nitrogen- and Oxygen-derived Reactive Species-- The mechanisms by which NO and H₂O₂ induce tumor cytotoxicity were further investigated in experiments in which B16M cells were directly exposed to NO and/or H₂O₂ at concentrations that reflect those found in co-cultures of HSE and B16M cells (see Table II). As shown in Table V, NO displayed a limited cytotoxicity (e.g. an ~18% increase in HD B16M cells compared with basal medium-treated cells), whereas H₂O₂ did not alter B16M cell viability as compared with controls. However, when NO and H₂O₂ were added together to the cultures, we observed a dramatic increase in tumor cytotoxicity (e.g. >70% in HD B16M cells). These results show that the NO-mediated loss of tumor cell viability depends on both NO and H₂O₂. Peroxynitrite also showed cytotoxic effects, but they were lower compared with those found in the presence of NO and H₂O₂ (Table V). Incubation of B16M cells in the presence of NO and superoxide dismutase, which removes O₂ anions and increases H₂O₂ levels, caused more B16M cell toxicity than NO alone (Table V). Furthermore, we used EGTA to remove metal ions present in the culture medium and found that, under these conditions, NO/H₂O₂-mediated cytotoxicity was decreased to values similar to those found in the presence of NO alone (Table V). Moreover, addition of NO, H₂O₂, and FeCl₃ to EGTA-pretreated B16M cells again increased tumor cytotoxicity to values similar to those induced by NO and H₂O₂. These results suggest, as previously proposed (45), a trace metal-catalyzed process within a mechanism where different reactions may take place, e.g. NO + H₂O₂, which generates NO + ^·OH + H⁺ via a Fenton process; Fe(III) + NO + H₂O₂, which generates Fe(II) + OONO + 2H⁺ (in the intact cells, other reducing agents, e.g. ascorbate, may be also utilized to reduce metals); ^·OH + H₂O₂, which generates ^·OOH + H₂O; or ^·OOH + NO, which generates OONO + H⁺. Chemical mechanisms that can produce potent oxidants, such as ^·OH and OONO, could contribute to the observed NO/H₂O₂-induced tumor cytotoxicity.

View this table: [in this window] [in a new window]   Table V B16M cytotoxicity of nitrogen- and/or oxygen-derived reactive species B16M cells were cultured for 12 h to low cellular density (LD cells) or for 72 h to high cellular density (HD cells). NO (10 µM), H2O2 (100 µM), superoxide dismutase (SOD; 100 units/ml), OONO (10 µM), EGTA (0.5 mM), or ebselen (10 µM) was added at 12 or 72 h of culture. FeCl3 (1 mM) was added 5 min after EGTA addition. Tumor cytotoxicity (see "Experimental Procedures") is expressed as the % of tumor cells that lost viability within a 1-h period of incubation. Data are means ± S.D. of eight to nine different experiments.

NO- and H₂O₂-induced Changes in the Antioxidant Potential of Tumor Cells-- Under normal conditions, cells utilize catalase and GSH peroxidase activities to maintain low intracellular H₂O₂ levels. However, as previously suggested in cell-free experiments (45), highly oxidant radical species may cause alterations in H₂O₂-catabolizing enzymes. In addition, S-nitrosation of many enzymes and proteins has been observed, and the chemical modification may affect activity (46). Moreover, NO can form nitroso adducts with different thiols, such as GSH and cysteine-containing proteins (47), and RSNO may also inactivate enzyme activities (46). GSH, the most abundant cytosolic thiol, is used in the GSH peroxidase-catalyzed reaction and easily reacts with NO, forming GSNO, and thus may play a key role in regulating tumor cell resistance to nitrosative and oxidative stress. We explored these mechanisms by measuring GSH status, formation of RSNO, catalase activity, and the GSH peroxidase/GSH reductase system activity in cultured B16M cells exposed to NO and/or H₂O₂. As shown in Table VI, NO and H₂O₂ addition induced a decrease in GSH levels and an increase in GSSG levels in the B16M cells, implying decreases in the GSH redox status (GSH/GSSG ratio) to ~10 and 6% of controls in LD and HD B16M cells, respectively. When comparing LD and HD B16M cells, the different extent of GSH depletion is not surprising if one takes into account that GSH synthesis is much more active in HD cells (25). In addition, NO promoted an increase in RSNO formation, which was higher in B16M cells with higher GSH content (LD cells) (Table VI). Moreover, the increase in nitrosative and oxidative stress-related molecular markers was associated with a significant decrease in catalase activity in both B16M cell subsets, whereas the GSH peroxidase/GSH reductase system (Table VI) or the total superoxide dismutase (2.1 ± 0.3 units/10⁶ LD cells and 1.5 ± 0.2 units/10⁶ HD cells; n = 5) activity remained unaffected. In agreement with these findings, we observed that ebselen, a GSH peroxidase mimic (48), was able to prevent the increase in NO toxicity induced by H₂O₂ (Table V). These facts suggest that, during their interaction with the HSE, B16M cells depend mainly on the GSH peroxidase/GSH reductase system to catabolize H₂O₂ and thus to prevent cytotoxic damage induced by highly oxidant radical species. Thus, the low GSH content found in HD B16M cells may be the cause of their higher sensitivity to nitrosative and oxidative attacks.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intravascular oxidative stress elicited by leukocytes and endothelial cells contributes to the elimination of circulating and capillary-arrested cancer cells (49, 50). Indeed, direct in vitro lysis of metastatic tumor cells by cytokine-activated murine vascular endothelial cells has been shown (4). In agreement with this are the findings that B16M cells pretreated with the lipophilic antioxidant -tocopherol (vitamin E) in vitro increased their survival in the hepatic sinusoids (51), that an increase in B16M cell GSH content upon hydroxyurea treatment also transiently increased metastasis (52), that capillary survival decreased in GSH-depleted B16M cells (24), and that B16M cells with high GSH content showed higher metastatic activity in the liver than those with lower GSH content (25). Nevertheless, how some metastatic cells survive their interaction with endothelial cells is a mechanism that is poorly understood.

Despite lethal effects on metastatic cells, oxidative stress may additionally create a pro-metastatic microenvironment at target organs by increasing cell adhesion molecule expression in both endothelial (53) and cancer (6) cells, activation of the early growth response-1 transcription factor gene (54), and activation of cancer and endothelial cell metalloproteinases (55). In consequence, although the antioxidant capacity of cancer cells may determine their intravascular survival, surviving cells may benefit from oxidative stress-promoting metastatic mechanisms.

By regulating vasodilatation, platelet aggregation, angiogenesis, production of prostaglandins, or leukocyte proliferation or by mediating tumor cytotoxicity (56), NO can affect tumor cell arrest in capillaries and metastasis. NO reversibly binds to cytochrome c oxidase and inhibits the respiration of tumor cells, an effect that can be suppressed by GSH (32). By interacting with their iron centers or forming RSNO, NO can affect the activity of membrane-bound, cytosolic, and nuclear proteins, including the N-methyl-D-aspartic acid receptor, hemoglobin, and transcription factors such as nuclear factor-B and SoxR; and in addition, by inducing S-nitrosylation of caspases and tissue transglutaminase, NO may regulate the balance between apoptosis and necrosis (57).

This study was undertaken to determine the role of NO and H₂O₂ in the HSE-induced cytotoxic effect on B16M cells during their adhesion process. Cytokine-mediated activation of endothelial cells during the metastatic process is a mechanism that must be expected to occur in vivo (see references cited in the "Introduction"). Thus, comparative studies in the presence and absence of cytokines appeared necessary. Our results reveal that inhibition of cytokine-activated endothelial NO production abrogates HSE-induced tumor cytotoxicity without altering endothelial H₂O₂ production (Table III). However, NO-induced tumor cytotoxicity was significantly decreased by catalase-mediated removal of extracellular H₂O₂ (Table III). Interestingly, previous observations showed that when NO was added in vitro to a superoxide-generating system, catalase inhibited the production of singlet oxygen, whereas superoxide dismutase enhanced it (58). In addition, it was shown that H₂O₂ enhanced the tumoricidal activity of NO in rat hepatoma cells, although no evidence was found for an interplay of NO with O₂ in cytotoxicity (21). We explored the chemical mechanisms by which NO and H₂O₂ are cytotoxic and found that a major part of the effect requires the presence of trace metals capable of generating highly oxidant radicals, likely ^·OH and OONO (see "Results" and Table V). However, under physiological conditions, the intracellular concentrations of free iron and H₂O₂ are kept very low by iron-binding proteins and the catalase/GSH peroxidase activities. Nevertheless, NO and O₂ can also release iron from proteins (59). In addition, NO and OONO can increase intracellular H₂O₂ by inhibiting the mitochondrial respiratory chain (32, 60) or through the inhibition of catalase (Table VI). Therefore, it is possible that NO may induce the release of catalytic amounts of free iron and increase H₂O₂ levels in the intact tumor cells.

Despite the fact that HSE-derived NO and H₂O₂ cause B16M cytotoxicity (Table III) and although findings in other cell systems support this cooperative action (e.g. Refs. 20, 21, 45, and 61), others have suggested that NO protects against reactive oxygen species (62, 63). A plausible explanation for this apparent paradox is that both NO and H₂O₂ can show very different effects depending on their concentrations. Indeed, recent experiments in isolated mitochondria showed that NO reversibly inhibited permeability transition pore opening with an IC₅₀ of 11 nM; however, at higher concentrations (>2 µM), NO accelerated pore opening (64). Identically, low levels of H₂O₂ (3-15 µM) may cause a significant mitogenic response in mammalian cells, whereas higher concentrations (>100 µM) may cause growth arrest and cell damage (65). Therefore, considering the amount of NO and H₂O₂ released by the HSE during the process of cancer cell adhesion (Table II), tumor cytotoxicity may be expected, as it indeed occurs (Table III).

Catalase activity was much lower in the presence of NO/H₂O₂ than in the presence of NO. This suggests the involvement of NO- and H₂O₂-derived radical species. In fact, we observed that OONO (10 µM) induced a decrease in catalase activity (data not shown) similar to that induced by NO and H₂O₂ (Table VI). In consequence, since no inhibition of the GSH peroxidase/GSH reductase system was induced by NO and/or H₂O₂, B16M cells must rely on this system to metabolize H₂O₂ and thereby to prevent lethal toxicity. This is in agreement with the resistance to macrophage-induced oxidative stress shown by tumor cells with high GSH peroxidase activity, a fact that has no correlation with catalase activity (66).

NO forms RSNO (Table VI) with different thiols, such as GSH, which decreases the levels of free NO and thereby its biological activity. GSNO, a slow releaser of NO, may also inhibit the respiration of tumor cell mitochondria, but its effect is much lower than that of NO (32). Nevertheless, NO-induced RSNO formation was not increased by H₂O₂ (Table VI), which suggests that RSNO formation is not required in the mechanism of NO- and H₂O₂-induced cooperative tumor cytotoxicity (see Tables III and IV). In addition, our results are also in agreement with previous reports showing that depletion of cellular GSH reduces the viability of cells exposed to NO (67). Indeed, GSH is implicated in the regulation of the mitochondrion-based mechanisms that lead to cell death (68). In conclusion, our results show that NO and H₂O₂ are critical factors involved in the tumoricidal activity of the HSE toward potentially metastatic cells. The fact that B16M cells with high GSH content (LD cells) are much less susceptible to NO- and H₂O₂-induced toxicity (see "Results") establishes a close relationship between intracellular GSH levels and cancer resistance against oxidative and nitrosative stress within a metastatic microenvironment. The biochemical mechanisms described in this report may explain, at least in part, how some cancer cells survive during their interaction with the endothelium and possibly with other cells such as macrophages and intravascular granulocytes.

	FOOTNOTES

^* This work was supported in part by Grants SAF99-112 and 1FD97-548 from the Comisión Interministerial de Ciencia y Tecnología (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.

^§ Supported by a fellowship from the Generalitat Valenciana.

^¶ Supported by a Carmen and Severo Ochoa fellowship from the Ayuntamiento de Valencia.

Supported by fellowships from the Ministerio de Ciencia y Tecnología (Spain).

To whom correspondence should be addressed: Dept. de Fisiología, Facultad de Medicina y Odontología, Av. Blasco Ibañez 17, 46010 Valencia, Spain. Tel.: 34-963864-646; Fax: 34-963864-642; E-mail: jose.m.estrela@uv.es.

Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M101148200

	ABBREVIATIONS

The abbreviations used are: B16M, B16 melanoma; HSE, hepatic sinusoidal endothelium; IL-1, interleukin-1; TNF-, tumor necrosis factor-; eNOS, endothelial nitric-oxide synthetase; L-NAME, N^G-nitro-L-arginine methyl ester; LD cells, cells cultured to low density; HD cells, cells cultured to high density; BCECF-AM, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; IFN-, interferon-; NO_x, nitrite plus nitrate; RSNO, S-nitrosothiols; GSNO, S-nitrosoglutathione; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate-buffered saline; BSO, L-buthionine (RS)-sulfoximine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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