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J. Biol. Chem., Vol. 282, Issue 5, 2880-2890, February 2, 2007
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From the Department of Physiology, University of Valencia, 46010 Valencia, Spain
Received for publication, June 21, 2006 , and in revised form, November 8, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Analysis of the bcl-2 family of genes revealed that B16M-F10 cells (high metastatic potential), compared with B16M-F1 cells (low metastatic potential), overexpress bcl-2 preferentially (7). t-PTER increases expression of pro-death bax (
2.2-fold) and decreases expression of anti-death bcl-2 (2.0-fold) (6), whereas QUER increases expression of different pro-death genes (bax, bak, bad, and bid; 1.5–2.5-fold) and decreases expression of all anti-death genes analyzed (bcl-2 (7.3-fold), bcl-w (1.5-fold), and bcl-xL (2-fold)) (6). bcl-2 overexpression prevents the QUER- and t-PTER-dependent increase in metastatic B16M-F10 cell death caused by the HSE in vivo (6), thus suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-induced damage. In agreement with this idea, we also observed that antisense bcl-2 therapy potentiates tumor necrosis factor-
-induced oxidative stress and death in B16M-F10 cells (7). However, the molecular mechanisms that link polyphenol signaling with bcl-2 expression are unclear.
In vitro, t-RESV-driven apoptosis of chronic leukemic B cells has been shown to correlate with activation of caspase-3, a drop in the mitochondrial membrane potential, and reduction in the expression of inducible nitric-oxide synthetase (iNOS) (8, 9). NO is a potential bioregulator of apoptosis because high concentrations of NO or peroxynitrite can induce apoptotic death in different cells types, including tumor cells (10), although, on the other hand, NO may also act as an anti-apoptotic signal associated with, for example, suppression of mitochondrial cytochrome c release, ceramide generation, and caspase activation (10). Indeed, NO donors can elevate bcl-2 expression both at the mRNA and protein levels and prevent apoptotic cell death in vitro (11), whereas, paradoxically, also in vitro, NO-induced apoptosis of K-1735 melanoma cells (12) or human myeloid leukemia U937 cells (13) is associated with down-regulation of Bcl-2 and caspase activation. Therefore, NO, which may either prevent or induce apoptosis, can also increase or decrease Bcl-2 levels. These apparently controversial facts suggest that different intracellular NO levels may likely determine opposite effects. Whether natural polyphenols such as t-PTER and QUER also cause reduction in iNOS gene expression in metastatic cells (and consequently a decrease in their intracellular NO levels) and whether this is linked to changes in bcl-2 expression is unknown.
On the other hand, t-RESV, as well as other polyphenols (e.g. black tea polyphenols), can increase endothelial nitric-oxide synthetase (eNOS) activity and induce accumulation of p53 and p21WAF1/CIP1 in cultured pulmonary artery endothelial cells (14, 15). Thus, it is possible that natural polyphenols may also alter NO levels in the metastatic microenvironment during interaction of cancer and endothelial cells, which is important because endothelial NO generation was found to be essential in the mechanism of tumor cytotoxicity during B16M-F10 cell adhesion to the vascular endothelium (16).
The aim of this study was to investigate the possible relationship between NO and the effect t-PTER and/or QUER (at in vivo bioavailable concentrations) on bcl-2 expression in B16M-F10 cells. Our results show that this polyphenolic association decreases NO production in isolated B16M-F10 cells and increases NO release from the vascular endothelium during B16M-F10/endothelial cell interaction. At both steps, changes in NO levels trigger Bcl-2 down-regulation and activation of death mechanisms in metastatic B16M-F10 cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Measurement of H2O2, Nitrite, and Nitrate—The assay of H2O2 production was based, as reported previously (16), on the H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) to a highly fluorescent dimer (2,2'-dihydroxydiphenyl-5,5'-diacetic acid), which is mediated by horseradish peroxidase (18). Nitrite and nitrate determinations were performed as described previously (16) and based on the methodology of Braman and Hendrix (19). Total NOx (
plus 
) determinations were made by monitoring NO evolution from a measured sample placed into a boiling VCl3/HCl solution (which will reduce both and to NO). Quantitation was accomplished using a standard curve made up of known amounts of and .
Isolation, Identification, and Culture of the HSE—Male C57BL/6J mice (10–12 weeks old) were from Charles River Laboratories, Inc. (Barcelona, Spain). The HSE was separated and identified as described previously (20). Sinusoidal cells were separated in a 17.5% (w/v) metrizamide gradient. HSE cultures were established and maintained in pyrogen-free DMEM supplemented as described above for the B16M-F10 cells. Differential adhesion of endothelial cells to the collagen matrix and washing allowed complete elimination of other sinusoidal cell types (Kupffer and stellate cells and lymphocytes) from the culture flasks.
B16M/Endothelial Cell Adhesion and Cytotoxicity Assays—B16M-F10 cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (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 x 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 adherent tumor cells was quantified by arbitrary fluorescence units based on the percentage of the initial number of B16M cells added to the HSE culture (21). Damage to B16M cells during their in vitro adhesion to the HSE was measured as described previously (22) using tumor cells loaded with calcein acetoxymethyl ester (Molecular Probes). Other reagents used in experiments of tumor cytotoxicity were from Sigma.
Reverse Transcription-PCR and Detection of mRNA Expression—Total RNA was isolated using the TRIzol kit (Invitrogen) following the manufacturer's instructions. cDNA was obtained using a random hexamer primer and a MultiScribe reverse transcription kit (TaqMan reverse transcription reagents, Applied Biosystems, Foster City, CA) as described by the manufacturer. A PCR Master Mix and AmpliTaq Gold DNA polymerase (Applied Biosystems) containing specific primers (synthesized by Integrated DNA Technologies according to published sequences available from the GenBankTM Data Bank) were then added: iNOS, 5'-CGGATATCTCTTGCAAGTCCAAA (forward) and 5'-AAGTATGTGTCTGCAGATATG (reverse); eNOS, 5'-CACCAGGAAGAAGACCTTTAAGGA (forward) and 5'-CACACGCTTCGCCATCAC (reverse); bcl-2, 5'-CTCGTCGCTACCGTCGTGACTTCG (forward) and 5'-CAGATGCCGGTTCAGGTACTCAGTC (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5'-CCTGGAGAAACCTGCCAAGTATG (forward) and 5'-GGTCCTCAGTGTAGCCCAAGATG (reverse). Real-time quantitation of the mRNA relative to glyceraldehyde-3-phosphate dehydrogenase was performed with a SYBR Green I assay and a iCycler detection system (Bio-Rad). Target cDNA was amplified as follows: 10 min at 95 °C and then 40 cycles of amplification (denaturation at 95 °C for 30 s and annealing and extension at 60 °C for 1 min per 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.
Bcl-2 Analysis—Bcl-2 protein was quantitated in the soluble cytosolic fraction by enzyme immunoassay (23) using a monoclonal antibody-based assay from Sigma (1 unit of Bcl-2 is defined as the amount of Bcl-2 in 1000 non-transfected B16M-F10 cells).
eNOS-deficient Mice—Generation of eNOS-deficient mice was carried out as described previously (24). 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 described previously (24). Briefly, 20-µg samples were digested with BamHI, separated on 1.0% agarose gels, and transferred to nylon-supported nitrocellulose. The blots were then hybridized using a random primer-labeled 1.4-kb eNOS cDNA probe (24). A 5.3-kb fragment was diagnostic of the endogenous eNOS locus, and a 6.4-kb fragment was diagnostic of the targeted allele.
Measurement of iNOS Activity in B16M-F10 Cells—Conversion of L-arginine to L-citrulline was measured by a modification of a previously described methodology (25). The assay was carried out by adding 100 µg of sample protein to 150 µl of assay buffer (50 mM HEPES, 1 mM dithiothreitol, 1 mM MgCl2, 5 mg/liter pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, and 3 mg/liter aprotinin, pH 7.4) containing 70 µM arginine, 250,000 dpm L-[3H]arginine, (GE Healthcare, Little Chalfont, UK), 2 mM NADPH, 5 µM tetrahydrobiopterin, 5 µM FAD, and 0.5 mM CaCl2 to measure total NOS activity or in the presence of 1 mM EGTA (without calcium) to determine Ca2+-independent iNOS activity. After 30 min at 37 °C, the reaction was stopped with 100 µl of 1 M trichloroacetic acid. The samples were adjusted to pH 4.6 by adding 500 µl of 20 mM HEPES and applied to Dowex AG 50W-X8 resin columns. L-[3H]Citrulline was eluted and separated by thin-layer chromatography. Radioactivity was counted with an AR-2000 scanner detector (Bioscan, Inc., Washington, D. C.). The results are expressed as pmol of L-[3H]citrulline/mg of protein/min.
Western Blot Analysis—Cultured cells were harvested as indicated above and then washed twice with ice-cold Krebs-Henseleit bicarbonate buffer, pH 7.4. Whole cell extracts were made by freeze-thaw cycles in buffer containing 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin, pH 7.4. Fifty µg of protein (as determined by the Bradford assay (68)) were boiled in Laemmli buffer and resolved by 12.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and subjected to Western blotting with anti-cAMP-response element-binding protein (CREB) or anti-phospho-CREB (Ser133) monoclonal antibody (Chemicon International Inc., Temecula, CA). Blots were developed using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL system, Amersham Biosciences).
Gene Silencing—Small interfering RNA (siRNA) transfection experiments were performed with double-stranded RNA designed and synthesized by Eurogentec (Seraing, Belgium). A CREB-specific siRNA sense orientation strand (5'-UACAGCUGGCUAACAAUGGdTdT-3') was used. Cells were transfected with the siRNA delivery reagent jetSITM (Eurogentec) at 3µl/µgof siRNA according to the manufacturer's instructions. Transfection efficiency in cells plated on coverslips was determined with fluorescein isothiocyanate-labeled siRNA and determined by cell counting using a TCS-SP2 confocal microscope (Leica Microsystems, Bensheim, Germany) to be 90–95% after 24 and 48 h.
Guanylate Cyclase Assay—Enzyme activity (soluble and particulate) was determined as described previously (26, 27). Briefly, the reaction mixture contained 7.5 pmol of Tris-HCl, pH 7.6, 0.75 µmol of creatine phosphate, 0.5 µmol of cGMP, 0.45 µmol of MgCl2, 1.2 µmol of theophylline, 0.6 units of creatine kinase, and 37.5 nmol of [8-3H]GTP (10–12 mCi/mmol; PerkinElmer Life Sciences) in a total reaction volume of 150 µl. The radioactive cGMP produced was isolated as described previously (28) and measured using a Packard Tri-Carb 2700TR Varisette analyzer (PerkinElmer Life Sciences).
Assay of Sphingomyelinases—This procedure was a modification of a previously reported methodology (29). B16M-F10 cells (5 x 106) were separated from the co-cultured HSE as described previously (30), washed twice with ice-cold phosphate-buffered saline, and homogenized in 0.4 ml of lysis buffer (10 mM HEPES/KOH, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 µM pepstatin, 0.15 units/ml aprotinin, and 50 mg/ml leupeptin) containing 0.1% Triton X-100. The homogenate was passed through a 25-gauge needle and then centrifuged at 100,000 x g for 1 h at 4°C. The supernatant was used as an enzyme source. The mixture used to assay magnesium-dependent neutral sphingomyelinase (SMase) contained 0.1 M Tris-HCl, pH 7.5, 50 nmol of [N-methyl-14C]sphingomyelin (specific activity, 55 mCi/mmol; GE Healthcare), 10 mM MgCl2, 0.1% Triton X-100, and 200 µg of protein in a final volume of 0.2 ml. For magnesium-independent neutral SMase, MgCl2 was removed from the reaction mixture. For acid SMase, 0.1 M sodium acetate, pH 5.0, was used instead of Tris-HCl. Incubation was carried out at 37 °C for 30 min. The reaction was stopped by adding 1.25 ml of chloroform/methanol (2:1). Then, 0.25 ml of double-distilled water were added to the tubes and vortexed. The tubes were centrifuged at 1000 x g for 6 min to separate the two phases. The clear upper phase was removed, placed in a glass scintillation vial, and counted with a scintillation counter (Packard Tri-Carb 2700TR Varisette).
C16-ceramide and C16-dihydroceramide Analysis—The amounts of ceramide and dihydroceramide in B16M-F10 cells were measured as described previously (31). Briefly, cancer cell suspensions (5 x 106 cells in 200 µl of DMEM) were vigorously mixed with 4 ml of chloroform/methanol (2:1, v/v) for 20 min. Then, 0.8 ml of distilled water were added to the mixture, and the sample was vortexed and centrifuged. The lower layer was collected, and the chloroform was allowed to evaporate. The residue was dissolved in a solvent and subjected to liquid chromatography-mass spectrometry analysis using a Quattro micro triple-quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with a Shimadzu LC-10ADVP pump and SCL-10AVP controller system with an SIL-10ADVP autoinjector. Samples were analyzed by reverse-phase high pressure liquid chromatography using a Phenomenex ODS column (35 x 2 mm) with 5-µm particle size. In all cases, 40 µl were injected onto the column. The temperature of the column was maintained at 25 °C. Mobile phases were as follows: mobile phase A, 5 mM ammonium formate/methanol/tetrahydrofuran (5:2:3, v/v); and mobile phase B, 5 mM ammonium formate/methanol/tetrahydrofuran (1:2:7, v/v) containing 0.01% formic acid. Elution was carried out at a flow rate of 0.2 ml/min with 70% mobile phase A and 100% mobile phase B for 6.3 min in a linear gradient mode.
Cell Death Analysis—Apoptotic and necrotic cell death were distinguished by fluorescence microscopy (32). For this purpose, isolated cells were incubated with Hoechst 33342 (10 µM; which stains all nuclei) and propidium iodide (10 µM; which stains nuclei of cells with disrupted plasma membranes) for 3 min and analyzed using a Nikon Diaphot 300 fluorescence microscope with excitation at 360 nm. Nuclei of viable, necrotic, and apoptotic cells were observed as blue round nuclei, pink round nuclei, and fragmented blue or pink nuclei, respectively. About 1000 cells were counted each time. DNA strand breaks in apoptotic cells were assayed using a direct terminal transferase dUTP nick end labeling assay (Roche Applied Science) and fluorescence microscopy following the manufacturer's methodology.
Assay for in Vitro Invasion of the Hepatic Endothelial Cell Monolayer by B16M-F10 Cells—Invasion of the endothelial cell monolayer by B16M-F10 cells was assayed following the method by Ohigashi et al. (33) with some modifications. HSE cells were seeded on 1% gelatin-coated tissue culture dishes with grids. When the cells reached confluency, the culture medium was replaced with fresh medium. After a 2-h incubation, the cultures were washed with DMEM, and then HSE cells were overlayered with B16M-F10 cells and cocultured for 5 days. The invasion capacity of B16M-F10 cells was measured by counting the number of colonies/1 cm2 formed under the HSE monolayer using a phase-contrast microscope.
Statistical Analysis—Data are presented as the means ± S.D. for the indicated number of different experiments. Statistical analyses were performed using Student's t test, and p values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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35%) when both polyphenols were present (Table 1).
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Furthermore, QUER undergoes rapid systemic metabolism once it is absorbed in the body. Its major metabolite, QUER 3'-sulfate, can be found in substantially higher concentrations than the parent compound in the plasma (38). However, it remains unclear whether QUER 3'-sulfate exhibits any activity upon NO production. To clarify this, QUER metabolites were analyzed in plasma after administration of 20 mg of QUER/kg to B16M-F10-bearing mice as described (6). Extraction of QUER metabolites from plasma and HPLC analysis were performed exactly as described by De Pascual-Teresa et al. (38). The HPLC profile showed a main peak that was identified as QUER 3'-sulfate by its tandem mass spectra. A calibration curve was obtained using a standard of QUER 3'-sulfate (0.01–50 µM; Extrasynthèse S.A., Bordeaux, France), which was found to be linear with a correlation coefficient of >0.99. The limits of detection and quantitation for our method were 0.02 µM. QUER 3'-sulfate levels in plasma were <0.1, 1 ± 0.2, 3 ± 0.6, 10 ± 1.7, 6 ± 0.9, 1 ± 0.3, and <0.1 µM at 5, 10, 20, 30, 60, 90, and 120 min, respectively, after intravenous administration of QUER. Cultured B16M-F10 cells were exposed to 8 µM QUER 3'-sulfate (approximate mean value of the plasma concentrations measured at 30–60 min after intravenous administration of 20 mg of QUER/kg) for 30 min using the same approach used for QUER and t-PTER that respects bioavailable concentrations (see Table 1). However, in the presence of QUER 3'-sulfate, NOx and H2O2 values were not significantly different from controls ("Basal medium" in Table 1). Therefore, we conclude that, although bioavailable concentrations of QUER decrease NO generation by the cancer cells, its main metabolite does not.
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, interleukin-1B, and interleukin-18) and NO/H2O2 release, VCAM-1-dependent adherence, and melanoma growth factor release by the HSE (e.g. Ref. 40). Endothelium-derived reactive nitrogen and oxygen species and –OONO radicals in particular are harmful and lethal for a high percentage of adherent melanoma cells (16). Thus, because t-RESV can increase eNOS activity (14), in the second set of experiments, we used an in vitro B16M-F10/endothelial cell adhesion assay to investigate the effect of t-PTER and QUER on NO and H2O2 production during the process of tumor cell attachment to the HSE. As shown in Table 2, during B16M-F10/HSE interaction, QUER and t-PTER/QUER significantly increased NO release to the extracellular medium, whereas H2O2 generation was not affected. Reverse transcription-PCR expression analysis revealed that the t-PTER- or t-PTER/QUER-induced increase in NO generation by B16M-F10 cells was associated with an increase in eNOS gene expression (Fig. 2). This is important because, as reported previously (16), just an increase in the metastatic microenvironment NO levels is enough to increase cancer cell death.
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or in the following 4 h.) Similar results were obtained when NOx produced by the HSE was calculated as follows: NOx produced by co-cultured HSE and B16M cells minus NOx produced by cultured B16M-F10 cells alone (data not shown). The percentage of H2O2 generated by the HSE was calculated by substracting H2O2 produced by co-cultured HSE and B16M-F10 cells minus H2O2 produced by cultured B16M-F10 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 2). Preculture of endothelial cells in the presence of NG-nitro-L-arginine methyl ester did not alter the percentage of B16M cell adhesion (data not shown) compared with controls (see the legend to Table 2).
Effect of t-PTER and QUER on Melanoma Cell Adhesion, Viability, and Invasive Growth upon Interaction with the Vascular Endothelium—The potential increase in the rate of metastatic cell death induced by the polyphenols was further investigated and correlated with NO and H2O2 generation, the percentage of tumor cell adhesion to the HSE, and the rate of invasion through HSE monolayers. These experiments were performed using control HSE cells and HSE cells isolated from eNOS-deficient (eNOS–/–) mice to abolish eNOS-dependent NO production. Because the effects of t-PTER and QUER on NOx generation either by the B16M-F10 cells (Table 1) or during cancer and endothelial cell interaction (Table 2) were practically additive, in the next experiments, we focused on the polyphenol association. As shown in Table 3, the percentage of B16M-F10 cell adhesion to the endothelium decreased in the presence of polyphenols, in agreement with the previously reported PTER-induced inhibition of VCAM-1 expression in the HSE (6). In the presence or absence of polyphenols, NO generation was almost abolished in eNOS–/– HSE cells (Table 3), implying that the t-PTER- and QUER-induced increase in NO production is fully dependent on eNOS. The t-PTER- and QUER-induced increase (5–6-fold) in the percentage of B16M-F10 cell death (mainly apoptotic) during cancer and endothelial cell interaction was also NO-dependent (Table 3). Furthermore, when assaying the in vitro invasion of hepatic endothelial cell monolayers by B16M-F10 cells, we found a marked decrease (83%) in the number of penetrated colonies in the presence of t-PTER and QUER (Table 3); however, when eNOS–/– HSE cells instead of control eNOS+/+ HSE cells were used, the number of colonies increased by 2-fold, and the inhibition elicited by the polyphenols almost disappeared (Table 3). To further prove that NO is indeed involved in the mechanism of tumor cell killing, we added a NO scavenger (300 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt, Affiniti Research Products, Mamhead, UK) (42) to the co-culture medium. During HSE and B16M-F10 cell interaction in either the presence or absence of t-PTER and QUER (as in Table 3), NOx values remained <0.1 nmol/106 cells in the presence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt. Under these conditions, tumor cell cytotoxicity was 1% in either the presence or absence of t-PTER and QUER.
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The mechanism of CREB transcriptional activation depends on phosphorylation of Ser133, which may be mediated by different kinases (e.g. Ref. 47). Thus, we investigated this possibility in B16M-F10 cells and found that t-PTER and QUER caused inhibition of CREB phosphorylation (Fig. 3). This effect was similar to the NO shortage-dependent inhibition of CREB phosphorylation observed in the presence of AMG (Fig. 3). The t-PTER- and QUER-induced decrease in phospho-CREB was prevented in the presence of 0.3 mM SNAP (Fig. 3), thus indicating a direct link between NO generation and CREB phosphorylation. Therefore, a NO shortage-dependent decrease in phospho-CREB could be, at least in part, the mechanism involved in the polyphenol-induced decrease in bcl-2 expression. To test this hypothesis further, B16M-F10 cells were loaded by electroporation (Bio-Rad system, 1.0 kV/cm with a time constant of 50 ms) with anti-phospho-CREB monoclonal antibody (see "Experimental Procedures" and Ref. 48 for technical details). A decrease in bcl-2 expression (80% compared with controls; data not shown), not significantly different from that induced by 2 mM AMG (Table 4), was observed in the presence of anti-CREB antibody. Moreover, silencing of CREB using specific siRNA was associated with lower bcl-2 expression in B16M-F10 cells (Fig. 4). In addition, PTER/QUER-induced bcl-2 down-regulation was reversed by a guanylate cyclase activator (YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, Yung Shin Pharmaceutical Industry Co., Ltd., Taichung, Taiwan) (Fig. 5). These results prove the direct connection of PTER/QUER to CREB and to bcl-2 expression. bcl-2 expression and phospho-CREB levels returned to control values (as in Fig. 4) (data not shown) in surviving B16M-F10 cells after 12 h of co-culture with endothelial cells, in agreement with the data showing that the effect of t-PTER and QUER could be reversed by NO directly (Table 4).
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2-fold) in ceramide within the cancer cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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As stated in the Introduction, paradoxically, NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others (see Ref. 10 for a review). NO-induced apoptosis in cells has been linked to high (supraphysiological) concentrations of NO (10) and appears to be regulated by the redox state and by formation of transition metal complexes (16, 54, 55). This concept is in agreement with the idea that activation of iNOS gene expression may facilitate cancer cell death and inhibition of metastases (56). On the other hand, anti-apoptotic signaling has been generally correlated with low or physiological NO levels (10). However, it was reported recently that AMG-induced inhibition of endogenous NO synthesis does not affect cell cycle progression of human melanoma cells, but leads to cell death by apoptosis (34). In addition, it has been shown that, in vitro, t-RESV induces the apoptosis of leukemic B cells and simultaneously inhibits the production of NO through iNOS gene down-regulation (57). Nevertheless, the effects of t-RESV on 48-h cultured leukemic B cells are unrealistic: e.g. 3–50 µM t-RESV, constantly present in the culture medium, increases the number of fluorescein isothiocyanate-labeled annexin V-positive cells by 10–50%; 10–50 µM t-RESV decreases iNOS gene expression by 50–90%; and 50 µM t-RESV decreases Bcl-2 content by 25–60% (57). Bioavailability of natural polyphenols is a critical issue that must be investigated before correlating in vitro observations and biochemical effects (2). After intravenous administration to rabbits of 20 mg of t-RESV/kg (a dose that represents, for an adult human weighing 70 kg, 140 times the maximum amount of total RESV found in 1 liter of red wine), its highest concentration in plasma (42.8 ± 4.4 µM at 5 min after administration) decreases very rapidly to 0.9 ± 0.2 µM (0.2 mg/liter) at 60 min and to <0.1 µM at 120 min (5). Following a similar protocol, we calculated a half-life of t-RESV in mouse plasma of 10.2 min (6) and in human plasma of 11.5 min.3 If the same amount of t-RESV (20 mg/kg) is administered orally, the highest concentration in plasma (2–3 µM in mice and 1 µM in rabbits or rats) is found within the first 5 min after administration, followed by a decrease to <0.1 µM at 60 min (5). On the other hand, after intravenous administration to mice of 20 mg of t-PTER or QUER/kg (a dose that represents, for an adult human weighing 70 kg, 1000 times the maximum amount of PTER found in 1 kg of dark grapes and 20 times the maximum daily intake of QUER), their mean values of plasma concentrations measured within the first hour are 40 and 20 µM, respectively (6). Both t-PTER and QUER decrease bcl-2 expression (6). Moreover, both polyphenols show synergic effects on NO generation by B16M-F10 and endothelial cells (Tables 1, 2, 3). Therefore, it is obvious that the association has advantages over each single polyphenol. Our present findings show that a link between iNOS down-regulation in malignant cells and polyphenols can be firmly established using the association of t-PTER and QUER at bioavailable concentrations (short-term exposure at mean plasma concentrations). However, for this purpose, concentrations of t-PTER and QUER obtained after intravenous administration were necessary.
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60–100 times stronger as an antifungal agent, shows similar anticarcinogenic properties (58). t-PTER is present, for example, in extracts of the heartwood of Pterocarpus marsupium, used in Ayurvedic medicine for the treatment of diabetes, and in dark-skinned grapes (although quantitative studies have shown that, for every 10 parts t-RESV, there are only 1–2 parts t-PTER) (Ref. 58 and references therein). Human intake of flavones and flavonols (the most common flavonoids) is 3–70 mg/day (mainly QUER, 60–75%; major sources include tea, wine, berries, apples, and onions) (2). Flavonoids are among the most potent antioxidants because they show one or more of the following structural elements: an o-diphenolic group, a 2,3-double bond conjugated with 4-oxo function, and OH groups at positions 3 and 5. QUER combines three properties, and previous research has confirmed that it also exhibits anti-tumor properties (2, 59). However, all bioactive polyphenols studied so far undergo conjugation in the intestinal tract of man and rodents. This conjugation is rapid and extensive. It is in this form that they are absorbed, with very little of the free polyphenol gaining access to the blood (60–63); in fact, the latter is so small that, theoretically, it could even arise from limited hydrolysis of the conjugates because blood is quite rich in glucuronidase, sulfatase, and other deconjugating enzymes. Orally administered polyphenols are unlikely to be biologically effective unless their biological effects are not inactivated by conjugation and/or the free polyphenol can be released by hydrolysis of conjugates and can be taken up by target tissues in amounts to generate pharmacologically active concentrations. There is no evidence so far that either of the above possibilities is true. These facts imply the need for intravenous administration to obtain pharmacologically active concentrations of natural polyphenols, at least for therapy applications in the case of systemically disseminated cancers. Nevertheless, doses required to inhibit metastatic growth in vivo may possibly depend on the tumor cell type. CREB and its associated proteins act as survival factors for human melanoma cells (44). A NO shortage-induced decrease in CREB activity (Fig. 3) interferes with the cGMP-dependent protein kinase, the most important intracellular signaling pathway activated by NO (64). In addition, high levels of NO (released by the endothelium) promote an increase in ceramide formation (Table 5), a potential apoptotic activator. Moreover, in cells expressing functional Bcl-2, the mechanism of death action for ceramide may also involve, at least in part, a p53-dependent suppression of bcl-2 expression (65, 66). Recently, Mihara et al. (67) also showed that p53 promotes permeabilization of the outer mitochondrial membrane by forming complexes with the protective Bcl-xL and Bcl-2 proteins. Moreover, the tumor suppressor p53 may also transactivate the expression of pro-apoptotic genes such as bax and of the cyclin-dependent kinase inhibitor p21 (65). Thus, theoretically, t-PTER and QUER may act against the tumor under in vivo conditions at different steps and by different mechanisms. Fig. 6 summarizes these mechanisms and their possible inter-relationships.
In conclusion, the more relevant and novel findings reported in this study are as follows. (a) At bioavailable concentrations, polyphenol-induced Bcl-2 down-regulation (via guanylate cyclase/cGMP-dependent protein kinase-mediated CREB phosphorylation) in pre-invasive B16M-F10 cells is NO-dependent; (b) by enhancing eNOS expression (HSE), polyphenols increase NO release from the vascular endothelium upon interaction with metastatic B16M-F10 cells; and (c) in the presence of polyphenols, NO-mediated neutral SMase activation and increased ceramide generation are the main cell death-activated mechanisms in Bcl-2-depleted metastatic B16M-F10 cells. Our results demonstrate that, at different steps, very low and high NO levels act in parallel polyphenol-activated mechanisms, leading to metastatic cell death.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Physiology, Faculty of Medicine and Odontology, University of Valencia, 17 Av. Blasco Ibañez, 46010 Valencia, Spain. Tel.: 34-963-864-649; Fax: 34-963-864-642; E-mail: jose.m.estrela{at}uv.es.
2 The abbreviations used are: t-RESV, trans-resveratrol; t-PTER, trans-pterostilbene; QUER, quercetin; B16M-F10, B16 melanoma F10; VCAM-1, vascular adhesion molecule 1; HSE, hepatic sinusoidal endothelium; iNOS, inducible nitric-oxide synthetase; eNOS, endothelial nitric-oxide synthetase; DMEM, Dulbecco's modified Eagle's medium; BCECF-AM, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; CREB, cAMP-response element-binding protein; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SMase, sphingomyelinase; AMG, aminoguanidine; HPLC, high pressure liquid chromatography; SNAP, S-nitroso-N-acetyl-DL-penicillamine.
3 J. M. Estrela, M. Asensi, and P. Ferrer, unpublished data.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-actin. Lane 1, control; lane 2, AMG; lane 3, t-PTER and QUER; lane 4, t-PTER, QUER, and SNAP.
