Involvement of Oxidative Stress in Tumor Cytotoxic Activity of Hepatocyte Growth Factor/Scatter Factor*

Naokatu ArakakiDagger §, Takehiro KajiharaDagger , Rieko Arakaki, Tomokazu OhnishiDagger , Jamil Ahsan KaziDagger , Hideki Nakashima, and Yasushi DaikuharaDagger parallel

From the Departments of Dagger  Biochemistry and  Microbiology and Immunology, Kagoshima University Dental School, Kagoshima 890-8544, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In this study, we show that N-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin traps N-t-butyl-alpha -phenylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2',7'-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Hepatocyte growth factor (HGF),1 which was initially isolated as a potent mitogen of hepatocytes in primary culture (1, 2), is now known to be a broad-spectrum mitogen of a variety of cell types including melanocytes and endothelial and epithelial cells (3). HGF has been found to be identical with the "scatter factor" (4), which dissociates and increases the motility of epithelial cells. In addition to these activities, HGF induces branching tubule formation of Madin-Darby canine kidney epithelial cells in a three-dimensional collagen gel matrix (5). HGF is a potent angiogenic factor in vivo (6, 7) and is involved in organ regeneration (8) and tumorigenesis (9). All these effects of HGF are mediated by the HGF receptor, identified as the product of the c-met proto-oncogene (10, 11). During embryogenesis, c-met is expressed in epithelial components of various organs, while hgf is expressed in the adjacent mesenchyme (12). Embryos of mutant mice homozygous for the hgf gene die due to abnormal development of the placenta or both the placenta and liver (13, 14). Likewise, c-met-deficient embryos lack muscles of the limbs, diaphragm, and tip of the tongue, all derived from migratory precursors (15). Thus, HGF is now understood as a multifunctional cytokine that mediates epithelial-mesenchymal interactions.

In addition to its mitogenic activity, HGF has tumor cytotoxic activity, which suppresses growth of several tumor cell lines including Sarcoma 180 cells, Meth A mouse sarcoma cells, and Hep G2 human hepatoblastoma cells (16-18). Moreover, HGF has been shown to inhibit the growth of hepatocellular carcinoma cells in vivo and c-myc-induced hepatocarcinogenesis in a transgenic mouse model co-expressing c-myc and hgf (19-21). However, little is known about the signaling pathway for the tumor cytotoxic activity of HGF. Recently, we found that HGF activates the apoptosis signaling pathway by increasing the activity of caspase-3, a member of the interleukin 1beta -converting enzyme family, without affecting caspase-1 activity or the expressions of the apoptosis suppressors Bcl-2 and Bcl-x in Sarcoma 180 cells (22). We also showed that HGF-suppressed growth was partially restored when the apoptosis signaling pathway was completely inhibited by a caspase-3 inhibitor. This suggested the existence of other pathways, besides that for induction of apoptosis, for the tumor cytotoxic activity of HGF.

Reactive oxygen species (ROS) such as H2O2 are produced during many physiological intracellular reactions and in response to external stimuli such as UV radiation. They are, in general, considered to be cytotoxic, but several lines of evidence indicate that regulation of the intracellular redox status is a versatile control mechanism in gene expressions and signal transductions, including those mediating apoptosis (23, 24). Tumor necrosis factor-alpha (TNF-alpha ) is known to induce apoptosis (25). Recently, it was shown that TNF-alpha induces intracellular ROS and that free radical scavengers inhibit TNF-alpha -mediated tumor cell killing (26-28). In addition, many of the chemical and physical stimuli that elicit programmed cell death also generate ROS such as H2O2 and OH- (29). Low doses of H2O2 induce apoptosis in a variety of cell types, again indicating a role of oxidative stress in regulating cell death (23, 24). The involvement of oxidative stress is also suggested in apoptosis induced by p53, transforming growth factor-beta , and Fas (30-32). Thus, it is likely that oxidative stress is a common signal transducer for diverse cell death-inducing stimuli.

The aim of this study was to evaluate the possible implication of oxidative stress in the mechanism by which HGF induces tumor cell killing. The present data provide direct evidence that HGF enhances the production of ROS in Sarcoma 180 cells, and suggest that this is a cause of the tumor cytotoxic activity of HGF.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Reagents-- The sources of materials used in this work were as follows: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) from Research Organics, Inc., Cleveland, OH; reduced form of glutathione (GSH), N-t-butyl-alpha -phenylnitrone (PBN), 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TEMPO), ascorbic acid, and superoxide dismutase from Sigma; acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-H) and Ac-DEVD-alpha -(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA) from the Peptide Institute, Osaka, Japan; Dulbecco's modified Eagle's medium from Nissui Pharmaceutical Co., Tokyo, Japan; Sarcoma 180 and Madin-Darby canine kidney (MDCK) cells from RIKEN Cell Bank, Tsukuba, Japan; Meth A mouse sarcoma cells were kindly provided by Dr. Eiichi Gohda, Faculty of Pharmaceutical Sciences, Okayama University, Japan; DNA molecular weight markers III and IX, and catalase from Roche Molecular Biochemicals, Mannheim, Germany; 2',7'-dichlorofluorescein diacetate (DCFH-DA) from Molecular Probes, Eugene, OR; N-acetyl-L-cysteine (NAC), NG-monomethyl-L-arginine monoacetate, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxyde (carboxy-PTIO), vitamin E (alpha -tocopherol), and (p-amidinophenyl)methanesulfonyl fluoride hydrochloride from Wako Pure Chemical Industrials, Osaka; monoclonal antibody PY20 against phosphotyrosine and polyclonal antibodies against mouse c-Met from Santa Cruz Biotechnology, Santa Cruz, CA. Recombinant human HGF was prepared and purified as described previously (33). Other materials used in this study were described previously (2, 22).

Assay of Tumor Cytotoxic Activity-- The tumor cytotoxic activity of HGF was determined by an MTT assay (34). Sarcoma 180 cells (2 × 105 cells/ml) were plated on Nunc 24-well plastic dishes (Roskilde, Denmark) in 0.4 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (basal medium) and cultured as described previously (22). After 24 h, the cells were preincubated with antioxidants for 1 h at 37 °C, then cultured with or without HGF at the indicated concentrations for 48 h. Because NAC itself is a reductant and interferes with the MTT assay, incubated medium with or without NAC was removed after incubation and then 400 µl of basal medium containing MTT (0.5 mg/ml) was added. This procedure almost completely rules out the reducing effect of NAC in the MTT assay. After 30 min, this medium was removed, formazan products were solubilized in 400 µl of acidified isopropyl alcohol (0.04 N HCl in isopropyl alcohol) and the absorbance of the MTT formazan products was measured at 570 nm using a reference wavelength of 650 nm. For Meth A cells, the cells (104 cells/ml) were plated on 24-well plastic dishes in 0.4 ml of RPMI 1640 (ICN Biomedicals, Aurora, OH) supplemented with 10% fetal calf serum and cultured with or without NAC in the presence or absence of HGF as described above for 96 h. After incubation, the cells were transferred to an Eppendorf tube and precipitated by centrifugation. The cells were washed once with 1 ml of NAC-free medium by centrifugation, and the MTT assay was performed as described above.

Preparation of DNA Fragments-- Cells were cultured as described above except for use of 10 ml of medium/plate in 8.5-cm plastic plates (Nunc). After incubation, the cells were collected and washed, and about 5 × 106 cells were lysed in 200 µl of 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA, 0.5% Triton X-100, and 0.1% SDS for 10 min at 4 °C as described previously (22). The lysate was centrifuged at 12,000 × g for 20 min, and the supernatant containing fragmented DNA was treated with RNase A (100 µg/ml) for 60 min at 37 °C, and then digested with proteinase K (100 µg/ml) for 60 min at 37 °C. Fragmented DNA was precipitated with isopropyl alcohol, washed with 70% ethanol and solubilized in 50 µl of 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. Then 10-µl samples were subjected to electrophoresis in 1.2% agarose gel containing 0.1 µg/ml ethidium bromide.

Assay of Caspase-3 Activity-- Caspase-3 activity was measured essentially as described by Enari et al. (35). Briefly, after incubation of cells in 8.5-cm plastic plates with or without additives as indicated, they were collected, washed as described above, and homogenized in 0.5 ml of extraction buffer (10 mM HEPES-KOH, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 100 µM p-(amidinophenyl)methanesulfonyl fluoride hydrochloride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A) in a Dounce homogenizer (type A pestle). The cell extracts (80-90 µg of protein) obtained as described above were diluted with 1 ml of reaction buffer (50 mM HEPES-KOH, pH 7.4, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, 0.1 mg/ml ovalbumin), and incubated for 30 min at 30 °C with 10 µM fluorescent substrate, Ac-DEVD-MCA, in the presence or absence of 10 µM of a specific inhibitor of caspase-3, Ac-DEVD-H. The fluorescence of the cleaved substrate was determined with a spectrofluorometer (Type 850 Hitachi spectrofluorometer, Hitachi, Tokyo) at an excitation wavelength of 380 nm and emission wavelength of 460 nm. Caspase-3-like activity was determined by subtracting the values obtained in the presence of inhibitor. One unit of enzyme activity corresponds to the activity that cleaves 1 pmol of the substrate at 30 °C in 1 min/mg of protein.

Measurement of Intracellular ROS by Flow Cytometry-- A peroxide-sensitive fluorescent probe DCFH-DA (36) was used to assess levels of net intracellular generation of ROS. Cells treated with HGF for the indicated times were incubated with 5 µM DCFH-DA for 30 min. After incubation, cells were washed once with Mg2+- and Ca2+-free phosphate buffered-saline, detached by trypsinization, collected by centrifugation as described above, and suspended in ice-cold phosphate-buffered saline. Levels of intracellular ROS were measured with a FACScan (Becton Dickinson, Mountain View, CA). In each analysis, 10,000 events were recorded.

Assay for Tyrosine Kinase Activity of c-Met-- After preincubation of Sarcome 180 cells (2 × 105/ml) in 8.5-cm plastic plates with or without 5 mM NAC for 1 h, they were incubated with 30 ng/ml HGF for 5 or 10 min. After incubation, they were collected, washed, lysed, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to Western blotting analysis as described previously (22). Membranes were probed with anti-phosphotyrosine or anti-mouse c-Met antibodies, and located with an ImmunoStar kit (Wako Pure Chemical Industrials, Osaka), according to the manufacturer's instructions.

Assay for Scatter Activity-- The scatter activity was assayed according to Stoker et al. (37). Briefly, MDCK cells were seeded at a density of 2 × 104 cells/ml of Dulbecco's modified Eagle's medium, containing 10% fetal calf serum in a 24-well plastic dish (0.4 ml/well), and they were allowed to attach for 24 h. The cells were preincubated with or without NAC (5 mM) for 1 h, and then incubated with or without HGF (30 ng/ml). The cells were further incubated for 24 h.

Other Methods-- Protein was determined with BCA Protein Assay Reagent (Pierce, Rockford, IL). Levels of the reduced form of glutathione were determined in cell extracts by the method of Hissin and Hilf (38), measuring the fluorescence produced by o-phthalaldehyde as fluorescent reagent at an excitation wavelength of 350 nm and emission wavelength of 420 nm. Student's t test for unpaired samples was used to compare mean values of groups according to Snedecor and Cochran (39).

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ABSTRACT
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Prevention of HGF-suppressed Growth of Sarcoma 180 Cells and Meth A Cells by Antioxidants-- To study the role of oxidative stress on the tumor cytotoxic activity of HGF, we first examined the effect of the glutathione precursor NAC, which is widely used as an intracellular free radical scavenger and has been shown to prevent diverse cell death-inducing stimuli (30, 31), on HGF-suppressed growth of Sarcoma 180 cells. As described previously (22), incubation of the cells with HGF (30 ng/ml) for 48 h resulted in reduction of viability to about 50% of that of control cells (Fig. 1). As shown in Fig. 1A, when the cells were incubated with NAC, the HGF-suppressed growth was restored to nearly the control level. NAC was effective at 1 mM and 5 mM NAC restored HGF-suppressed viability to about 85% of that of control cells. NAC itself (over 2 mM) had weak cytotoxic activity on the cells and 5 mM NAC decreased the viability about 16%. These results indicate that NAC completely prevented HGF-suppressed growth of the cells. In addition, we found that NAC did not shift the concentration of HGF required for growth suppression. As shown in Fig. 1B, 100 ng/ml HGF decreased the viability of the cells about 60% and the growth suppression restored to the control level by NAC treatment. The preventive effect of NAC was also confirmed by cell counts. When the cells were incubated with HGF (30 ng/ml) for 48 h, cell numbers decreased to about 65% of that of control cells but the cell numbers again were restored to the control level by NAC (5 mM) treatment. Recently, HGF has been shown to induce apoptosis of Meth A cells (40). Therefore, we examined whether NAC blocks HGF-induced growth suppression of Meth A cells as Sarcoma 180 cells. As shown in Fig. 1C, HGF caused reduction in the viability of the cells about 30%, and NAC (5 mM) almost completely restored the HGF-suppressed viability.


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Fig. 1.   Effect of NAC on HGF-induced growth suppression of Sarcoma 180 (A and B) and Meth A (C) cells. Cells were preincubated with indicated concentrations (A) or 5 mM (B and C) NAC for 1 h and then cultured with 30 ng/ml (A and C) or the indicated concentrations (B) of HGF for 48 h (A and B) or 96 h (C). Cell viability was analyzed by MTT assay as described under "Experimental Procedures." Data are mean ± S.D. for three independent experiments. *, p < 0.01; **, p < 0.001 (versus with HGF alone).

Other antioxidants and free radical trappers, e.g. GSH, ascorbic acid, superoxide dismutase, catalase, vitamin E, PBN, and TEMPO, are also reported to prevent cell death induced by several cell death-inducing stimuli (30, 31, 41-43). Therefore, we next examined the effects of these compounds on HGF-suppressed growth of the cells. As shown in Fig. 2, GSH prevented the effect of HGF in a dose-dependent manner as effectively as NAC. Ascorbic acid partially prevented the effect of HGF, but other compounds at the concentrations tested did not have protective effects. PBN and TEMPO had cytotoxic effects on the cells. When the cells were incubated with 5 mM NAC for 48 h, the intracellular GSH level increased and the mean GSH contents (n = 2) were 5.4, 12.1, and 11.1 µg/mg protein, in cell extracts without NAC, with NAC, and with NAC plus HGF (30 ng/ml), respectively. These results suggest a role of intracellular GSH in the protective effect of NAC against HGF-induced growth suppression.


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Fig. 2.   Effects of antioxidants on HGF-induced growth suppression of Sarcoma 180 cells. Cells were preincubated with antioxidants at the indicated concentrations for 1 h and then cultured with (closed bars) or without (open bars) 30 ng/ml HGF for 48 h. Viability of the cells was analyzed as described in the legend to Fig. 1. Data are mean ± S.D. for three independent experiments. VE, vitamin E; SOD, superoxide dismutase; *, p < 0.001; **, p < 0.02 (versus value with HGF alone).

Prevention of HGF-induced Morphological Changes and Apoptosis in Sarcoma 180 Cells by NAC-- HGF also caused morphological changes of the cells (Fig. 3). Sarcoma 180 cells without HGF had a flattened morphology during culture (Fig. 3A). But when they were incubated with HGF (30 ng/ml) for 48 h, many cells became condensed and rounded with an irregular plasma membrane (Fig. 3B). Detachment of degenerated cells was always observed. These HGF-induced morphological changes were completely inhibited by co-incubation with 5 mM NAC (Fig. 3C), the cells showing a normal morphology, indicating the involvement of oxidative stress in HGF-induced morphological changes.


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Fig. 3.   Morphological changes of Sarcoma 180 cells induced by HGF. Cells were preincubated with (C and D) or without (A and B) 5 mM NAC for 1 h, and then cultured in the presence (B and C) or absence (A and D) of 30 ng/ml HGF for 48 h. Then they were examined by phase-contrast microscopy (magnification, × 200). Note that when the cells were cultured with 30 ng/ml HGF for 48 h, many of them have condensed and rounded showing an irregular shaped plasma membrane (B), but that these morphological changes were inhibited by co-incubation with 5 mM NAC (C). Similar results were obtained in four independent experiments.

Previously, we found that HGF activates apoptosis in the cells, as assessed by DNA fragmentation and an increase in caspase-3 activity (22). As shown in Fig. 4A, incubation of the cells with HGF (30 ng/ml) for 48 h resulted in the formation of high molecular weight DNA fragments of about 20 kilobase pairs, as well as oligonucleosomal DNA fragments. HGF also increased caspase-3 activity to about 2.4 times the control value after 48 h incubation (Fig. 4B). Both HGF-induced DNA fragmentation and caspase-3 activity were almost completely inhibited by NAC (5 mM), again establishing a role of oxidative stress in regulating HGF-induced apoptosis. The results also indicated that HGF-increased caspase-3 activity may be mediated directly or indirectly by a redox-sensitive pathway, as suggested by others (44, 45).


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Fig. 4.   Inhibition of HGF-induced apoptosis of Sarcoma 180 cells by NAC. Panel A, cells were preincubated with (lanes 3 and 4) or without (lanes 1 and 2) 5 mM NAC for 1 h, and cultured with (lanes 2 and 3) or without (lanes 1 and 4) 30 ng/ml HGF for 48 h. DNA fragments were prepared, and analyzed as described in "Experimental Procedures." Panel B, cells were preincubated with or without 5 mM NAC for 1 h, and cultured in the presence or absence of 30 ng/ml HGF for 48 h. Cell extracts were prepared and caspase-3 activity was measured as described under "Experimental Procedures." Values are mean ± S.D. for three independent experiments. *, p < 0.001 (versus without HGF); **, p < 0.002 (versus HGF alone).

Enhancement of Formation of Intracellular ROS in Sarcoma 180 Cells by HGF-- We next investigated the effect of HGF on the formation of intracellular ROS by flow cytometric analysis using a peroxide-sensitive fluorescent probe DCFH-AD (36). This probe primarily measures hydrogen peroxide and the hydroxy radical. As shown in Fig. 5A, HGF caused an increase in the intracellular ROS level within 7 h incubation and increase in the intracellular ROS level was completely suppressed by co-incubation with 5 mM NAC (Fig. 5B). Increase in ROS was also observed at 24 h (Fig. 5C) and 48 h (data not shown) after HGF treatment. NAC suppressed this increase (Fig. 5D, data not shown for 48 h).


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Fig. 5.   Intracellular ROS production in Sarcoma 180 cells by HGF and its inhibition by NAC. Cells were preincubated with (B and D) or without (A and C) 5 mM NAC for 1 h, and cultured with (black area) or without (white area) 30 ng/ml HGF for 7 (A and B) or 24 h (C and D). Cells were treated with the peroxide-sensitive fluorescent probe DCFH-DA (5 µM) during the final 30 min of each treatment. Relative peroxide concentrations in the cells were then quantitated by flow cytometry. Representative results for four experiments are shown.

Effect of NAC on HGF-stimulated Scattering of MDCK Cells-- Finally, we investigated whether NAC prevents the HGF-induced scattering (motility) of MDCK cells, which is one of well known biological activities of HGF (4, 8). As shown in Fig. 6B, HGF treatment induced scattering in MDCK cells after 24 h of incubation. However, NAC did not prevent the HGF-induced scattering (Fig. 6C). NAC itself had no affect the scattering of the cells (Fig. 6D).


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Fig. 6.   Effect of NAC on HGF-stimulated scattering of MDCK cells. Cells were preincubated with (C and D) or without (A and B) 5 mM NAC for 1 h, and then cultured in the presence (B and C) or absence (A and D) of 30 ng/ml HGF for 24 h. Then they were examined by phase-contrast microscopy (magnification, × 100).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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HGF has diverse biological and physiological functions including mitogenic, motogenic (scatter factor activity), morphogenic, and tumor cytotoxic activities (8). To date, however, many studies have been focused on understand the signaling pathways of HGF in mitogenic, motogenic, and morphogenic activities (46-49), but little is known about the mechanism of the signaling pathway in the tumor cytotoxic activity of HGF. In this study, we obtained the first direct evidence that HGF enhances the production of ROS in Sarcoma 180 cells (Fig. 5). The present finding that the antioxidant NAC, which increases GSH, thereby providing a substrate for the reduction of peroxide by glutathione peroxidase (50), almost completely prevented HGF-induced growth suppression, morphological changes, apoptosis, and ROS production, suggests the involvement of oxidative stress in the tumor cytotoxic activity of HGF.

HGF is a heterodimeric protein consisting of heavy and light chains with molecular masses of 54-65 and 31.5-34.5 kDa, respectively, linked together by a disulfide bond (2) and its biological activity was disappeared when it was reduced with reducing agents such as 2-mercaptoethanol (51). Because NAC is also a reductant, we examined whether HGF was reduced by NAC during incubations, thereby destroying HGF activity. When HGF (30 ng/ml) was treated with 5 mM NAC in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C for 48 h, the concentration of HGF decreased to about 18 ng/ml (60% of the initial concentration), as determined by an enzyme-linked immunosorbent assay which detects only the heterodimeric form of HGF, but not the reduced form of HGF (52). However, the decrease alone in the HGF content cannot explain all the protective effects of NAC on HGF-induced growth suppression of Sarcoma 180 cells reported in this paper, because of that (i) 10 ng/ml HGF decreased the viability of the cells more than 40% (22) and (ii) 5 mM NAC restored HGF-suppressed viability to about 85% of that of control cells (Fig. 1). In addition, we showed that growth suppression of Sarcoma 180 cells induced by 100 ng/ml HGF was also restored to the control level by NAC treatment. In this condition, it can be presumed that about 60 ng/ml HGF may still remain in the medium after 48 h incubation with 5 mM NAC, as described above, and this concentration of HGF should decrease the viability of the cells by at least 50% of that of control cells (Fig. 3B). These observations indicate that NAC does not shift the concentration of HGF required for growth suppression and suggest that the effects of NAC on the effects of HGF are mediated mostly through its antioxidant effect, although it remains to be determined whether NAC affects directly other molecules such as HGF receptor c-Met or intracellular signal transducing molecules. Interestingly, however, we found that NAC could not prevent the HGF-induced scattering of MDCK cells (Fig. 6). These results further support the involvement of oxidative stress on tumor cytotoxic activity of HGF. It also suggests the presence of other mechanism(s) than oxidative stress on the scattering activity of HGF. We recently found that HGF enhances expression of the cyclin-dependent kinase inhibitor p21waf1, cLip1, SDI1 within 7 h in Sarcoma 180 cells and that this effect was not suppressed by 5 mM NAC treatment.2 Thus, HGF seems to activate diverse signaling pathways, NAC-sensitive and -insensitive pathways, involved in growth suppression of the cells.

Extracellularly added superoxide dismutase and catalase have been shown to protect against cell death induced by several stimuli inducing cell death (30, 31, 41-43). However, these enzymes did not have protective effects on HGF-induced growth suppression of Sarcoma 180 cells under the conditions used (Fig. 2). Although these enzymes have been shown to be taken up by liver cells by an endocytosis-dependent mechanism (53, 54), one possible explanation for their inability to prevent the effect of HGF is that their transports into Sarcoma 180 cells are limited. They may be instable in our system. The present results also suggest that membrane lipid peroxidation is not involved in HGF-induced growth suppression of Sarcoma 180 cells, since vitamin E, a lipophilic free radical scavenger, showed no protection from the effect of HGF.

Nitric oxide (NO) is a free radical species with multiple biological functions and has been shown to be important in the cytotoxic activity of macrophages on tumor cells and microbial pathogens (55-58). Because NAC has protective effects on NO-mediated cytotoxicity and induction of DNA single-strand breakage (57, 58) as well as oxidative damage, we examined whether NO mediates HGF-induced cytotoxicity in Sarcoma 180 cells. NG-Monomethyl-L-arginine monoacetate, which is a competitive inhibitor of NO synthases, and a NO scavenger, carboxy-PTIO, were used to test the role of NO. However, NG-monomethyl-L-arginine monoacetate (up to 3 mM) and carboxy-PTIO (up to 100 µM) did not have protective effects on HGF-suppressed growth of the cells, although carboxy-PTIO itself slightly increased the viability of the cells (data not shown), suggesting that NO is not involved in HGF-induced growth suppression.

As defense systems, mammalian cells possess antioxidative enzymes including three types of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase, which play roles in protecting cells against oxidative damage. Kayanoki et al. (59) recently reported that transforming growth factor-beta suppresses the expressions of these antioxidative enzyme genes in adult rat hepatocytes, thereby showing that production of ROS is increased in transforming growth factor-beta -treated cells. Therefore, we investigated whether HGF affects the expressions of antioxidative enzymes by Western blotting and found that incubation of the cells with HGF (30 ng/ml) for 7 h resulted in a decrease in the expression of catalase to about 40% of that of control cells, but it did not affect the expression of Cu2+, Zn2+-superoxide dismutase (data not shown). When the cells were treated with 30 ng/ml HGF for 7 h, glutathione peroxidase activity in the cell extracts was also decreased to about 80% of that of control cells (data not shown). Thus it seems likely that HGF induces intracellular ROS by suppression of the expressions of some antioxidative enzymes in Sarcoma 180 cells. We are now examining the effects of HGF on the activities and gene expressions of other antioxidative enzymes.

HGF shows opposite biological activities in regulating cell death. It induces growth suppression of some tumor cell lines (Refs. 19-22 and this study), but prevents apoptosis induced by interferon-gamma in cultured mouse hepatocytes (60). The mechanism by which HGF exerts these opposite effects is not known. In this regard, it has been suggested that alterations in number or structure, with gain of function, of growth factor receptors may result in deregulated activation of the signaling pathway possibly involved in growth suppression. An unusually high density of epidermal growth factor (EGF) receptor in A431 cells has been suggested to be related to an increased phosphotyrosine content and inhibition of proliferation of EGF-treated cells (61, 62). In this connection, it is noteworthy that Bae et al. (63) recently reported that EGF enhances the generation of intracellular ROS in A431 cells, although the role of ROS in EGF-suppressed growth of the cells has not been investigated. On the other hand, mutations of fibroblast growth factor receptor 3 found in human skeletal disorders (64) were shown to have constitutive tyrosine kinase activity and expression of mutant fibroblast growth factor receptor 3 in 293T cells caused activation of the transcription factor STAT 1 (signal transducer and activator of transcription 1), which may negatively regulate cell growth by inducing the cyclin-dependent kinase inhibitor p21 (65). Like EGF and fibroblast growth factors, all of the biological effects of HGF are transduced through its receptor, Met, by the activation of its tyrosine kinase activity (10, 11). Therefore, we examined tyrosine phosphorylation of Met in the presence of HGF and NAC. However, we found that Met in Sarcoma 180 cells was constitutively activated even in the absence of HGF as mutant fibroblast growth factor receptor 3 and EGF receptors in A431 cells (data not shown). Although treatment with HGF alone for 5-10 min slightly activated (about 2-fold) its tyrosine kinase activity in Sarcoma 180 cells, NAC had not affected the tyrosine phosphorylation of Met on the cells in the presence or absence of HGF (data not shown). These results suggest that constitutive tyrosine kinase activity of growth factor receptors is one common mechanism for deregulated activation of the signaling pathway involved in growth suppression, although HGF only slightly activated (about 2-fold) its kinase activity. Further studies are necessary for complete understanding of the mechanism by which HGF exerts its diverse biological functions as well as on how HGF enhances intracellular ROS generation.

    ACKNOWLEDGEMENTS

We thank Dr. Takehisa Ishii, Mitsubishi Chemical Corp., for providing recombinant human HGF. We also thank Dr. Kyoko Kakimoto for determination of HGF and Yoko Amita for secretarial assistance.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for a Priority Area (Cancer) and for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.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.

§ Present address: Faculty of Pharmaceutical Sciences, The University of Tokushima, 770-8505, Japan.

parallel To whom correspondence should be addressed: Dept. of Biochemistry, Kagoshima University Dental School, 35-1 Sakuragaoka-8, Kagoshima 890-8544, Japan. Tel.: 81-99-275-6130; Fax: 81-99-275-6138; E-mail: daiku{at}dentc.hal.kagoshima-u.ac.jp.

2 N. Arakaki, T. Kajihara, T. Ohnishi, and Y. Daikahara, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: HGF, hepatocyte growth factor; ROS, reactive oxygen species; TNF-alpha , tumor necrosis factor-alpha ; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; GSH, reduced form of glutathione; PBN, N-t-butyl-alpha -phenylnitrone; TEMPO, 3,3,5,5-tetramethyl-1-pyrroline-1-oxide; Asc, ascorbic acid; Ac-DEVD-H, acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-DEVD-MCA, Ac-DEVD-alpha -(4-methyl-coumaryl-7-amide); MDCK, Madin-Darby canine kidney; DCFH-DA, 2',7'-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; NO, nitric oxide; EGF, epidermal growth factor; STAT, signal transducer and activator of transcription; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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