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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 220-226, 2002
© 2002 American Thoracic Society

A Two-way Interaction between Hepatocyte Growth Factor and Interleukin-6 in Tissue Invasion of Lung Cancer Cell Line

Yasuo To, Makoto Dohi, Kunio Matsumoto, Ryoichi Tanaka, Atsushi Sato, Kazuyuki Nakagome, Toshikazu Nakamura and Kazuhiko Yamamoto

Pulmonary Section, Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, Tokyo; and Division of Molecular Regenerative Medicine, Department of Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

Address correspondence to: Makoto Dohi, M.D., Ph.D., Pulmonary Section, Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongoh, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: DOHI-PHY{at}h.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although both hepatocyte growth factor (HGF) and interleukin (IL)-6 play important roles in invasion of cancer cells, interaction between these two critical factors has not been well elucidated. In the present study we demonstrated a two-way interaction between HGF and IL-6 in in vitro invasion of a lung cancer cell line. A549 lung adenocarcinoma cells were stimulated with IL-6, and this treatment induced an upregulation of c-Met/HGF receptor mRNA expression in the cells. In addition, IL-6 enhanced the HGF-induced in vitro cell invasion. This effect was abolished by pretreatment of the cells with either anti–IL-6 neutralizing antibody or with anti–c-Met/HGF receptor blocking antibody. We also found that HGF upregulated the expression of IL-6 receptor mRNA in the same cell line, and that this upregulation enhanced the IL-6–induced cell invasion. Finally, costimulation with HGF and IL-6 showed an additive effect on invasion, and this effect was mediated by production of matrix metalloproteinase (MMP)-2 and MMP-9. These results suggest that HGF and IL-6 upregulate each other's receptors, and thus would cooperatively enhance tissue invasion. They also suggest an "autocrine circuit" among cytokines and growth factors in certain cancer cells which functions to accelerate their biologic activities such as metastatic property.

Abbreviations: fetal calf serum, FCS • glycerylaldehyde-3-phosphate dehydrogenase, GAPDH • hepatocyte growth factor, HGF • interleukin, IL • matrix metalloproteinase, MMP • phosphate-buffered saline, PBS • reverse transcriptase-polymerase chain reaction, RT-PCR • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue invasion and metastasis often determine the prognosis of patients with cancer, so their regulation is critical for the treatment of cancer. Some cytokines or growth factors play important roles in carcinogenesis as well as in normal tissue repair, and overexpression of these cytokine and growth factors sometimes causes carcinogenesis of the same cell or tissue. For example, hepatocyte growth factor (HGF) is a multipotential growth factor that plays an essential role in tissue repair and regeneration processes (1). As a therapeutic application, HGF has been tried for therapy of fibrosing diseases of liver, kidney, and lung (2, 3). On the other hand, HGF has various functions which could promote invasion and metastasis of cancer cells, including proliferation of epithelial cells (4), cell scattering, angiogenesis (1), and enhancing matrix metalloproteinase (MMP) activity (5). Actually, overexpression of HGF was reported in human non–small cell lung cancer and mesothelioma (6). HGF–c-Met receptor autocrine-loop enhances tumorigenecity in some lung cancers (7). An increased level of HGF was detected in the extract from tumor tissue of adenocarcinoma, diffuse type bronchioloalveolar cell carcinoma, and squamous cell carcinoma (8, 9). In addition, a high HGF level in tumor tissue correlated with poor prognosis (9). NK4, a four-kringle antagonist of HGF, suppressed tumor growth by inhibiting angiogenesis (10). These findings strongly suggest that the HGF/c-met signaling system plays an important role in the development of lung cancers by either autocrine or paracrine mechanisms.

On the other hand, interleukin (IL)-6 would also play some role in tumor cell growth and progression. IL-6 is associated with poor prognosis in cancer cells of bone, colon, and breast (11). For lung cancer, there are several studies that showed an increased concentration of IL-6 in the sera of lung cancer patients (12–14), which correlated with poor prognosis (12, 15). These findings support the idea that IL-6 also plays some role in the process of progression or invasion of cancer cells of the lung. However, the mechanism of IL-6 elevation in patients with cancer is not clear. At present, the effect of IL-6 on tumor is controversial. In mice, IL-6 showed an antitumor effect (16). IL-6 could have a direct inhibiting effect for solid tumor cell lines (17) and for experimental metastasis in mice (18). Therefore, IL-6 could be induced as a part of antitumor immunity (16, 19). However, in clinical trials with recombinant human IL-6, no clear results were obtained (20). In addition, IL-6 even stimulated tumor growth (21). Another study suggests that elevation of IL-6 might merely reflect an inflammatory reaction of the body (13). In the studies using the A549 lung cancer cell line, IL-6 stimulated (22) or inhibited (23) tumor cell growth. In other studies, IL-6 regulated differentiation of A549 cells (24). Hence, no clear conclusion has been drawn even for the same cell line.

As mentioned above, both HGF and IL-6 play important roles; there are only a few studies that examined the relation or interactions of these two factors. Injection of IL-6 increases the serum HGF concentration in patients with cancer (25). In another study, a synergistic effect of HGF and IL-6 was reported for proliferation of a cholangiocarcinoma cell line (26). These findings suggest some interaction might exist between these two factors.

The purpose of the present study is to elucidate the relation between HGF and IL-6 in the process of invasion of lung cancer cells. We found that both of them upregulated each other's receptors in a lung adenocarcinoma cell line. This effect enhanced the tissue invading capacity of the cells in vitro, and we also found that it was mediated by production of MMP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Line and Reagents
The A549 human adenocarcinoma cell line was obtained from the American Type Culture Collection (Rocksville, ML). Recombinant human (rh) IL-6 was purchased from GIBCO BRL (Gaithersburg, MD). Anti-human IL-6 antibody (made in goat) and anti-human HGF antibody (goat) were from Genzyme (Cambridge, MA). Anti-human c-Met/HGF receptor blocking antibody (goat) was obtained from R&D Systems, Inc. (Minneapolis, MN). The Superscript Preamplification System for First Strand cDNA Synthesis was from Life Technologies Inc., (Gaithersburg, MD). Jumpstart REDTaq DNA polymerase was obtained from Sigma-Aldrich Inc. (St. Louis, MO). The 48-well chemotaxis (Boyden) chamber was from Neuro Probe, Inc. (Gaithersburg, MD). The polycarbonate filter (8 µm pore size) was from Nucleopore (Costar, CA). Matrigel was obtained from Collaborative Research, Inc. (Bedford, CA). The ELISA kit for human IL-6 was from Genzyme-Techne (Minneapolis, MN). The Gelatin-Zymo electrophoresis Kit was from Yagai Central Institute (Yamagata, Japan). All other reagents were of analytical grade.

Cell Culture and Stimulation with Cytokines
The A549 cells were cultured in Ham's F-12 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml), as reported previously (27). Cells were grown to confluence in complete media, then washed with phosphate-buffered saline (PBS) and changed to serum-free medium. Fresh serum-free medium was added 24 h before the experimental period unless otherwise indicated. Then each dish was stimulated with various concentrations of IL-6 in serum-free medium (0–300 ng/ml) individually. In another group of experiments, cells were stimulated with various concentrations of HGF (0–100 ng/ml). In designated experimental groups, anti-human IL-6 antibody (100 µg/ml) or anti-HGF neutralizing antibody (100 µg/ml), or anti–c-Met/HGF receptor antibody (100 µg/ml) was also added into serum-free medium according to the study protocols.

Reverse Transcriptase–Polymerase Chain Reaction
Total cellular RNAs were isolated by acid guanidine thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene Co. Ltd., Tokyo, Japan). Then, 1.5 µg of each RNA was reverse-transcripted at 37°C for 50 min using Superscript II Reverse Transcriptase and Oligo (dT) primer. Reverse transcriptase was then inactivated by heating at 70°C for 15 min, and RNase was added to remove the template RNA. PCR was performed with 1x PCR Gold buffer (Perkin-Elmer, Norwalk, CT), 2.5 mM of each dNTPs and 1.25 units of Taq DNA polymerase with GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA). PCR for c-Met consisted of 1 min of denaturation at 94°C, 1 min of annealing at 48°C, and 1 min of extension at 72°C for 30 cycles. PCR for IL-6 receptor (IL-6R) consisted of 30 s of denaturation at 94°C, 30 s of annealing at 60°C, and 30 s of extension at 72°C for 30 cycles. PCR for IL-6 consisted of 40 s of denaturation at 94°C, 90 s of annealing at 57°C, and 90 s of extension at 72°C for 35 cycles. PCR for GAPDH consisted of 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1 min of extension at 72°C for 30 cycles. Each set of the sense and anti-sense primers for PCR is presented in Table 1 (28). Aliquots of PCR products (10 µl) were electrophoresed in a 2.5% agarose gel. The results were visualized by ethidium bromide staining. Densitometric analysis of these mRNA expressions was performed using NIH Image software (National Institutes of Health, Bethesda, MD). Results are expressed as a ratio of mRNA of the gene examined to the mRNA of GAPDH for semiquantitative analysis. The values presented are a ratio of c-Met (or IL-6, IL-6R)/GAPDH from each experimental sample to the c-Met (or IL-6, IL-6R)/GAPDH ratio from the control sample. Each experiment was repeated at least four times, and values were expressed as means ± SEM.


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  		TABLE 1 PCR primer sequences

 
In Vitro Invasion Assay with Matrigel
In vitro invasion assay of A549 cells was performed by the Boyden chamber using filters coated with Matrigel, essentially following the methods described previously (29). Briefly, both the upper and lower sides of polycarbonate filters were coated with Matrigel and placed in the Boyden chambers. A549 cells were pretreated with rhIL-6 (0, 10, 30, 100, and 300 ng/ml, respectively) for 24 h. Each cell group was resuspended in serum-free medium (1.0 x 105 cells/ml). Forty-five microliters of the cell suspensions were placed into the upper chamber wells. The lower chamber wells were filled with either 25 µl of growth medium that contained 30 ng/ml of rhHGF or medium alone. After loading the cell samples, the chambers were incubated for 6 h at 37°C in 5% carbon dioxide containing air. At the end of incubation, cells that remained on upper surface of the filter were gently removed with a rubber swab. Then, the cells that had migrated to the lower surface of the filter were stained with Diff-Quik staining solutions (Baxter, McGraw Park, IL), and counted using a light microscope. The number of the cells were counted for 10 microscopic fields (magnification: x200) and the examination was triplicated. In another group of experiments, the cells were stimulated with rhHGF (30 ng/ml), and were placed in the upper chamber wells. In the lower wells, growth medium with 100 ng/ml of rhIL-6 or medium alone was placed. Then, the invasion cell assay was performed and analyzed in the same way as described above.

Measuring IL-6 in Culture Supernatants
Concentrations of IL-6 in the supernatant were measured using the ELISA kit, following the manufacturer's instruction. Briefly, a flat-bottomed immunoassay plate was precoated with murine anti-human IL-6 monoclonal antibody, and then incubated with nondiluted supernatants or standard human IL-6. After incubating the plate with an IL-6 conjugate for 2 h at room temperature, it was washed, and a substrate solution was added. The reaction was stopped with 2 N sulfuric acid. A Microplate Reader at 450 nm measured the plate, and the data were analyzed with Microplate Manager III, Version 1.45 for Macintosh (Bio-Rad Laboratories, Hercules, CA).

Gelatin Zymography
SDS-PAGE gelatin zymography was performed with Gelatin-Zymo electrophoresis Kit. After stimulation with IL-6 or HGF, 40 µl of each culture supernatant diluted with equal amounts of the loading buffer attached to the Kit was electrophoresed with polyacrylamide gels containing gelatin for 20 min at 10 V and for 80 min at 20 V. After washing with the washing buffers, gels were incubated at 37°C for 40 h and then stained with the staining buffer for 30 min. Then the gel was destained in a buffer containing 5% acetic acid glacial, and 10% methanol in distilled water. Finally, the gel was dried. Gelatinolytic activity was identified as white bands on a blue background. The experiment was repeated at least three times to confirm its reproducibility.

Statistical Analysis
Values were expressed as the means ± SEM. The statistical analysis was performed using the StatView Ver. 4.5J software for Macintosh (Abacus Concepts, Inc., Berkeley, CA). Two-way ANOVA was used for evaluation, and P < 0.05 was recognized as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IL-6 on the c-Met mRNA Expression in A549 Cells
In a previous study we found that interferon (IFN)-{gamma} upregulated c-Met mRNA expression of A549 cells, but other cytokines such as IL-1, IL-4, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, platelet-derived growth factor, tumor necrosis factor (TNF)-{alpha}, and transforming growth factor-ß did not (27). In addition, HGF itself did not upregulate c-Met receptor mRNA expression in an autocrine manner. In the present study, a pilot study found that IL-6 upregulated c-Met mRNA expression. Then we determined the optimal concentration and optimal stimulation time of IL-6 at which the c-Met mRNA reached the maximal expression (Figures 1A and 1B) . The optimal concentration of IL-6 was 100 ng/ml, and the optimal time for stimulation was 24 h. The effect of IL-6 was blocked by neutralizing antibody (Figure 1A).



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  		Figure 1. Effect of IL-6 on c-Met mRNA expression of A549 cells. (A) A549 cells were stimulated with IL-6 (0–300 ng/ml) for 24 h and total RNA was extracted. In designated samples, anti–IL-6 neutralizing antibody (100 µg/ml) was added to the culture medium. (B) The cells were stimulated with IL-6 (100 ng/ml) for 0–48 h. From each sample total RNA was extracted, then cDNA was synthesized and RT-PCR was performed for detecting the c-Met gene. Results were analyzed semiquantitatively as relative densitometric graph (ratio of c-Met to GAPDH). The values presented are a ratio of c-Met/GAPDH from each dosage or time point to the c-Met/GAPDH ratio from control samples (medium alone or incubated for 0 h). Values represent the means ± SEM for groups of four individual experiments. *P < 0.05 compared with the baseline ratio without IL-6 (A) or without incubation (B) (ANOVA). Representative bands in gel-electrophoresis are presented.

 
Effect of IL-6 on HGF-Induced Cell Invasion
To confirm that the c-Met receptor that was upregulated by IL-6 would be biologically functional at protein level, we next performed an in vitro cell invasion assay with Matrigel. As shown in Figure 2 , pretreatment of the cells with IL-6 for 24 h significantly enhanced the migration activity of A549 cells toward HGF. Similar to the results of c-Met mRNA expression, this invasion activity was optimal with 100 ng/ml of rhIL-6. Pretreatment of the cells with anti–c-Met antibody (100 µg/ml) inhibited the IL-6–mediated enhancement of HGF-induced migration, suggesting that the enhancement of the function was mediated through the c-Met receptor. In this assay, IL-6–treated cells alone (without HGF in the lower wells) showed a slight migration activity in a dose-dependent manner, which might be caused by an effect of IL-6 such as chemokinesis other than that through the HGF/c-Met pathway. Pretreatment of the cells with anti-human IL-6 neutralizing antibody (100 µg/ml) completely inhibited the whole effect of IL-6.



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  		Figure 2. Effect of IL-6 treatment on HGF-induced in vitro cell invasion of A549 cells. The cells were pretreated with IL-6 (0–300 ng/ml) for 24 h. After washing, the cells were placed into the upper chambers of the Boyden's chamber. The lower chambers were filled with the Ham's F-12 medium with or without 30 ng/ml of HGF. The whole chamber was incubated at 37°C for 6 h. The cells that had migrated to the lower surface of the filter were stained with Diff-Quik staining solutions, and were counted using a light microscope. Values represent the means ± SEM for groups of four to five samples in each group. In designated groups, during stimulation with IL-6, the cells were co-incubated with anti–c-Met antibody or with anti–IL-6 neutralizing antibody (100 µg/ml, respectively). **P < 0.01 compared with the results from the group without IL-6 pretreatment or those from the antibody-treated groups. #P < 0.05 compared with the results from the group without HGF in the lower chamber (ANOVA).

 
Effect of HGF on IL-6R mRNA Expression of A549 Cells
To study the possibility of an interaction between IL-6 and HGF, we next studied whether HGF may have some effect on IL-6R expression in A549 cells. As shown in Figure 3 , HGF upregulated cell surface IL-6R mRNA expression. The optimal dose and time of stimulation with rhHGF was 30 ng/ml and 24 h, respectively.



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  		Figure 3. Effect of HGF on IL-6R mRNA expression on A549 cells. (A) The cells were stimulated with rhHGF (0–100 ng/ml) for 24 h. (B) The cells were stimulated with rhHGF (30 ng/ml) for 0–48 h. From each sample total RNA was extracted, then cDNA was synthesized and RT-PCR was performed for detection of the IL-6R gene. The results were analyzed semiquantitatively as a relative densitometric graph (ratio of IL-6R to GAPDH). The values presented are a ratio of IL-6R/GAPDH from each dosage or time point to the IL-6R/GAPDH ratio from control samples (medium alone or incubated for 0 h). The values are results from four individual experiments. *P < 0.05 compared with the baseline ratio (ANOVA). Representative bands in gel-electrophoresis are also presented.

 
Effect of HGF on IL-6–Induced Invasion of A549 Cells
We next performed an in vitro invasion assay with Matrigel using A549 cells prestimulated with HGF (Figure 4) . Stimulation with HGF brought a marked increase in cell migration toward IL-6 compared with that of controls. Anti-HGF neutralizing antibody or anti–c-Met antibody abolished the IL-6–induced migration that was enhanced by HGF.



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  		Figure 4. Effect of HGF on IL-6–induced in vitro cell invasion of A549 cells. The cells were pretreated with rhHGF (30 ng/ml) for 24 h. In designated samples, cells were co-incubated with anti-HGF neutralizing antibody or with anti–c-Met antibody (100 µg/ml, respectively). After washing, the cells were placed into the upper chambers of the Boyden's chamber. The lower chambers were filled with the Ham's F-12 medium with 100 ng/ml of rhIL-6. The whole chamber was incubated at 37°C for 6 h. The cells that had migrated to the lower surface of the filter were stained with Diff-Quik staining solutions, and were counted using a light microscope. Values represent the means ± SEM for groups of four to five samples in each group. **P < 0.01.

 
Effect of HGF on IL-6 mRNA and Protein Expression of A549 Cells
We also studied the effect of HGF on the expression of IL-6 itself (Figure 5) . HGF upregulated the mRNA expression of IL-6 with an optimal concentration and incubation time of 100 ng/ml and 24 h, respectively (Figures 5A and 5B). The result of ELISA also confirmed an increased production of IL-6 protein in the culture supernatant (Figure 5C). A significant difference was detected between samples without HGF and samples with 30 or with 100 ng/ml of HGF (P < 0.01). On the other hand, IL-6 alone did not upregulate the IL-6 mRNA nor IL-6R mRNA expression in an autocrine manner (data not shown).



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  		Figure 5. Effect of HGF on IL-6 mRNA and protein expression of A549 cells. (A) The cells were stimulated with rhHGF (0–100 ng/ml) for 24 h. (B) The cells were stimulated with rhHGF (30 ng/ml) for 0–48 h. From each sample total RNA was extracted, then cDNA was synthesized and RT-PCR was performed for detecting the IL-6 gene. The results were analyzed semiquantitatively as a relative densitometric graph (ratio of IL-6 to GAPDH). The values presented are a ratio of IL-6/GAPDH from each dosage or time point to the IL-6/GAPDH ratio from control samples (medium alone or incubated for 0 h). (C) The cells were stimulated with rhHGF (0–100 ng/ml) for 24 h. IL-6 concentration in the supernatant was measured by ELISA. Values represent the means ± SEM for groups of four individual experiments. *P < 0.05 and **P < 0.01 compared with the baseline ratio (ANOVA). Representative bands in gel-electrophoresis are also presented.

 
Additive Effect by IL-6 and HGF on In Vitro Cell Invasion
Based on the findings that IL-6 and HGF upregulated each other's surface receptors, we studied the effect of costimulation with these two factors on in vitro cell invasion (Figure 6) . Costimulation of the factors potentially enhanced cell invasion. Simultaneous treatment of the cells with anti–IL-6 antibody and with anti-Met antibody significantly abolished this effect.



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  		Figure 6. Additive effect of IL-6 and HGF on in vitro cell invasion. A549 cells were incubated with rhIL-6 (100 ng/ml) and/or rhHGF (30 ng/ml) for 24 h. Then the cells were washed and resuspended in serum-free medium. The cell suspension was placed in the upper chamber wells. In the lower chamber wells, 25 µl of medium containing rhIL-6 (100 ng/ml) and rhHGF (30 ng/ml) was applied. In some experimental groups, cells were co-incubated with anti–IL-6 antibody and anti-c-Met antibody (100 µg/ml, respectively) during the stimulation. Then, in vitro cell invasion assay with Matrigel was performed as described in MATERIALS AND METHODS. **P < 0.01. *P < 0.05.

 
Additive Effect by IL-6 and HGF on Gelatinolytic Activity of A549 Cells
Finally, to elucidate the mechanism to enhance the cell invasion, we performed the gelatin zymography assay with culture supernatant of the stimulated cells (Figure 7) . Treatment of the A549 cells with either HGF or with IL-6 enhanced the gelatinolytic activity of both MMP-2 and MMP-9. They also enhanced the production of the active form of MMP-2. Simultaneous treatment of the cells with HGF and IL-6 further upregulated the production of these MMPs. Pretreatment of the cells with anti–c-Met antibody and anti–IL-6 antibody suppressed this activity. These results suggested that the enhanced cell invasion activity by HGF or IL-6 was at least partly mediated by the production of MMP-2 and MMP-9.



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  		Figure 7. Gelatin zymography of MMPs in the culture supernatant. A549 cells were stimulated with rhIL-6 (100 ng/ml) and/or rhHGF (30 ng/ml) for 24 h. In designated samples, cells were co-incubated with anti–IL-6 antibody and anti–c-Met antibody (100 µg/ml, respectively). After stimulation, 40 µl of each culture supernatant was electrophoresed with polyacrylamide gels containing gelatin. After washing with the washing buffers, gels were incubated at 37°C for 40 h and stained with the staining buffer, then destained. The experiment was repeated at least three times to confirm its reproducibility, and the figure shown is a representative case of the repeated experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study clearly showed that HGF and IL-6 worked cooperatively and accelerated invasion-stimulatory activity by upregulating each others surface receptors, and that this effect was at least partly mediated by production of MMPs. This would be the first study that clearly demonstrated the additive effect of HGF with other cytokines in the process of cell invasion in lung cancer cells.

There are several studies that elucidated the effects of growth factors and cytokines on the invasion of cancer cells. Bredin and coworkers reported that insulinlike growth factor-I and -II, HGF, epidermal growth factor, and stem cell factor independently showed an invasion-stimulating capacity in a Matrigel assay system (28). In breast cancer carcinoma cell lines, HGF, IL-6, IL-8, and IL-11 demonstrated invasion-stimulating capacity in a similar system (11). However, the interaction among these factors has not been studied.

Although both HGF and IL-6 have been recognized as a cytokine or a growth factor that plays an important role in tissue invasion of cancer cells, there are limited number of studies reporting a relation between these two factors. These previous studies were mainly on liver tumors (30, 31). By in situ hybridization, Ho and coworkers demonstrated the coexpression of HGF and IL-6 in 14 liver and 4 kidney malignant tissues (30). IL-6 induces HGF production in patients with breast cancer or non–small cell lung cancer (25). In hepatocellular carcinoma cell lines, both HGF and IL-6 induce c-Met receptor expression (31). In another study, both HGF and IL-6 induced the phosphorylation of c-Met receptor in a cholangiocarcinoma cell line or primary culture of biliary epithelial cells (26). The current results demonstrated that a similar cooperative relation between HGF and IL-6 would exist in lung cancer cells. In addition, we clarified that one mechanism of this cooperation was upregulating each other's surface receptors. Furthermore, they would suggest that there might be "an autocrine growth control circuit" (26) that is accelerated by upregulating their receptors as a mechanism of cancer cells to potentiate their property for invasion. This possibility should be further investigated in other cancer cells.

In the present study we used the A549 adenocarcinoma cell line. Crestani and coworkers reported that IL-1ß, TNF-{alpha}, or combinations of IFN-{gamma} plus LPS induced IL-6 gene and protein expression in this cell line (32). Their results and those of the present study suggest that alveolar macrophage secretary proteins such as IL-1ß, TNF-{alpha}, and HGF play an important role in the regulation of intra-alveolar immune response or carcinogenesis through the production of IL-6 from alveolar epithelial cells. On the other hand, the effect of IL-6 on A549 cell proliferation is controversial. One study demonstrated that IL-6 inhibits the growth of A549 cells (23), whereas another showed the opposite result (22). This should be further elucidated. In addition, differences in the mechanisms of cell proliferation and of cell invasion should also be considered.

In the present study, we found that both HGF and IL-6 induced production of MMPs (MMP-2 and MMP-9). Both of them produced active forms of MMP-2 as well as an inactive pro–MMP-2, although production of the active form of MMP-9 was not clear in our experiments (Figure 7). It is widely recognized that HGF produces MMPs and membrane type (MT)-1 MMP in various cancer cell lines such as hepatocellular carcinoma, prostate cell cancer, glioma cells, rectal carcinoma cells, and squamous cell carcinoma (33). This production plays a critical role in the process of invasion of the cells. This effect of HGF on MMP production was also reported in the case of endothelial cells, which might play an important role in angiogenesis (34). The present findings were basically similar to the results of these previous studies.

On the other hand, studies on the effect of IL-6 on MMP production from normal or cancer cells are limited to those of hematopoietic origin (35). In a human non-Hodgkin's lymphoma cell line, IL-6 induced MMP-2 and MMP-9, and the cell invasion capacity of the Raji and Jarkat cells assessed with the Matrigel assay was significantly increased (35). In the present study, we found a similar effect of IL-6 on MMP activity in a lung cancer cell line.

In summary, the results of the current study demonstrated that by upregulating each other's receptors, HGF and IL-6 cooperatively work to enhance tissue invasion through production of MMPs in a lung cancer cell line. Furthermore, taking the results of previous studies on other cancer cells and those of the present study together, there might be a so called "an autocrine growth control circuit" that is accelerated by upregulating their receptors as a mechanism of cancer cells to potentiate their ability for invasion. This should be further investigated, and it may lead to more effective approaches for cancer therapy.


   Acknowledgments
 
The authors thank Ms. Mayumi Katakawa and Ms. Eri Ogawa for excellent technical support. This work was supported by grants from the Japan Ministry of Health, Labor, and Welfare (No. 12670549) and from the Manabe Medical Foundation.

Received in original form December 21, 2001

Received in final form March 29, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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