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Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion
BBRC Biochemical and Biophysical Research Communications 321 (2004) 783–787 www.elsevier.com/locate/ybbrc
Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion q Daisuke Kudo a, Atsushi Kon a, Shuichi Yoshihara b, Ikuko Kakizaki a, Mutsuo Sasaki b, Masahiko Endo a, Keiichi Takagaki a,* a
Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Surgery, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Received 22 June 2004 Available online 28 July 2004
Abstract Hyaluronan (HA) is a ubiquitous, major component of the extracellular matrix. It is involved in cell adhesion and locomotion, and hence in tumor metastasis. We have previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and may be a useful tool for examining the functions of HA. We here demonstrate that the formation of cell surface HA by melanoma cells and its release into the culture medium are inhibited by MU. Adhesion and locomotion assays revealed that the adhesion and locomotion of melanoma cells were dose-dependently inhibited by MU. Conversely, treatment with exogenous HA enhanced both adhesion and locomotion. Thus, preventing the formation of cell surface HA reduced both the adhesion and locomotion of melanoma cells, suggesting that MU may act as an inhibitor of tumor metastasis. 2004 Elsevier Inc. All rights reserved. Keywords: Hyaluronan; 4-Methylumbelliferone; B16F-10 melanoma cell; Adhesion; Locomotion; Metastasis
Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) of high molecular mass, which consists of repeated b-1,4-GlcUA-b-1,3-GlcNAc disaccharide units [1]. It is present in many tissues as a major component of the extracellular matrix. HA provides a highly hydrated, gel-like environment due to its physiochemical properties, and maintains the extracellular spaces [2,3]. In addition to its structural function, HA plays an important role in tissue organization, wound healing, and inflammation [4–6]. Several studies have also demonstrated that HA participates in various phases of tumor metastasis, i.e., in cell adhesion and locomotion [7,8]. q Abbreviations: HA, hyaluronan; MU, 4-methylumbelliferone; GAG, glycosaminoglycan; FCS, fetal calf serum; DMSO, dimethyl sulfoxide; HAS, hyaluronan synthase; PBS, phosphate-buffered saline; UGT, UDP-glucuronyltransferase. * Corresponding author. Fax: +81-172-39-5016. E-mail address: [email protected] (K. Takagaki).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.041
Elevated levels of HA in tumor tissue frequently lead to metastasis and hence to a worse prognosis [9–11]. We have previously reported that HA synthesis by cultured human dermal fibroblasts was inhibited by the addition of 4-methylumbelliferone (MU) to the culture medium [12]. Moreover, we examined the mechanism by which MU inhibits HA synthesis in Streptococcus equi FM100 and demonstrated that MU did not affect expression of the HA synthase (HAS) gene but altered the lipid environment of the membrane, especially the distribution of different cardiolipin species, surrounding HAS [13]. In addition, recently we demonstrated a novel mechanism of the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UDP-glucuronyltransferase (UGT) resulting in a depletion of UDP-GlcUA using rat 3Y1 fibroblasts stably expressing HA synthase 2 (HAS2) [14]. These data indicated that MU may be a useful tool in examining the functions of HA. Indeed,
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using MU, Kosaki et al. investigated the influence of cell surface HA on the anchorage-dependent proliferation of human myofibrosarcoma cells [15], and Sohara et al. [16] studied the effect of cell surface HA on the migration of rat fibroblast cells. Malignant tumor cells are known to leave the primary lesion, enter the vascular system, and finally reach remote tissues. Various kinds of malignant tumor cells, including mesothelioma and WilmÕs tumor, produce large amounts of HA, and HA overproduction by tumor cells enhances their metastatic potential [17]. Therefore, it has long been suggested that HA may be a risk factor for tumor metastasis. Based on this concept, as a suppressor of HAS activity, MU may have an inhibitory effect on the metastasis of malignant tumor cells. In the present study, we examined the effect of MU on the formation of cell surface HA in a highly malignant tumor cell line, B16F-10 melanoma cells. We also investigated the effect of MU on the adhesion and locomotion of the melanoma cells, since these properties are important during the early stages of metastasis.
Materials and methods Materials. MU and actinase E were purchased from Sigma Chemical (St. Louis, MO) and Kaken Pharmaceutical (Tokyo, Japan), respectively. HA and Streptomyces hyaluronidase were obtained from Seikagaku (Tokyo, Japan). 2-Aminopyridine was purchased from Wako Pure Chemical (Osaka, Japan) and recrystallized from hexane. All other reagents were of analytical grade and obtained from commercial sources. Tumor cells. B16F-10 melanoma cells (henceforth referred to as melanoma cells) were kindly provided by the Cell Resource Center of the Biochemical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The cells were maintained in RPMI-1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS), L -glutamine, and antibiotics. Addition of MU to the medium was carried out as described previously [12]. Cell growth assay. Cell proliferation was determined using the Alamar Blue assay [18]. Ninety microliters of medium containing 6.25 · 102 cells in the growth phase and 1 · 104 cells in the plateau phase was dispensed into 96-well plates (Iwaki, Tokyo, Japan). After 12 h, serial dilutions of MU dissolved in dimethyl sulfoxide (DMSO) were added to a final volume of 100 ll. The concentration of DMSO in the medium did not exceed 0.1%. An MU-free well was used as a control, and a blank well containing no MU or melanoma cells was also included. After incubation periods of 24, 48, and 72 h, 10 ll of Alamar Blue dye (Sensititre/Alamar, Westlake, OH) was added to each well. The plates were incubated for a further 3 h and then read on a microplate fluorescence reader (Fluoroscan II, Labsystems, Helsinki, Finland) at excitation and emission wavelengths of 544 and 590 nm, respectively. All assays were replicated five times. Assay of HA release into the culture medium. Melanoma cells suspended at a density of 2.5 · 105/ml of culture medium were placed into 10-cm dishes. After 12 h, serial dilutions of MU were added and the dishes were incubated for a further 48 h. The medium was withdrawn and centrifuged to remove the cells. The supernatant was subjected to ethanol precipitation. After labelling with a fluorescent regent, 2-aminopyridine, by reductive amination, the HA in the precipitates was analyzed by ion-exchange high-performance liquid chromatography (HPLC) as described previously [19].
Visualization of cell surface HA. Cell surface HA was visualized using a particle-exclusion assay [20]. Fixed sheep erythrocytes (Sigma, UK) were reconstituted in phosphate-buffered saline (PBS) at a density of 5 · 108 cells/ml. Melanoma cells were cultured in 35-mm dishes. Pericellular HA was visualized by adding 0.75 ml of the erythrocyte suspension to the growth medium with or without MU and viewing under a light microscope. To demonstrate that the pericellular halo was composed of HA, MU-free dishes were preincubated with 1.0 U/ml of Streptomyces hyaluronidase prior to the assay. Adhesion assay. Melanoma cells that had been preincubated with or without MU for 48 h were released using trypsin, washed with RPMI1640 medium containing 10% FCS and suspended at 5 · 105 cells/ml in the same medium. The cell suspension (2 ml) was added to a HAcoated or HA-uncoated culture dish and incubated for 20 min at 37 C. Any non-adherent cells were removed by two gentle washes with PBS. The adherent cells were removed using trypsin, resuspended in medium, and counted with a hematocytometer. All assays were performed in triplicate. The effect of MU on cell adhesion was expressed as the relative adhesion (% of control) and was calculated as follows: relative adhesion = (number of adherent cells in experimental dishes)/(number of adherent cells in control dishes) · 100%. Locomotion assay. The locomotion assay was performed using the Matrigel Invasion Chamber (BD Bioscience, Franklin Lakes, NJ). Melanoma cells that had been preincubated with MU were suspended at 1 · 106 cells/ml in culture medium without FCS, and 500 ll of the cell suspension was placed in the upper chamber. Various concentrations of HA in 750 ll RPMI-1640 containing 5% FCS were added to the lower chamber. After incubation for 5 h at 37 C in 5% CO2, the filters were stained with hematoxylin and the cells migrating through the membrane were counted by microscopy at 100· magnification. The cells were counted in triplicate fields on three separate membranes. The effect of MU on cell invasion was expressed as the relative locomotion (% of control) and was calculated as follows: relative locomotion = (number of cells migrating under experimental conditions)/ (number of cells migrating under control conditions) · 100%.
Results and discussion MU has no significant cytotoxic effect on melanoma cell growth To determine whether MU has any direct effect on cell growth, melanoma cells were incubated with 0– 0.5 mM MU for various incubation times (24, 48, and 72 h), then their growth was assessed using the Alamar Blue assay, as described in ‘‘Materials and methods.’’ Neither the growth phase (Fig. 1A) nor the plateau phase (Fig. 1B) of cell growth was inhibited by MU. The DMSO added to the medium to dissolve the MU also had no influence on either phase of cell growth (Fig. 1). Thus, MU did not have any significant cytotoxic effect on melanoma cell growth. MU inhibits HA synthesis by melanoma cells We have previously shown that MU specifically inhibits HA synthesis in cultured dermal fibroblasts [12]. To determine whether MU also inhibits HA synthesis by melanoma cells, B16F-10 cells were incubated with various concentrations of MU for 48 h, and the HA released into the culture medium was analyzed by ion-ex-
D. Kudo et al. / Biochemical and Biophysical Research Communications 321 (2004) 783–787
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Fig. 3. Effect of MU on the synthesis of cell surface HA by cultured melanoma cells. Cell surface HA was visualized using a particleexclusion assay, as described in ‘‘Materials and methods.’’ Melanoma cells were cultured in the absence (A) or presence (B) of 0.5 mM MU. Some melanoma cells were incubated with 1.0 U/ml of Streptomyces hyaluronidase prior to the exclusion assay (C). Fig. 1. Effect of MU on the growth of melanoma cells. Melanoma cells were incubated with 0–0.5 mM MU for various incubation times. The growth of cells in the growth phase (A) and plateau phase (B) at each incubation time was determined using the Alamar Blue assay as described in ‘‘Materials and methods.’’ Each point represents the mean of five replicated experiments.
change HPLC. As shown in Fig. 2, the amount of HA released into the culture medium was decreased in a dose-dependent manner when MU was present. This reduction became apparent in cells treated with 0.1 mM of MU and maximal reduction (40%) was achieved at 0.5 mM. Next, we examined the effect of MU on the formation of cell surface HA by a particle-exclusion assay, as described in ‘‘Materials and methods.’’ When the cells were incubated without MU, a pericellular halo could be observed (Fig. 3A). On the other hand, no such halo
Fig. 2. Effect of MU on HA release from melanoma cells. Melanoma cells were incubated with various concentrations of MU for 48 h, and the HA released into the culture medium was analyzed by ionexchange HPLC. Each bar represents the mean + SD of triplicate experiments.
was apparent in the presence of 0.5 mM MU (Fig. 3B). Cells treated with 1.0 U/ml of Streptomyces hyaluronidase prior to the particle-exclusion assay also showed no halo (Fig. 3C). These results suggest that MU inhibits both the formation of cell surface HA by melanoma cells and its retention on the cell surface. HA promotes the adherence and locomotion of melanoma cells HA surrounding the cell surface is eventually released and contributes to the formation of the extracellular matrix. Several studies have shown that high HA levels in the extracellular matrix enhance the metastatic potential of malignant tumor cells [17]. Therefore, we investigated the effect of HA on adhesion and locomotion, which are characteristic properties of malignant tumor cells involved in metastasis. A suspension of melanoma cells was added to a HA-coated culture dish and incubated for 20 min at 37 C. After removing any non-adherent cells by washing, the adherent cells were counted with a hematocytometer. As shown in Fig. 4A, exogenous HA increased the relative adhesion of the melanoma cells in a dose-dependent manner. The maximal value was obtained when the cells were added to a culture dish coated with 0.5 mg/cm2 of HA. We also performed a locomotion assay using the Matrigel Invasion Chamber, as described in ‘‘Materials and methods.’’ Melanoma cells suspended in culture medium without FCS were placed in the upper chamber, and culture medium containing various concentrations of HA was added to the lower chamber.
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Fig. 4. Effect of exogenous HA on the adhesion and locomotion of melanoma cells. The influence of HA on adhesion (A) and locomotion (B) was investigated using immobilized-HA dishes and an improved model of BoydenÔs chamber, respectively. Each bar represents the mean + SD of triplicate experiments.
After incubation for 5 h, the filters were stained and the cells migrating through the membrane were counted by microscopy. As shown in Fig. 4B, the relative locomotion was also dose-dependently increased by HA. Taken together, these results indicate that HA promotes both the adherence of melanoma cells and their locomotion into the surrounding extracellular space. This further suggests that HA may accelerate metastasis to remote tissue. MU inhibits the adhesion and locomotion of melanoma cells Based on the results shown in Fig. 4, we investigated alterations in the adhesion and locomotion of melanoma cells when the formation of cell surface HA was inhibited by MU. Adhesion assay revealed that pretreatment with MU inhibited the adhesion of melanoma cells. This inhibition was dose-dependent; it became apparent in cells treated with 0.1 mM MU, and maximal inhibition (49%) was observed when the cells were preincubated with 0.5 mM MU (Fig. 5A). The locomotion assay revealed that MU also inhibits the locomotion of melanoma cells in a dose-dependent manner. This inhibition was observed in melanoma cells pretreated with as little as 0.1 mM MU, and maximal
Fig. 5. Effect of MU on the adhesion and locomotion of melanoma cells. Melanoma cells that had been preincubated with different concentrations of MU were subjected to adhesion (A) and locomotion (B) assays, as described in ‘‘Materials and methods.’’ Each bar represents the mean + SD of triplicate experiments.
inhibition (37%) was achieved after pretreatment with 0.5 mM MU (Fig. 5B). Thus, MU has inhibitory effects on both the adherence and locomotion of melanoma cells. In this study, we examined the effects of MU on HA synthesis by, and the adhesion and locomotion of, B16F-10 melanoma cells. The major findings were as follows. First, the formation of cell surface HA and its release into the culture medium were inhibited by MU. This is in agreement with our previous studies, which showed that MU specifically inhibits HA synthesis in cultured dermal fibroblasts [12]. Moreover, we have previously demonstrated that MU decreases the enzymatic activity of HAS in Streptococcus equi FM100. This inhibitory effect is achieved indirectly, by altering the distribution of one of the membrane phospholipids of S. equi FM100, cardiolipin, which in turn destabilizes HAS activity [13]. Furthermore, another inhibitory mechanism of MU that depends on the endogenous UGT activity was found using transfectants expressing mouse HAS2 [14]. Thus, MU-mediated inhibition of the formation and subsequent release of cell surface HA occur in various types of cell and may not be cell-type-specific. Second, we demonstrated that HA increases both the adhesion and the locomotion of melanoma cells, and that these increases are inhibited by treatment with MU. HA-rich matrices in malignant tumor tissues are associated with a worse prognosis because of their higher metastatic potential [17,21,22]. Furthermore, several studies have shown that the metastatic potency of malignant tumor cells is enhanced by transfection with a HAS expression construct [17,23]. In addition, injection of nude mice with HAS gene-transfected cells produces tumors smaller than those formed by control cells [15]. Conversely, inhibiting HAS expression in tumor cells with an antisense HAS construct diminishes not only HA synthesis and the formation of cell surface HA but also cell adhesion [24,25]. Thus, HA-rich matrices provide a suitable environment for metastatic changes such as vascularization and invasion [26,27]. Taken together with these earlier findings, the results of our present study strongly suggest that MU-mediated inhibition of adhesion and locomotion may inhibit the metastasis of melanoma cells, which are accelerated by HA. In conclusion, using the novel approach of inhibiting HA synthesis with MU, we demonstrated that the formation of cell surface HA is necessary for the adhesion and locomotion of melanoma cells. Since these properties are characteristic of tumor cells involved in the early stages of metastasis, our results suggest that MU may act as an inhibitor of metastasis. Therefore, MU is not only a useful tool for investigating the various functions of HA but also has potential as a novel therapeutic agent that may control the metastasis of melanoma cells.
D. Kudo et al. / Biochemical and Biophysical Research Communications 321 (2004) 783–787
Acknowledgments This work was supported by Grants-in-Aid (Nos. 08457032, 09240202, 09358013, 114700290, 12680603, and 12793010) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion q Daisuke Kudo a, Atsushi Kon a, Shuichi Yoshihara b, Ikuko Kakizaki a, Mutsuo Sasaki b, Masahiko Endo a, Keiichi Takagaki a,* a
Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b Department of Surgery, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Received 22 June 2004 Available online 28 July 2004
Abstract Hyaluronan (HA) is a ubiquitous, major component of the extracellular matrix. It is involved in cell adhesion and locomotion, and hence in tumor metastasis. We have previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and may be a useful tool for examining the functions of HA. We here demonstrate that the formation of cell surface HA by melanoma cells and its release into the culture medium are inhibited by MU. Adhesion and locomotion assays revealed that the adhesion and locomotion of melanoma cells were dose-dependently inhibited by MU. Conversely, treatment with exogenous HA enhanced both adhesion and locomotion. Thus, preventing the formation of cell surface HA reduced both the adhesion and locomotion of melanoma cells, suggesting that MU may act as an inhibitor of tumor metastasis. 2004 Elsevier Inc. All rights reserved. Keywords: Hyaluronan; 4-Methylumbelliferone; B16F-10 melanoma cell; Adhesion; Locomotion; Metastasis
Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) of high molecular mass, which consists of repeated b-1,4-GlcUA-b-1,3-GlcNAc disaccharide units [1]. It is present in many tissues as a major component of the extracellular matrix. HA provides a highly hydrated, gel-like environment due to its physiochemical properties, and maintains the extracellular spaces [2,3]. In addition to its structural function, HA plays an important role in tissue organization, wound healing, and inflammation [4–6]. Several studies have also demonstrated that HA participates in various phases of tumor metastasis, i.e., in cell adhesion and locomotion [7,8]. q Abbreviations: HA, hyaluronan; MU, 4-methylumbelliferone; GAG, glycosaminoglycan; FCS, fetal calf serum; DMSO, dimethyl sulfoxide; HAS, hyaluronan synthase; PBS, phosphate-buffered saline; UGT, UDP-glucuronyltransferase. * Corresponding author. Fax: +81-172-39-5016. E-mail address: [email protected] (K. Takagaki).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.041
Elevated levels of HA in tumor tissue frequently lead to metastasis and hence to a worse prognosis [9–11]. We have previously reported that HA synthesis by cultured human dermal fibroblasts was inhibited by the addition of 4-methylumbelliferone (MU) to the culture medium [12]. Moreover, we examined the mechanism by which MU inhibits HA synthesis in Streptococcus equi FM100 and demonstrated that MU did not affect expression of the HA synthase (HAS) gene but altered the lipid environment of the membrane, especially the distribution of different cardiolipin species, surrounding HAS [13]. In addition, recently we demonstrated a novel mechanism of the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UDP-glucuronyltransferase (UGT) resulting in a depletion of UDP-GlcUA using rat 3Y1 fibroblasts stably expressing HA synthase 2 (HAS2) [14]. These data indicated that MU may be a useful tool in examining the functions of HA. Indeed,
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using MU, Kosaki et al. investigated the influence of cell surface HA on the anchorage-dependent proliferation of human myofibrosarcoma cells [15], and Sohara et al. [16] studied the effect of cell surface HA on the migration of rat fibroblast cells. Malignant tumor cells are known to leave the primary lesion, enter the vascular system, and finally reach remote tissues. Various kinds of malignant tumor cells, including mesothelioma and WilmÕs tumor, produce large amounts of HA, and HA overproduction by tumor cells enhances their metastatic potential [17]. Therefore, it has long been suggested that HA may be a risk factor for tumor metastasis. Based on this concept, as a suppressor of HAS activity, MU may have an inhibitory effect on the metastasis of malignant tumor cells. In the present study, we examined the effect of MU on the formation of cell surface HA in a highly malignant tumor cell line, B16F-10 melanoma cells. We also investigated the effect of MU on the adhesion and locomotion of the melanoma cells, since these properties are important during the early stages of metastasis.
Materials and methods Materials. MU and actinase E were purchased from Sigma Chemical (St. Louis, MO) and Kaken Pharmaceutical (Tokyo, Japan), respectively. HA and Streptomyces hyaluronidase were obtained from Seikagaku (Tokyo, Japan). 2-Aminopyridine was purchased from Wako Pure Chemical (Osaka, Japan) and recrystallized from hexane. All other reagents were of analytical grade and obtained from commercial sources. Tumor cells. B16F-10 melanoma cells (henceforth referred to as melanoma cells) were kindly provided by the Cell Resource Center of the Biochemical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The cells were maintained in RPMI-1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS), L -glutamine, and antibiotics. Addition of MU to the medium was carried out as described previously [12]. Cell growth assay. Cell proliferation was determined using the Alamar Blue assay [18]. Ninety microliters of medium containing 6.25 · 102 cells in the growth phase and 1 · 104 cells in the plateau phase was dispensed into 96-well plates (Iwaki, Tokyo, Japan). After 12 h, serial dilutions of MU dissolved in dimethyl sulfoxide (DMSO) were added to a final volume of 100 ll. The concentration of DMSO in the medium did not exceed 0.1%. An MU-free well was used as a control, and a blank well containing no MU or melanoma cells was also included. After incubation periods of 24, 48, and 72 h, 10 ll of Alamar Blue dye (Sensititre/Alamar, Westlake, OH) was added to each well. The plates were incubated for a further 3 h and then read on a microplate fluorescence reader (Fluoroscan II, Labsystems, Helsinki, Finland) at excitation and emission wavelengths of 544 and 590 nm, respectively. All assays were replicated five times. Assay of HA release into the culture medium. Melanoma cells suspended at a density of 2.5 · 105/ml of culture medium were placed into 10-cm dishes. After 12 h, serial dilutions of MU were added and the dishes were incubated for a further 48 h. The medium was withdrawn and centrifuged to remove the cells. The supernatant was subjected to ethanol precipitation. After labelling with a fluorescent regent, 2-aminopyridine, by reductive amination, the HA in the precipitates was analyzed by ion-exchange high-performance liquid chromatography (HPLC) as described previously [19].
Visualization of cell surface HA. Cell surface HA was visualized using a particle-exclusion assay [20]. Fixed sheep erythrocytes (Sigma, UK) were reconstituted in phosphate-buffered saline (PBS) at a density of 5 · 108 cells/ml. Melanoma cells were cultured in 35-mm dishes. Pericellular HA was visualized by adding 0.75 ml of the erythrocyte suspension to the growth medium with or without MU and viewing under a light microscope. To demonstrate that the pericellular halo was composed of HA, MU-free dishes were preincubated with 1.0 U/ml of Streptomyces hyaluronidase prior to the assay. Adhesion assay. Melanoma cells that had been preincubated with or without MU for 48 h were released using trypsin, washed with RPMI1640 medium containing 10% FCS and suspended at 5 · 105 cells/ml in the same medium. The cell suspension (2 ml) was added to a HAcoated or HA-uncoated culture dish and incubated for 20 min at 37 C. Any non-adherent cells were removed by two gentle washes with PBS. The adherent cells were removed using trypsin, resuspended in medium, and counted with a hematocytometer. All assays were performed in triplicate. The effect of MU on cell adhesion was expressed as the relative adhesion (% of control) and was calculated as follows: relative adhesion = (number of adherent cells in experimental dishes)/(number of adherent cells in control dishes) · 100%. Locomotion assay. The locomotion assay was performed using the Matrigel Invasion Chamber (BD Bioscience, Franklin Lakes, NJ). Melanoma cells that had been preincubated with MU were suspended at 1 · 106 cells/ml in culture medium without FCS, and 500 ll of the cell suspension was placed in the upper chamber. Various concentrations of HA in 750 ll RPMI-1640 containing 5% FCS were added to the lower chamber. After incubation for 5 h at 37 C in 5% CO2, the filters were stained with hematoxylin and the cells migrating through the membrane were counted by microscopy at 100· magnification. The cells were counted in triplicate fields on three separate membranes. The effect of MU on cell invasion was expressed as the relative locomotion (% of control) and was calculated as follows: relative locomotion = (number of cells migrating under experimental conditions)/ (number of cells migrating under control conditions) · 100%.
Results and discussion MU has no significant cytotoxic effect on melanoma cell growth To determine whether MU has any direct effect on cell growth, melanoma cells were incubated with 0– 0.5 mM MU for various incubation times (24, 48, and 72 h), then their growth was assessed using the Alamar Blue assay, as described in ‘‘Materials and methods.’’ Neither the growth phase (Fig. 1A) nor the plateau phase (Fig. 1B) of cell growth was inhibited by MU. The DMSO added to the medium to dissolve the MU also had no influence on either phase of cell growth (Fig. 1). Thus, MU did not have any significant cytotoxic effect on melanoma cell growth. MU inhibits HA synthesis by melanoma cells We have previously shown that MU specifically inhibits HA synthesis in cultured dermal fibroblasts [12]. To determine whether MU also inhibits HA synthesis by melanoma cells, B16F-10 cells were incubated with various concentrations of MU for 48 h, and the HA released into the culture medium was analyzed by ion-ex-
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Fig. 3. Effect of MU on the synthesis of cell surface HA by cultured melanoma cells. Cell surface HA was visualized using a particleexclusion assay, as described in ‘‘Materials and methods.’’ Melanoma cells were cultured in the absence (A) or presence (B) of 0.5 mM MU. Some melanoma cells were incubated with 1.0 U/ml of Streptomyces hyaluronidase prior to the exclusion assay (C). Fig. 1. Effect of MU on the growth of melanoma cells. Melanoma cells were incubated with 0–0.5 mM MU for various incubation times. The growth of cells in the growth phase (A) and plateau phase (B) at each incubation time was determined using the Alamar Blue assay as described in ‘‘Materials and methods.’’ Each point represents the mean of five replicated experiments.
change HPLC. As shown in Fig. 2, the amount of HA released into the culture medium was decreased in a dose-dependent manner when MU was present. This reduction became apparent in cells treated with 0.1 mM of MU and maximal reduction (40%) was achieved at 0.5 mM. Next, we examined the effect of MU on the formation of cell surface HA by a particle-exclusion assay, as described in ‘‘Materials and methods.’’ When the cells were incubated without MU, a pericellular halo could be observed (Fig. 3A). On the other hand, no such halo
Fig. 2. Effect of MU on HA release from melanoma cells. Melanoma cells were incubated with various concentrations of MU for 48 h, and the HA released into the culture medium was analyzed by ionexchange HPLC. Each bar represents the mean + SD of triplicate experiments.
was apparent in the presence of 0.5 mM MU (Fig. 3B). Cells treated with 1.0 U/ml of Streptomyces hyaluronidase prior to the particle-exclusion assay also showed no halo (Fig. 3C). These results suggest that MU inhibits both the formation of cell surface HA by melanoma cells and its retention on the cell surface. HA promotes the adherence and locomotion of melanoma cells HA surrounding the cell surface is eventually released and contributes to the formation of the extracellular matrix. Several studies have shown that high HA levels in the extracellular matrix enhance the metastatic potential of malignant tumor cells [17]. Therefore, we investigated the effect of HA on adhesion and locomotion, which are characteristic properties of malignant tumor cells involved in metastasis. A suspension of melanoma cells was added to a HA-coated culture dish and incubated for 20 min at 37 C. After removing any non-adherent cells by washing, the adherent cells were counted with a hematocytometer. As shown in Fig. 4A, exogenous HA increased the relative adhesion of the melanoma cells in a dose-dependent manner. The maximal value was obtained when the cells were added to a culture dish coated with 0.5 mg/cm2 of HA. We also performed a locomotion assay using the Matrigel Invasion Chamber, as described in ‘‘Materials and methods.’’ Melanoma cells suspended in culture medium without FCS were placed in the upper chamber, and culture medium containing various concentrations of HA was added to the lower chamber.
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Fig. 4. Effect of exogenous HA on the adhesion and locomotion of melanoma cells. The influence of HA on adhesion (A) and locomotion (B) was investigated using immobilized-HA dishes and an improved model of BoydenÔs chamber, respectively. Each bar represents the mean + SD of triplicate experiments.
After incubation for 5 h, the filters were stained and the cells migrating through the membrane were counted by microscopy. As shown in Fig. 4B, the relative locomotion was also dose-dependently increased by HA. Taken together, these results indicate that HA promotes both the adherence of melanoma cells and their locomotion into the surrounding extracellular space. This further suggests that HA may accelerate metastasis to remote tissue. MU inhibits the adhesion and locomotion of melanoma cells Based on the results shown in Fig. 4, we investigated alterations in the adhesion and locomotion of melanoma cells when the formation of cell surface HA was inhibited by MU. Adhesion assay revealed that pretreatment with MU inhibited the adhesion of melanoma cells. This inhibition was dose-dependent; it became apparent in cells treated with 0.1 mM MU, and maximal inhibition (49%) was observed when the cells were preincubated with 0.5 mM MU (Fig. 5A). The locomotion assay revealed that MU also inhibits the locomotion of melanoma cells in a dose-dependent manner. This inhibition was observed in melanoma cells pretreated with as little as 0.1 mM MU, and maximal
Fig. 5. Effect of MU on the adhesion and locomotion of melanoma cells. Melanoma cells that had been preincubated with different concentrations of MU were subjected to adhesion (A) and locomotion (B) assays, as described in ‘‘Materials and methods.’’ Each bar represents the mean + SD of triplicate experiments.
inhibition (37%) was achieved after pretreatment with 0.5 mM MU (Fig. 5B). Thus, MU has inhibitory effects on both the adherence and locomotion of melanoma cells. In this study, we examined the effects of MU on HA synthesis by, and the adhesion and locomotion of, B16F-10 melanoma cells. The major findings were as follows. First, the formation of cell surface HA and its release into the culture medium were inhibited by MU. This is in agreement with our previous studies, which showed that MU specifically inhibits HA synthesis in cultured dermal fibroblasts [12]. Moreover, we have previously demonstrated that MU decreases the enzymatic activity of HAS in Streptococcus equi FM100. This inhibitory effect is achieved indirectly, by altering the distribution of one of the membrane phospholipids of S. equi FM100, cardiolipin, which in turn destabilizes HAS activity [13]. Furthermore, another inhibitory mechanism of MU that depends on the endogenous UGT activity was found using transfectants expressing mouse HAS2 [14]. Thus, MU-mediated inhibition of the formation and subsequent release of cell surface HA occur in various types of cell and may not be cell-type-specific. Second, we demonstrated that HA increases both the adhesion and the locomotion of melanoma cells, and that these increases are inhibited by treatment with MU. HA-rich matrices in malignant tumor tissues are associated with a worse prognosis because of their higher metastatic potential [17,21,22]. Furthermore, several studies have shown that the metastatic potency of malignant tumor cells is enhanced by transfection with a HAS expression construct [17,23]. In addition, injection of nude mice with HAS gene-transfected cells produces tumors smaller than those formed by control cells [15]. Conversely, inhibiting HAS expression in tumor cells with an antisense HAS construct diminishes not only HA synthesis and the formation of cell surface HA but also cell adhesion [24,25]. Thus, HA-rich matrices provide a suitable environment for metastatic changes such as vascularization and invasion [26,27]. Taken together with these earlier findings, the results of our present study strongly suggest that MU-mediated inhibition of adhesion and locomotion may inhibit the metastasis of melanoma cells, which are accelerated by HA. In conclusion, using the novel approach of inhibiting HA synthesis with MU, we demonstrated that the formation of cell surface HA is necessary for the adhesion and locomotion of melanoma cells. Since these properties are characteristic of tumor cells involved in the early stages of metastasis, our results suggest that MU may act as an inhibitor of metastasis. Therefore, MU is not only a useful tool for investigating the various functions of HA but also has potential as a novel therapeutic agent that may control the metastasis of melanoma cells.
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Acknowledgments This work was supported by Grants-in-Aid (Nos. 08457032, 09240202, 09358013, 114700290, 12680603, and 12793010) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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