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Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by blocking cell adhesion and invasion
International Immunopharmacology 6 (2006) 697 – 702 www.elsevier.com/locate/intimp
Preliminary report
Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by blocking cell adhesion and invasion Sang-Bae Han, Chang Woo Lee, Jong Soon Kang, Yeo Dae Yoon, Ki Hoon Lee, Kiho Lee, Song-Kyu Park *, Hwan Mook Kim * Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oundong, Yusong, Taejon 305-333, South Korea Received 18 August 2005; received in revised form 3 October 2005; accepted 14 October 2005
Abstract The acidic polysaccharide (PL) from Phellinus linteus is an immunostimulator that has therapeutic activity against cancers. Here, we show that PL markedly inhibits melanoma cell metastasis in mice, and report that PL directly inhibits cancer cell adhesion to and invasion through the extracellular matrix, but that it has no direct effect on cancer cell growth. In addition, we found that PL increased macrophage NO production. These results suggest that PL has two antimetastatic functions, i.e., it acts as an immunopotentiator and as a direct inhibitor of cancer cell adhesion. D 2005 Elsevier B.V. All rights reserved. Keywords: Phellinus linteus; Metastasis; Invasion; Adhesion
1. Introduction Metastasis is one of the major causes of cancerassociated mortality [1]. Moreover, the treatment of metastasis is still far from satisfactory, due in part to a lack of effective drugs. Cancer metastasis has been described as a complex series of cell movements, i.e., cancer cell adhesion, invasion, migration, and circulation in blood and lymph [2–4]. Of these events, invasion probably is the most crucial, and it depends on specific cell-to-cell and cell-to-extracellular matrix (ECM) interactions, which are mediated directly by specific cell-surface adhesion receptors.
* Corresponding authors. Tel.: +82 42 860 4660; fax: +82 42 860 4605. E-mail addresses: [email protected] (S.-K. Park), [email protected] (H.M. Kim). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.10.003
It had been well established that many polysaccharides extracted from mushrooms act as immunomodulators, and that several of these compounds act to prevent and can be used to treat cancers [5–7]. More specifically, five mushroom preparations have shown to have significant positive effects on cancers in humans and mice, i.e., lentinan from Lentinus edodes, D-fraction from Grifola frondosa, schizophyllan from Schzophyllum commune, PSK from Trametes versicolor, and PSP from Trametes versicolor. We have previously shown that polysaccharide preparation (PL) from Phellinus linteus has inhibitory effects on tumor growth and metastasis in a murine model [8]. To date investigations on the anticancer mechanisms of these polysaccharide preparations have focused for the large part on the functional activations of immune cells. And, it has been reported that they stimulate T cells, B cells, natural killer cells, dendritic cells, and macrophages, which all have the capability to eliminate transformed cancer
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cells [2,5,6,9–13]. However, to date little attention has been paid to the direct effects of polysaccharides on cancer cells. In the present study, we examined the inhibitory effects of PL on cancer cell metastasis in B16F10 melanoma cell model. Moreover, we investigated the direct effects of PL on melanoma cells with respect to cell adhesion and invasion. 2. Materials and methods 2.1. Materials Female C57BL/6 mice were purchased from Daehan Biolink Co. Ltd (Chungbuk, Korea) and maintained under specific pathogen free conditions until required. The experimental procedures used in this study were approved by the KRIBB Animal Experimentation Ethics Committee. Lipopolysaccharide (LPS) and adriamycin were purchased from Sigma (St. Louis, MO). B16F10 murine melanoma cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (HyClone, Logan, UT). Polysaccharide (PL) was prepared from a mycelial culture of the mushroom Phellinus linteus [14]. In brief, PL was extracted with boiled water for 12 h and this was followed by ethanol precipitation, DEAE-cellulose, and gel permeation chromatography. The PL obtained contained 13.2% protein and 82.5% carbohydrate, and had a molecular weight of 153 kDa by gel permeation HPLC. It was heat-stable and its biological activity was unchanged by proteinases K treatment. 2.2. Tumor metastasis For in vivo experimental pulmonary metastasis assays, B16F10 melanoma cells (5 105 cells/mouse) were injected into a tail vein of C57BL/6 mice (n = 10) on day 0. On day 14, lungs were excised and metastatic nodules were counted [15]. 2.3. Adhesion assay Cancer cell adhesion was assayed by incubating B16F10 cells in matrigel-coated plates. Twenty-four well plates were coated with matrigel (5 Ag/well) overnight at 4 8C, were washed three times with PBS, blocked with 1% BSA in PBS for 2 h, and rewashed with PBS. B16F10 melanoma cells were resuspended in DMEM containing 0.1% BSA, added at 1 104 cells/well, and incubated for 1 h. After removing unattached cells by washing, attached cells were detached with trypsin-EDTA and counted [15]. 2.4. Invasion assay Invasion assays were performed using BD BioCoatk Matrigelk Invasion Chambers (BD Biosciences, MA) con-
taining a matrigel-coated membrane (8 Am pore size). Lower chamber compartments were filled with fibroblast-conditioned medium, which functioned as a chemoattractant. B16F10 cells and PL were loaded into upper compartments. After incubation for 24 h, filters were collected and cells adhering to lower membrane surfaces were fixed with methanol, stained with 0.5% crystal violet for 30 min, and counted in four randomly selected fields [16]. 2.5. Nitrite production assay Peritoneal macrophages were isolated from the abdominal cavities of C57BL/6 mice and adherent macrophages were enriched by removing non-adherent cells. Macrophages were plated at 5 105 cells/ml, and then stimulated with PL for 24 h. Isolated supernatants were mixed with an equal volume of Griess reagent (Sigma) and then incubated at room temperature for 10 min. Nitrite production was determined by measuring absorbance at 540 nm versus a NaNO2 derived standard curve. We also used RAW 264.7 macrophage cell lines, which showed responses to PL that were similar to those of peritoneal macrophages [17]. 2.6. Polyclonal IgM production assay Total spleen cells were cultured with 30~300 Ag/ml of PL for 3 days. The number of antibody forming cells (AFCs) was counted by using a Plaque Forming Cell (PFC) assay, and results are expressed as AFCs/106 cells [17]. 2.7. Cytotoxicity assay The cytotoxicities of PL and adriamycin against B16F10 cells were evaluated using sulforhodamine (SRB) B assays [8]. Briefly, cancer cells were inoculated into 96 well plates at 4 104 cells/well and cultured in RPMI 1640 medium containing 5% serum for 24 h. After adding adriamycin or PL, cells were further incubated for 48 h. Cells were fixed by adding 50 Al of 50% trichloroacetic acid (TCA) for 2 h at 4 8C. Then, plates were washed with tap water three times and air-dried. The TCA-fixed cells so obtained were then stained for 30 min with SRB (0.4% in 1% acetic acid), unbound dye was washed out with 1% acetic acid, and cells were then air-dried. Tris base (100 Al, 10 mM, pH 10.5) was the added to each well, and optical densities were measured using a microplate reader at 570 nm. 2.8. Statistics In vivo results represent samples from 10 mice per experiment, whereas in vitro results represent the mean values of triplicate samples. All experiments were performed at least three times. Standard deviations (SDs) and p values were calculated using the StudentTs t test in Microsoft ExcelR [18].
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3. Results
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3.1. Inhibition of tumor metastasis by PL In metastasis studies, B16F10 melanoma cells were injected into a tail vein on day 0, and they formed many metastatic lung colonies by day 14 in control mice were administered PBS (Fig. 1). Lung metastasis frequency was significantly reduced by 30, 77, and 80% in mice given PL at 10, 30, and 100 mg/kg, respectively.
Attached cell number/well
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3.2. Inhibition of cancer cell adhesion and invasion by PL
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We next investigated whether PL inhibits tumor metastasis by directly preventing tumor cell adhesion. B16F10 cells were treated with PL for 1 h during adhesion assays and for 24 h during invasion assays. In adhesion assays, PL significantly and dose-dependently inhibited B16F10 cell adhesion to matrigel (Fig. 2); PL at 10, 30, and 100 Ag/ml reduced cell adhesion by 28, 62, and 75%, respectively. To examine the effect of PL on B16F10 cell invasion through the extracellular matrix, an in vitro matrigel invasion assay system was employed. As shown in Fig. 3, PL at 10, 30, and 100 Ag/ml strongly reduced B16F10 cell invasion through matrigel by 23, 79, and 89%, respectively. 3.3. Activation of macrophages and B cells by PL Since it was suggested that the antitumor effect of PL is mediated via immunostimulation, we examined the effect of PL on macrophages and B cells. As we found previously [13], PL increased NO production by peritoneal macrophages and
10
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PL (µg/ml) Fig. 2. Inhibition of cancer cell adhesion by PL. B16F10 melanoma cells were added to each well of matrigel-coated 24 well plates, and PL was treated at doses ranging between 10 and 100 Ag/ml for 1 h. Unattached cells were removed by washing with PBS and adherent cells were detached by treating with trypsin-EDTA for 10 min. Numbers of adherent cells were counted. Significance was determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
RAW 264.7 cells (Fig. 4A), and increased dose-dependently polyclonal IgM production by B cells (Fig. 4B). 3.4. Cytotoxicity of PL Finally, we evaluated the cytotoxic effect of PL on B16F10 melanoma cells, and found that PL was non-toxic at up to 100 Ag/ml. However, adriamycin (a cytotoxic anticancer drug) showed strong cytotoxicity (Fig. 5).
Invasive cell number/well
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Metastatic colonies/mouse
500
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PL (mg/kg) Fig. 1. Inhibition of tumor metastasis by PL. B16F10 melanoma cells were injected into a C57BL/6 mouse tail vein on day 0 (n = 10). Control mice were administered PBS. PL was administered i.p. at 10, 30, or 100 mg/kg daily for 13 days. On day 14, lungs were excised and metastatic foci were counted. Significance was determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
Fig. 3. Inhibition of cancer cell invasion by PL. HT-1080-conditioned medium was placed in the lower compartment of matrigel-coated 24well plates. B16F10 cells and PL at 10, 30, or 100 Ag/ml were added to the upper compartment. Control cells were treated with PBS. After incubating for 24 h, filters were fixed with methanol and stained with crystal violet. Cells that had penetrated filters were counted on bottom filter faces. Four different fields were randomly counted on 4 different filters for each test condition. Significance was determined using the StudentTs t-test versus vehicle (PBS)-treated controls (* p b 0.01).
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4. Discussion This study demonstrates that PL effectively prevents tumor metastasis. In terms of its mode of action, it is found that PL activates macrophage functions and inhibits cancer cell adhesion and invasion. These results provide several evidences concerning the antimetastatic mechanism of PL. First, PL might inhibit cancer cell metastasis by activating host immunity. Previous and present results show that PL activates macrophages, natural killer cells, cytotoxic T cells, B cells, and dendritic cells [9–13]. Several studies have also demonstrated that mushroom-derived polysaccharides, e.g., lentinan and schizophyllan have immunomodulatory effects [5]. It is recognized that nonspecific host defense functions by
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Concentrations (µg/ml) Fig. 5. Direct cytotoxic effect of PL on B16F10 cells. B16F10 melanoma cells were inoculated onto 96-well plates. PL or adriamycin were treated at concentrations ranging from 0.03 to 30 Ag/ml, and cytotoxicities were determined by Sulforhodamine B assay. The mean optical density of the B16F10 vehicle controls was 2.655 F 0.047, which was taken as 100%. Results are expressed as means F SD of six separate determinations. Significances were determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
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B16F10 cell growth (%)
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0 0
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PL (µg/ml) Fig. 4. Effect of PL on macrophages and B cells. Peritoneal macrophages and RAW 264.7 cells were activated by PL from 10 to 100 Ag/ ml and nitrite generation in culture supernatant was determined 24 h later (A). Total spleen cells were cultured with 30~300 Ag/ml of PL for 3 days. The number of antibody forming cells (AFCs) was counted by using a Plaque Forming Cell (PFC) assay, and results are expressed as AFCs/106 cells. Significances were determined using the StudentTs t-test versus vehicle (PBS)-treated controls (* p b 0.01).
macrophages and natural killer cells are known to be main effectors against tumor cells [19,20]. Second, anti-metastatic effect of PL might be associated with a direct effect of PL on cancer cells. We found that PL inhibits cancer cell adhesion to and invasion through the ECM. It is known that metastasis requires interactions between cancer cells and the ECM [2–4]. After binding to the ECM, cancer cells invade the matrix, and migrate away from primary tumors, to bind to and penetrate blood vessel walls, migrate to new sites, and finally to form secondary tumors. However, although our in vitro data provides evidence that PL inhibits two important metastatic processes, i.e., adhesion and invasion, it remains unclear how PL inhibits B16F10 metastasis in our in vivo metastasis assay. We injected B16F10 melanoma cells intravenously to recipient mice and subsequently examined lung metastasis. It may be that PL inhibited cancer cell adhesion, transmigration through blood vessel walls, or secondary tissue invasion. Currently, we are devising experiments to examine how PL influences interactions between the integrins of cancer cells and the ECM, by following the co-localizations of fluorescence-labeled PL and dyelabeled antibodies to various B16F10 cell integrins. Currently, immunomodulatory polysaccharides are considered to be non-toxic agents, and in the present study, we also found that PL is non-toxic to B16F10 melanoma cells. However, other studies have shown that the extract from Phellinus linteus induced apoptotic death in SW480 human colon cancer and SKN-MC human neuroblastoma cells [21,22]. This ap-
S.-B. Han et al. / International Immunopharmacology 6 (2006) 697–702
parent contradiction may be due to that polysaccharide preparations are likely to differ. The biological activities of polysaccharides are influenced by their different solubility in water, molecular weights, degrees of branching, and by their different triple helical confirmations [7]. In addition, polysaccharide preparations generally include many trace elements and low molecular weight compounds, e.g., inhibitors of NF-nB [23], protein kinases [24], cell-cycle-related kinases [25], and DNA polymerases [26]. Unfortunately, it is unclear whether the PL used in the present study differs substantially in these respects from those used in other studies. Thus, studies on composition and structure-activity relationships (SARs) are warranted to investigate the effect of polysaccharide preparations on cancer cell survival. The present study shows that PL effectively inhibits B16F10 melanoma cell metastasis in our in vivo murine model. Moreover, in terms of mode of action of PL, this study demonstrates that PL has two functions, i.e., it acts as an immunopotentiator and as a direct inhibitor of cancer cell adhesion.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements [16]
This research was supported by a grant from the KRIBB Research Initiative Program. References [1] Kato R, Ishikawa T, Kamiya S, Oguma F, Ueki M, Goto S, et al. A new type of antimetastatic peptide derived from fibronectin. Clin Cancer Res 2002;8:2455 – 62. [2] Hannigan G, Troussard AA, Dedhar S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nat Rev Cancer 2005;5:51 – 63. [3] Playford MP, Schaller MD. The interplay between Src and integrins in normal and tumor biology. Oncogene 2004;23: 7928 – 46. [4] Stacker SA, Achen MG, Jussila L, Baldwin ME, Alitalo K. Lymphangiogenesis and cancer metastasis. Nat Rev Cancer 2002;2:573 – 83. [5] Borchers AT, Keen CL, Gershwin ME. Mushrooms, tumors, and immunity: an update. Exp Biol Med (Maywood) 2004;229: 393 – 406. [6] Brown GD, Gordon S. Fungal beta-glucans and mammalian immunity. Immunity 2003;19:311 – 5. [7] Zaidman BZ, Yassin M, Mahajna J, Wasser SP. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl Microbiol Biotechnol 2005;67:453 – 68. [8] Han SB, Lee CW, Jeon YJ, Hong ND, Yoo ID, Yang KH, et al. The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharmacology 1999;41:157 – 64. [9] Kim GY, Han MG, Song YS, Shin BC, Shin YI, et al. Proteoglycan isolated from Phellinus linteus induces toll-like receptors
[17]
[18]
[19]
[20]
[21]
[22]
[23]
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2-and 4-mediated maturation of murine dendritic cells via activation of ERK, p38, and NF-kappaB. Biol Pharm Bull 2004;27:1656 – 62. Kim GY, Oh WK, Shin BC, Shin YI, Park YC, Ahn SC, et al. Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+DC and type I helper T cell-dominant immune state. FEBS Lett 2004;576:391 – 400. Kim GY, Oh YH, Park YM. Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C. Biochem Biophys Res Commun 2003;309: 399 – 407. Kim GY, Park SK, Lee MK, Lee SH, Oh YH, Kwak JY, et al. Proteoglycan isolated from Phellinus linteus activates murine B lymphocytes via protein kinase C and protein tyrosine kinase. Int Immunopharmacol 2003;3:1281 – 92. Kim HM, Han SB, Oh GT, Kim YH, Hong DH, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus. Int J Immunopharmacol 1996;18:295 – 303. Song KS, Cho SM, Lee JH, Kim HM, Han SB, et al. Blymphocyte-stimulating polysaccharide from mushroom Phellinus linteus. Chem Pharm Bull (Tokyo) 1995;43: 2105 – 8. Hibino S, Shibuya M, Engbring JA, Mochizuki M, Nomizu M, et al. Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy. Cancer Res 2004;64:4810 – 6. Rothhammer T, Poser I, Soncin F, Bataille F, Moser M, et al. Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration. Cancer Res 2005;65:448 – 56. Han SB, Yoon YD, Ahn HJ, Lee HS, Lee CW, et al. Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus. Int Immunopharmacol 2003;3:1301 – 12. Han SB, Moratz C, Huang NN, Kelsall B, Cho H, et al. Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity 2005;22:343 – 54. Hong F, Hansen RD, Yan J, Allendorf DJ, Baran JT, et al. Betaglucan functions as an adjuvant for monoclonal antibody immunotherapy by recruiting tumoricidal granulocytes as killer cells. Cancer Res 2003;63:9023 – 31. Hong F, Yan J, Baran JT, Allendorf DJ, Hansen RD, Ostroff GR, et al. Mechanism by which orally administered beta-1,3glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models. J Immunol 2004;173:797 – 806. Choi YH, Huh MK, Ryu CH, Choi BT, Jeong YK. Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells. Int J Mol Med 2004; 14:227 – 32. Li G, Kim DH, Kim TD, Park BJ, Park HD, Park JI, et al. Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett 2004;216:175 – 81. Ariga A, Namekawa J, Matsumoto N, Inoue J, Umezawa K. Inhibition of tumor necrosis factor-alpha-induced nuclear translocation and activation of NF-kappa B by dehydroxymethylepoxyquinomicin. J Biol Chem 2002;277:24625 – 30.
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[24] Cassinelli G, Lanzi C, Pensa T, Gambetta RA, Nasini G, Cassinis S, et al. Clavilactones, a novel class of tyrosine kinase inhibitors of fungal origin. Biochem Pharmacol 2000;59: 1539 – 47. [25] Hu H, Ahn NS, Yang X, Lee YS, Kang KS. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Int J Cancer 2002;102:250 – 3.
[26] Mizushina Y, Takahashi N, Hanashima L, Koshino H, Esumi J, Uzawa J, et al. Lucidenic acid O and lactone, new terpene inhibitors of eukaryotic DNA polymerases from a basidiomycete, Ganoderma lucidum. Bioorg Med Chem 1999;7: 2047 – 52.
Preliminary report
Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by blocking cell adhesion and invasion Sang-Bae Han, Chang Woo Lee, Jong Soon Kang, Yeo Dae Yoon, Ki Hoon Lee, Kiho Lee, Song-Kyu Park *, Hwan Mook Kim * Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oundong, Yusong, Taejon 305-333, South Korea Received 18 August 2005; received in revised form 3 October 2005; accepted 14 October 2005
Abstract The acidic polysaccharide (PL) from Phellinus linteus is an immunostimulator that has therapeutic activity against cancers. Here, we show that PL markedly inhibits melanoma cell metastasis in mice, and report that PL directly inhibits cancer cell adhesion to and invasion through the extracellular matrix, but that it has no direct effect on cancer cell growth. In addition, we found that PL increased macrophage NO production. These results suggest that PL has two antimetastatic functions, i.e., it acts as an immunopotentiator and as a direct inhibitor of cancer cell adhesion. D 2005 Elsevier B.V. All rights reserved. Keywords: Phellinus linteus; Metastasis; Invasion; Adhesion
1. Introduction Metastasis is one of the major causes of cancerassociated mortality [1]. Moreover, the treatment of metastasis is still far from satisfactory, due in part to a lack of effective drugs. Cancer metastasis has been described as a complex series of cell movements, i.e., cancer cell adhesion, invasion, migration, and circulation in blood and lymph [2–4]. Of these events, invasion probably is the most crucial, and it depends on specific cell-to-cell and cell-to-extracellular matrix (ECM) interactions, which are mediated directly by specific cell-surface adhesion receptors.
* Corresponding authors. Tel.: +82 42 860 4660; fax: +82 42 860 4605. E-mail addresses: [email protected] (S.-K. Park), [email protected] (H.M. Kim). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.10.003
It had been well established that many polysaccharides extracted from mushrooms act as immunomodulators, and that several of these compounds act to prevent and can be used to treat cancers [5–7]. More specifically, five mushroom preparations have shown to have significant positive effects on cancers in humans and mice, i.e., lentinan from Lentinus edodes, D-fraction from Grifola frondosa, schizophyllan from Schzophyllum commune, PSK from Trametes versicolor, and PSP from Trametes versicolor. We have previously shown that polysaccharide preparation (PL) from Phellinus linteus has inhibitory effects on tumor growth and metastasis in a murine model [8]. To date investigations on the anticancer mechanisms of these polysaccharide preparations have focused for the large part on the functional activations of immune cells. And, it has been reported that they stimulate T cells, B cells, natural killer cells, dendritic cells, and macrophages, which all have the capability to eliminate transformed cancer
698
S.-B. Han et al. / International Immunopharmacology 6 (2006) 697–702
cells [2,5,6,9–13]. However, to date little attention has been paid to the direct effects of polysaccharides on cancer cells. In the present study, we examined the inhibitory effects of PL on cancer cell metastasis in B16F10 melanoma cell model. Moreover, we investigated the direct effects of PL on melanoma cells with respect to cell adhesion and invasion. 2. Materials and methods 2.1. Materials Female C57BL/6 mice were purchased from Daehan Biolink Co. Ltd (Chungbuk, Korea) and maintained under specific pathogen free conditions until required. The experimental procedures used in this study were approved by the KRIBB Animal Experimentation Ethics Committee. Lipopolysaccharide (LPS) and adriamycin were purchased from Sigma (St. Louis, MO). B16F10 murine melanoma cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (HyClone, Logan, UT). Polysaccharide (PL) was prepared from a mycelial culture of the mushroom Phellinus linteus [14]. In brief, PL was extracted with boiled water for 12 h and this was followed by ethanol precipitation, DEAE-cellulose, and gel permeation chromatography. The PL obtained contained 13.2% protein and 82.5% carbohydrate, and had a molecular weight of 153 kDa by gel permeation HPLC. It was heat-stable and its biological activity was unchanged by proteinases K treatment. 2.2. Tumor metastasis For in vivo experimental pulmonary metastasis assays, B16F10 melanoma cells (5 105 cells/mouse) were injected into a tail vein of C57BL/6 mice (n = 10) on day 0. On day 14, lungs were excised and metastatic nodules were counted [15]. 2.3. Adhesion assay Cancer cell adhesion was assayed by incubating B16F10 cells in matrigel-coated plates. Twenty-four well plates were coated with matrigel (5 Ag/well) overnight at 4 8C, were washed three times with PBS, blocked with 1% BSA in PBS for 2 h, and rewashed with PBS. B16F10 melanoma cells were resuspended in DMEM containing 0.1% BSA, added at 1 104 cells/well, and incubated for 1 h. After removing unattached cells by washing, attached cells were detached with trypsin-EDTA and counted [15]. 2.4. Invasion assay Invasion assays were performed using BD BioCoatk Matrigelk Invasion Chambers (BD Biosciences, MA) con-
taining a matrigel-coated membrane (8 Am pore size). Lower chamber compartments were filled with fibroblast-conditioned medium, which functioned as a chemoattractant. B16F10 cells and PL were loaded into upper compartments. After incubation for 24 h, filters were collected and cells adhering to lower membrane surfaces were fixed with methanol, stained with 0.5% crystal violet for 30 min, and counted in four randomly selected fields [16]. 2.5. Nitrite production assay Peritoneal macrophages were isolated from the abdominal cavities of C57BL/6 mice and adherent macrophages were enriched by removing non-adherent cells. Macrophages were plated at 5 105 cells/ml, and then stimulated with PL for 24 h. Isolated supernatants were mixed with an equal volume of Griess reagent (Sigma) and then incubated at room temperature for 10 min. Nitrite production was determined by measuring absorbance at 540 nm versus a NaNO2 derived standard curve. We also used RAW 264.7 macrophage cell lines, which showed responses to PL that were similar to those of peritoneal macrophages [17]. 2.6. Polyclonal IgM production assay Total spleen cells were cultured with 30~300 Ag/ml of PL for 3 days. The number of antibody forming cells (AFCs) was counted by using a Plaque Forming Cell (PFC) assay, and results are expressed as AFCs/106 cells [17]. 2.7. Cytotoxicity assay The cytotoxicities of PL and adriamycin against B16F10 cells were evaluated using sulforhodamine (SRB) B assays [8]. Briefly, cancer cells were inoculated into 96 well plates at 4 104 cells/well and cultured in RPMI 1640 medium containing 5% serum for 24 h. After adding adriamycin or PL, cells were further incubated for 48 h. Cells were fixed by adding 50 Al of 50% trichloroacetic acid (TCA) for 2 h at 4 8C. Then, plates were washed with tap water three times and air-dried. The TCA-fixed cells so obtained were then stained for 30 min with SRB (0.4% in 1% acetic acid), unbound dye was washed out with 1% acetic acid, and cells were then air-dried. Tris base (100 Al, 10 mM, pH 10.5) was the added to each well, and optical densities were measured using a microplate reader at 570 nm. 2.8. Statistics In vivo results represent samples from 10 mice per experiment, whereas in vitro results represent the mean values of triplicate samples. All experiments were performed at least three times. Standard deviations (SDs) and p values were calculated using the StudentTs t test in Microsoft ExcelR [18].
S.-B. Han et al. / International Immunopharmacology 6 (2006) 697–702
3. Results
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3.1. Inhibition of tumor metastasis by PL In metastasis studies, B16F10 melanoma cells were injected into a tail vein on day 0, and they formed many metastatic lung colonies by day 14 in control mice were administered PBS (Fig. 1). Lung metastasis frequency was significantly reduced by 30, 77, and 80% in mice given PL at 10, 30, and 100 mg/kg, respectively.
Attached cell number/well
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3.2. Inhibition of cancer cell adhesion and invasion by PL
400
*
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We next investigated whether PL inhibits tumor metastasis by directly preventing tumor cell adhesion. B16F10 cells were treated with PL for 1 h during adhesion assays and for 24 h during invasion assays. In adhesion assays, PL significantly and dose-dependently inhibited B16F10 cell adhesion to matrigel (Fig. 2); PL at 10, 30, and 100 Ag/ml reduced cell adhesion by 28, 62, and 75%, respectively. To examine the effect of PL on B16F10 cell invasion through the extracellular matrix, an in vitro matrigel invasion assay system was employed. As shown in Fig. 3, PL at 10, 30, and 100 Ag/ml strongly reduced B16F10 cell invasion through matrigel by 23, 79, and 89%, respectively. 3.3. Activation of macrophages and B cells by PL Since it was suggested that the antitumor effect of PL is mediated via immunostimulation, we examined the effect of PL on macrophages and B cells. As we found previously [13], PL increased NO production by peritoneal macrophages and
10
30
100
PL (µg/ml) Fig. 2. Inhibition of cancer cell adhesion by PL. B16F10 melanoma cells were added to each well of matrigel-coated 24 well plates, and PL was treated at doses ranging between 10 and 100 Ag/ml for 1 h. Unattached cells were removed by washing with PBS and adherent cells were detached by treating with trypsin-EDTA for 10 min. Numbers of adherent cells were counted. Significance was determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
RAW 264.7 cells (Fig. 4A), and increased dose-dependently polyclonal IgM production by B cells (Fig. 4B). 3.4. Cytotoxicity of PL Finally, we evaluated the cytotoxic effect of PL on B16F10 melanoma cells, and found that PL was non-toxic at up to 100 Ag/ml. However, adriamycin (a cytotoxic anticancer drug) showed strong cytotoxicity (Fig. 5).
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30
100
PL (mg/kg) Fig. 1. Inhibition of tumor metastasis by PL. B16F10 melanoma cells were injected into a C57BL/6 mouse tail vein on day 0 (n = 10). Control mice were administered PBS. PL was administered i.p. at 10, 30, or 100 mg/kg daily for 13 days. On day 14, lungs were excised and metastatic foci were counted. Significance was determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
Fig. 3. Inhibition of cancer cell invasion by PL. HT-1080-conditioned medium was placed in the lower compartment of matrigel-coated 24well plates. B16F10 cells and PL at 10, 30, or 100 Ag/ml were added to the upper compartment. Control cells were treated with PBS. After incubating for 24 h, filters were fixed with methanol and stained with crystal violet. Cells that had penetrated filters were counted on bottom filter faces. Four different fields were randomly counted on 4 different filters for each test condition. Significance was determined using the StudentTs t-test versus vehicle (PBS)-treated controls (* p b 0.01).
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4. Discussion This study demonstrates that PL effectively prevents tumor metastasis. In terms of its mode of action, it is found that PL activates macrophage functions and inhibits cancer cell adhesion and invasion. These results provide several evidences concerning the antimetastatic mechanism of PL. First, PL might inhibit cancer cell metastasis by activating host immunity. Previous and present results show that PL activates macrophages, natural killer cells, cytotoxic T cells, B cells, and dendritic cells [9–13]. Several studies have also demonstrated that mushroom-derived polysaccharides, e.g., lentinan and schizophyllan have immunomodulatory effects [5]. It is recognized that nonspecific host defense functions by
A
250
Nitrite (nmole/106 cells)
Peritoneal RAW
*
200
PL adriamycin
80
* 60
* 40 20
* *
0 -20 0.01
0.1
1
* 10
100
Concentrations (µg/ml) Fig. 5. Direct cytotoxic effect of PL on B16F10 cells. B16F10 melanoma cells were inoculated onto 96-well plates. PL or adriamycin were treated at concentrations ranging from 0.03 to 30 Ag/ml, and cytotoxicities were determined by Sulforhodamine B assay. The mean optical density of the B16F10 vehicle controls was 2.655 F 0.047, which was taken as 100%. Results are expressed as means F SD of six separate determinations. Significances were determined using the Student’s t-test versus vehicle (PBS)-treated controls (* p b 0.01).
* *
150
*
*
100
* *
50
*
0 0
1
3
10
30
100
PL (µg/ml)
B 1000 *
800
AFCs/106 cells
B16F10 cell growth (%)
100
*
600
*
400
200
0 0
30
100
300
PL (µg/ml) Fig. 4. Effect of PL on macrophages and B cells. Peritoneal macrophages and RAW 264.7 cells were activated by PL from 10 to 100 Ag/ ml and nitrite generation in culture supernatant was determined 24 h later (A). Total spleen cells were cultured with 30~300 Ag/ml of PL for 3 days. The number of antibody forming cells (AFCs) was counted by using a Plaque Forming Cell (PFC) assay, and results are expressed as AFCs/106 cells. Significances were determined using the StudentTs t-test versus vehicle (PBS)-treated controls (* p b 0.01).
macrophages and natural killer cells are known to be main effectors against tumor cells [19,20]. Second, anti-metastatic effect of PL might be associated with a direct effect of PL on cancer cells. We found that PL inhibits cancer cell adhesion to and invasion through the ECM. It is known that metastasis requires interactions between cancer cells and the ECM [2–4]. After binding to the ECM, cancer cells invade the matrix, and migrate away from primary tumors, to bind to and penetrate blood vessel walls, migrate to new sites, and finally to form secondary tumors. However, although our in vitro data provides evidence that PL inhibits two important metastatic processes, i.e., adhesion and invasion, it remains unclear how PL inhibits B16F10 metastasis in our in vivo metastasis assay. We injected B16F10 melanoma cells intravenously to recipient mice and subsequently examined lung metastasis. It may be that PL inhibited cancer cell adhesion, transmigration through blood vessel walls, or secondary tissue invasion. Currently, we are devising experiments to examine how PL influences interactions between the integrins of cancer cells and the ECM, by following the co-localizations of fluorescence-labeled PL and dyelabeled antibodies to various B16F10 cell integrins. Currently, immunomodulatory polysaccharides are considered to be non-toxic agents, and in the present study, we also found that PL is non-toxic to B16F10 melanoma cells. However, other studies have shown that the extract from Phellinus linteus induced apoptotic death in SW480 human colon cancer and SKN-MC human neuroblastoma cells [21,22]. This ap-
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parent contradiction may be due to that polysaccharide preparations are likely to differ. The biological activities of polysaccharides are influenced by their different solubility in water, molecular weights, degrees of branching, and by their different triple helical confirmations [7]. In addition, polysaccharide preparations generally include many trace elements and low molecular weight compounds, e.g., inhibitors of NF-nB [23], protein kinases [24], cell-cycle-related kinases [25], and DNA polymerases [26]. Unfortunately, it is unclear whether the PL used in the present study differs substantially in these respects from those used in other studies. Thus, studies on composition and structure-activity relationships (SARs) are warranted to investigate the effect of polysaccharide preparations on cancer cell survival. The present study shows that PL effectively inhibits B16F10 melanoma cell metastasis in our in vivo murine model. Moreover, in terms of mode of action of PL, this study demonstrates that PL has two functions, i.e., it acts as an immunopotentiator and as a direct inhibitor of cancer cell adhesion.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements [16]
This research was supported by a grant from the KRIBB Research Initiative Program. References [1] Kato R, Ishikawa T, Kamiya S, Oguma F, Ueki M, Goto S, et al. A new type of antimetastatic peptide derived from fibronectin. Clin Cancer Res 2002;8:2455 – 62. [2] Hannigan G, Troussard AA, Dedhar S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nat Rev Cancer 2005;5:51 – 63. [3] Playford MP, Schaller MD. The interplay between Src and integrins in normal and tumor biology. Oncogene 2004;23: 7928 – 46. [4] Stacker SA, Achen MG, Jussila L, Baldwin ME, Alitalo K. Lymphangiogenesis and cancer metastasis. Nat Rev Cancer 2002;2:573 – 83. [5] Borchers AT, Keen CL, Gershwin ME. Mushrooms, tumors, and immunity: an update. Exp Biol Med (Maywood) 2004;229: 393 – 406. [6] Brown GD, Gordon S. Fungal beta-glucans and mammalian immunity. Immunity 2003;19:311 – 5. [7] Zaidman BZ, Yassin M, Mahajna J, Wasser SP. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl Microbiol Biotechnol 2005;67:453 – 68. [8] Han SB, Lee CW, Jeon YJ, Hong ND, Yoo ID, Yang KH, et al. The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharmacology 1999;41:157 – 64. [9] Kim GY, Han MG, Song YS, Shin BC, Shin YI, et al. Proteoglycan isolated from Phellinus linteus induces toll-like receptors
[17]
[18]
[19]
[20]
[21]
[22]
[23]
701
2-and 4-mediated maturation of murine dendritic cells via activation of ERK, p38, and NF-kappaB. Biol Pharm Bull 2004;27:1656 – 62. Kim GY, Oh WK, Shin BC, Shin YI, Park YC, Ahn SC, et al. Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+DC and type I helper T cell-dominant immune state. FEBS Lett 2004;576:391 – 400. Kim GY, Oh YH, Park YM. Acidic polysaccharide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C. Biochem Biophys Res Commun 2003;309: 399 – 407. Kim GY, Park SK, Lee MK, Lee SH, Oh YH, Kwak JY, et al. Proteoglycan isolated from Phellinus linteus activates murine B lymphocytes via protein kinase C and protein tyrosine kinase. Int Immunopharmacol 2003;3:1281 – 92. Kim HM, Han SB, Oh GT, Kim YH, Hong DH, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus. Int J Immunopharmacol 1996;18:295 – 303. Song KS, Cho SM, Lee JH, Kim HM, Han SB, et al. Blymphocyte-stimulating polysaccharide from mushroom Phellinus linteus. Chem Pharm Bull (Tokyo) 1995;43: 2105 – 8. Hibino S, Shibuya M, Engbring JA, Mochizuki M, Nomizu M, et al. Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy. Cancer Res 2004;64:4810 – 6. Rothhammer T, Poser I, Soncin F, Bataille F, Moser M, et al. Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration. Cancer Res 2005;65:448 – 56. Han SB, Yoon YD, Ahn HJ, Lee HS, Lee CW, et al. Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus. Int Immunopharmacol 2003;3:1301 – 12. Han SB, Moratz C, Huang NN, Kelsall B, Cho H, et al. Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity 2005;22:343 – 54. Hong F, Hansen RD, Yan J, Allendorf DJ, Baran JT, et al. Betaglucan functions as an adjuvant for monoclonal antibody immunotherapy by recruiting tumoricidal granulocytes as killer cells. Cancer Res 2003;63:9023 – 31. Hong F, Yan J, Baran JT, Allendorf DJ, Hansen RD, Ostroff GR, et al. Mechanism by which orally administered beta-1,3glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models. J Immunol 2004;173:797 – 806. Choi YH, Huh MK, Ryu CH, Choi BT, Jeong YK. Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells. Int J Mol Med 2004; 14:227 – 32. Li G, Kim DH, Kim TD, Park BJ, Park HD, Park JI, et al. Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett 2004;216:175 – 81. Ariga A, Namekawa J, Matsumoto N, Inoue J, Umezawa K. Inhibition of tumor necrosis factor-alpha-induced nuclear translocation and activation of NF-kappa B by dehydroxymethylepoxyquinomicin. J Biol Chem 2002;277:24625 – 30.
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S.-B. Han et al. / International Immunopharmacology 6 (2006) 697–702
[24] Cassinelli G, Lanzi C, Pensa T, Gambetta RA, Nasini G, Cassinis S, et al. Clavilactones, a novel class of tyrosine kinase inhibitors of fungal origin. Biochem Pharmacol 2000;59: 1539 – 47. [25] Hu H, Ahn NS, Yang X, Lee YS, Kang KS. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Int J Cancer 2002;102:250 – 3.
[26] Mizushina Y, Takahashi N, Hanashima L, Koshino H, Esumi J, Uzawa J, et al. Lucidenic acid O and lactone, new terpene inhibitors of eukaryotic DNA polymerases from a basidiomycete, Ganoderma lucidum. Bioorg Med Chem 1999;7: 2047 – 52.