Applied Microbiology and Biotechnology

, Volume 85, Issue 6, pp 1977–1990

Chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from Pleurotus pulmonarius mycelium and fruiting bodies

Authors

  • Iris Lavi
    • Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality SciencesThe Hebrew University of Jerusalem
  • Dana Levinson
    • Department of Plant Pathology and MicrobiologyThe Hebrew University of Jerusalem
  • Irena Peri
    • Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality SciencesThe Hebrew University of Jerusalem
  • Yoram Tekoah
    • Department of BiotechnologyBen-Gurion University of the Negev
  • Yitzhak Hadar
    • Department of Plant Pathology and MicrobiologyThe Hebrew University of Jerusalem
    • Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality SciencesThe Hebrew University of Jerusalem
Applied Microbial and Cell Physiology

DOI: 10.1007/s00253-009-2296-x

Cite this article as:
Lavi, I., Levinson, D., Peri, I. et al. Appl Microbiol Biotechnol (2010) 85: 1977. doi:10.1007/s00253-009-2296-x

Abstract

Mushroom polysaccharides are potent substances that exhibit antitumor and immunomodulatory properties. Studies comparing the chemical composition and antitumor-related activities of polysaccharides released by fungal strains under different growth conditions are not available. Thus, the present study compared polysaccharides extracts produced by Pleurotus pulmonarius from mycelium grown in liquid culture (ME) or fruiting bodies (FBE). Polysaccharides of both ME and FBE had a relatively high molecular mass. NMR spectroscopy indicated that ME glucan is an α-glucan whereas FBE glucan is a mixture of both α- and β-glucans. Glucose and galactose where the most prominent monosaccharide in both glucans. Treatment of several colon cancer cell lines expressing varying amounts of galectin-3 with the two fungal glucans inhibited their viability and significantly reduced their ability to adhere to the key component of the extracellular matrix, fibronectin, and to a human umbilical vein endothelial cell monolayer, in a time- and dose-dependent manner mainly in those cell lines expressing high amounts of galectin-3. We conclude that ME and FBE glucans may exert a direct antiproliferative effect on cancer cells expressing high galectin-3 concentrations and concomitantly downregulate tumor cell adherence, the latter being directly related to cancer progression and metastasis.

Keywords

Pleurotus pulmonariusGlucansColon cancer cellsGalectin-3

Introduction

A large number of polysaccharides, including several α- and β-glucans, have been isolated from a wide variety of fungi and chemically characterized. Different bioactive polysaccharides can be isolated from the mycelium, the fruiting body, and the sclerotium, representing three different stages in the fungal life cycle. Polysaccharides differ in their primary structure (type of basic sugar), type of linkage (α, β, etc.), degree of branching, and molecular weight, among other parameters. Several therapeutic capacities have been attributed to fungal polysaccharides in general and to β-glucans in particular. Their antitumor effects have been shown to depend not only on the molecular weight of the polysaccharide but also on the structure of the polymeric backbone and on the degree of branching (Wasser 2002). Most polysaccharides are classified as nonspecific bioactive substances because their exact mode of action is still unknown, and the exact chain conformation of the active components is undefined. Polysaccharides with antitumor properties have been screened mostly in the fruiting bodies, less so in liquid culture medium and mycelium. There are two proposed mechanisms by which polysaccharides extracted from mushroom exert their antitumor effect: direct activity on the tumor cells or indirect activity by regulating their host’s immune system. The indirect pathway, in which the polysaccharide acts only as a mediator in the immune response, has been studied in detail (Adachi et al. 1994; Chihara 1992; Ho et al. 2004), while the direct pathway, in which the polysaccharide itself inhibits the cancerous growth, is more complex and has only been partially characterized (Chen et al. 2008; Li et al. 2004; Xie et al. 2006; Zaidman et al. 2005; Zhou et al. 2007).

In this regard, modified citrus pectin was shown to inhibit binding of the lectin galectin-3 to human umbilical vein endothelial cells (HUVECs) and to inhibit the adhesion of breast cancer MDA-MB-435 cells, which express galectin-3, to HUVECs in a dose-dependent manner (Nangia-Makker et al. 2002). d-galactose and arabinogalactan substantially inhibited the formation of experimental liver metastasis by L-1 sarcoma cells (Beuth et al. 1988). In another study, treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialyl-Le(x) and sialyl-Le(a). The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in decreased E-selectin-mediated adhesion of human colon cells to HUVECs and impaired adhesion of HT-29 cells to the β-galactoside-binding lectin, galectin-1 (Glinsky et al. 1996). More recently, it was reported that anti-galectin-3 antibody and lactose inhibit liver metastasis by the adenocarcinoma cell lines XK4A3 and RPMI4788 (Inufusa et al. 2001). We recently demonstrated that low molecular weight, hot-water-soluble (HWS) α-glucan extracted from Pleurotus ostreatus grown in submerged culture markedly inhibits proliferation of HT29, a colon cancer cell line, and induces apoptosis, whereas the dietary fiber neutral carboxymethyl cellulose (CMC), a natural polysaccharide, does not have such effect (Lavi et al. 2006). Pleurotus pulmonarius is an edible mushroom belonging to the basidiomycetes and appreciated for its flavor and nutritional value. Several therapeutic effects have been associated with polysaccharides isolated from Pleurotus species (Hu et al. 2006; Rout et al. 2005; Sarangi et al. 2006; Zhang et al. 2001; Zusman et al. 1997), but only a few reports specifically describe and characterize the role of polysaccharides extracted from P. pulmonarius. In the present study, a polysaccharide fraction was prepared from fruiting bodies of P. pulmonarius grown in a commercial farm and the chemical, structural, and biological properties were assessed and compared with those of a polysaccharide fraction extracted from a submerged cultured mycelium of P. pulmonarius. The biological activities of both polysaccharide fractions on several aspects of colon carcinogenesis were also determined.

Materials and methods

Preparation of P. pulmonarius extracts

Fruiting body extracts

P. pulmonarius was cultivated in a commercial farm (Ramot Meir, Israel) on wheat straw as substrate. The fresh fruiting bodies were processed within 2 h after harvesting. Dried powder was prepared from the fruiting bodies by freezing with liquid nitrogen and grinding with mortar and pestle. Three hundred grams of powdered fruiting bodies was extracted with 3,000 ml dH2O at 121°C for 30 min (in autoclave) and centrifuged at 13,000×g at 10°C for 15 min. Ethanol (EtOH) was added to the supernatant, to a final concentration of 50% (v/v), and the mixture was stored overnight at 4°C. The float was taken out, and the viscous fraction was removed and lyophilized. We refer to this fraction as fruiting body extract (FBE).

Mycelium extracts

P. pulmonarius mycelia were maintained on 2% agar basidiomycete synthetic medium (BSM) essentially as previously described (Lavi et al. 2006). BSM contains the following components in 1 l of liquid medium: glucose (5 g), K2HPO4 (1 g), l-asparagine (0.6 g), yeast extract (0.5 g), KCl (0.5 g), MgSO4·7H2O (0.5 g), FeSO4 (0.01 g), ZnNO3·4H2O (0.002 g), Ca(NO3)2·4H2O (0.05 g), CuSO4·5H2O (0.003 g). The medium was brought to pH 5.5. Cultures were incubated on a rotary shaker at 28°C and 120 rpm in 250-ml Erlenmeyer flasks containing 60 ml of liquid BSM for five consecutive days. The resulting mycelia were homogenized by Ultra-Turrax (Jank and Kunkel, Staufen, Germany) for 30 s. The homogenized mycelia provided the basis for more homogeneous inoculums which was later incubated in 500-ml Erlenmeyer flasks containing 200-ml liquid BSM for an additional 2 days at 28°C in a rotary shaker at 120 rpm; 12-ml aliquots of these cell cultures were used for further inoculation in 250-ml Erlenmeyer flasks containing 60-ml liquid BSM. After the inoculation, mycelia were grown for six more days. At the end of the incubation period, the biomass was collected while the extracellular medium was discarded. The methodology we provide concentrates in exopolysaccharides that are bound to the biomass, which are indeed different to those secreted to the media in terms of effectiveness (toxicity) towards cancer cells and structure linkage of α-glucosyl residues rather than linkage of β-glucosyl residues (Lavi et al. 2006). The biomass was washed twice with distilled water at 80°C for 2 h and centrifuged. The resulting wash water was collected, frozen and lyophilized, and designated mycelium extract (ME). The polysaccharide extraction methodology for FBE and ME is based essentially in the methodology we have previously published for extraction of polysaccharides from P. ostreatus (Lavi et al. 2006), a methodology based essentially on the one published by Kodama et al. (2001), with minor modifications. Since fruiting bodies are richer in proteins and unrelated molecules, the water temperatures used in isolation procedure are higher for fruiting bodies than for mycelium extract.

Monosaccharide analysis

For identification of monosaccharides from the lyophilized FBE or ME extracts, a method derived by Valent et al. (1980) was used, with minor modifications. In short, dried polysaccharide samples (4 mg) were hydrolyzed for 2 h at 100°C with 200 μl 2 M trifluoroacetic acid. The soluble fraction was evaporated to dryness at 40°C under a stream of nitrogen. This was repeated twice after each of two additions of 500 μl toluene. The sugar mixtures were reduced to respective alditols using a 250-μl solution of sodium borohydride (40 mg) in 1 M ammonium hydroxide (4 ml). After incubation for 1 h at room temperature, the excess borohydride was decomposed using two drops of glacial acetic acid and 500 μl of 10% glacial acetic acid in methanol (v/v), followed by evaporation under a stream of nitrogen. Additional evaporation was used to remove three consecutive portions of methanol (500 μl). Acetylation of alditols was performed in sealed tubes (20 min, 120°C) with a mixture of acetic anhydride (200 μl) and pyridine (200 μl). Toluene (1 ml) was added to the soluble mixture, followed by evaporation to dryness at 40°C under a stream of nitrogen. A 5-ml mixture of dichloromethane and water (1:1, v/v) was added to the samples and mixed, and the aqueous phase was removed. After repeating this last step twice, the organic phase was evaporated to dryness at 40°C under a stream of nitrogen, followed by the addition of 500 μl of acetonitrile and evaporation. Samples were redissolved in 1 ml acetone with diethyl phthalate as an internal standard and filtered (polytetrafluoroethylene, 0.45 μm), prior to injection into the gas chromatograph (GC) or gas chromatograph coupled with a mass spectrometer (GC–MS). Sugar composition of modified alditols was determined by GC with a Hewlett Packard HP 5890 Series II GC, equipped with a DB-225 capillary column (30 m, 0.25 mm, 0.25 μm) from JW Scientific (Folsom, CA, USA) and a flame ionization detector at 250°C. Samples (1 μl) of mixed alditols were separated at 220°C with helium as the carrier gas. MS confirmation of the derived sugars was determined by GC–MS with an Agilent 6,890 N GC connected to a 5,973-N single-quadrupole mass-selective detector and compared to an NIST02 MS internal database library (Agilent Technologies, Santa Clara, CA, USA). Separation of derived sugars was achieved through an HP-5 capillary column (30 m, 0.25 mm, 0.25 μm, Agilent Technologies), set at 150°C for 13 min, followed by an increase of 10°C/min, up to 220°C with helium as the carrier gas.

Magnetic resonance of polysaccharides

1H nuclear magnetic resonance (NMR) or 13C NMR spectra of polysaccharides in D2O were obtained with a 200- or 500-MHz Bruker NMR spectrometer (DPX-200 or DMX-500, respectively, Bruker Biospin GmbH, Rheinstetten, Germany).

Viscosity measurements

The viscosity of the two polysaccharide solutions (0.25%, w/v) was measured at 25 ± 0.1°C using a Brookfield programmable LVDV+2 viscometer (Brookfield Eng. Labs. Inc., Stoughton, MA, USA) spindle 18, at 30 rpm.

Size determination by size-exclusion chromatography

Determination of molecular weight and size distribution was performed by coupling, online, size-exclusion chromatography (SEC), a multiangle laser light scattering (MALLS) photometer, and a differential refractive index (DRI) detector. A phosphate buffer mobile phase (pH 7.00 at 25°C) was filtered through a 0.1-µm filter (Gelman VacuCap®, Pall Corporation, East Hills, NY, USA), carefully degassed (Waters™ In-Line Degasser, Waters Co., Milford, MA, USA), eluted at a flow rate of 0.5 ml/min (Waters 616 pumping system), and filtered online, through a 0.5-μm filter unit (Upchurch Scientific, Oak Harbor, WA, USA). Samples (500 µl) were injected via a Rheodyne 7725 syringe loading sample injector (Rheodyne, Rohnert Park, CA, USA) with a 100-µl loop. The SEC system consisted of a GPC-Guard Column PSS SUPREMA followed by two serial analytical GPC columns (PSS SUPREMA, 10 µm/100 Å and 1,000 Å, Polymer Standards Service, GmbH, Mainz, Germany) with a separation range from 1 to 5,000 kDa. The column packing was a macroporous cross-linked polymer gel, specially designed for the separation of water-soluble polymers. A DAWN DSP MALLS photometer, from Wyatt Technology Incorporation (Santa Barbara, CA, USA) was fitted with a K5 flow cell and a He–Ne laser (633 nm), installed online between the columns and the DRI detector [Optilab DSP, Wyatt Technology (Santa Barbara, CA, USA)]. A DRI detector operating at the same wavelength (633 nm) as the light scattering was used as a mass-sensitive detector. The Dawn DSP measures the angular distributions and intensity of light scattered simultaneously at 16 angles from 20o to 160o for each elution volume (Vi) of concentration (Ci) measured online by the DRI detector. The Astra v-4.90.07 software package was used to calculate molecular weight, and the radius of gyration (Rg) was from the extrapolation of the light scattered to 0° at each “slice” according to the Berry method (Berry 1966) with a first-order polynomial fit. The refractive index increment, dn/dc, of the sample was 0.15 ml/g. Prior to injection, all samples were rendered dust free by filtering through a 0.45-μm nylon filter (Whatman plc, Kent, England).

Size determination by MALDI-TOF MS and high-performance liquid chromatography

Polysaccharides were labeled with 2-aminobenzamide, and the total pool was separated for an estimation of the sizes of the various polysaccharide fractions by high-performance liquid chromatography (HPLC; Alliance 2695 separation module, Waters Co.), equipped with a Multi λ 2475 fluorescence detector (Waters Co.), using a modified method based on Kuster et al. (1997). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS of the polysaccharide samples was performed by MALDI MS in positive-ion mode with a Bruker REFLEX-IV instrument (Bruker Daltonics, Bremen, Germany). A nitrogen laser VSL-337ND (Laser Science, Inc., Franklin, MA) with an emission wavelength at 337 nm and 4-ns pulse duration was used. Samples (0.3 µl in water) were mixed with a saturated solution of 2,5-dihydroxybenzoic acid on the MALDI target and allowed to dry at room temperature. Each sample was then recrystallized from ethanol. The MALDI instrument was calibrated with dextran oligomers. Monoisotopic masses of the [M+Na]+ ions indicated molecular weight values of the various polysaccharide sizes in the total pool.

Western blot analyses

Cells were lysed, electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gels, transferred to nitrocellulose transfer membranes (Whatman, Schleicher, Schuell; Dassel, Germany), blocked in 1.0 mM Tris base and 0.1 M sodium chloride containing 5% dry nonfat milk, incubated with galectin-3 antibody (Abcam Inc., Cambridge, MA, USA, antibody catalog ab58086), and subsequently incubated with a secondary antibody coupled to horseradish peroxidase. Proteins were visualized using ECL kit (Amersham Biosciences, Buckinghamshire, UK). Effective transfer to nitrocellulose membrane was confirmed by washing with TBS containing 0.1% Tween-20 followed by staining with Ponceau. Blots were stripped and incubated with β-actin to confirm equal protein loading.

Cell culture, glucan preparation treatments

Colon cancer cells (HT-29, Caco2, HCT-116, LS174T, HM-7) and an RSB cell line obtained from a colonic tumor of an ulcerative colitis patient were cultured on Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Beit Haemek, Israel) containing penicillin, streptomycin, nystatin, 10% (v/v) fetal calf serum (FCS), and 1% (v/v) l-glutamine. Cells were plated at a density of 500,000 cells per well in six-well culture plates and allowed to adhere for 24 h. The medium was removed and replaced with medium containing filter-sterilized FBE or ME extract (0.20-µm nylon filters; Fisher Scientific, Pittsburgh, PA, USA). The negative controls were untreated cells cultured in medium alone. HUVECs were grown in M199 medium containing 10% FCS, penicillin, streptomycin, endothelial cell growth factor (ECGF), heparin, and filter-sterilized FBE or ME extract. Cell viability was assessed by the 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) assay as previously described (Schwartz et al. 1992). Statistical analysis of the results was performed by ANOVA and complemented by Tukey’s test.

Cells adhesion to fibronectin

Cell adhesion was performed essentially as described previously (Schwartz et al. 1992). Briefly, 96-well plates were precoated with 50 µl of 5 µg/ml fibronectin (FN) overnight at room temperature and blocked with 0.2 ml DMEM per well containing 3% (w/v) bovine serum albumin (BSA) for 1 h at 37°C. The treated HT29, Caco2, HCT, and LS174T cells, after 24 and 48 h, were resuspended in DMEM containing 0.1% BSA and counted, and 1 × 105 cells were added to each well. The plates were incubated at 37°C for 5 h under sterile conditions. The wells were washed twice with warm phosphate-buffered saline to remove the unattached cells, and the number of bound cells was analyzed by MTT assay as described in the previous paragraph.

Cell adhesion to HUVECs

HUVECs were grown to confluence on 24-well plates and exposed to FBE glucan or to ME glucan for 8 and 24 h. Prior to the cell–cell adhesion assay, the HUVEC monolayers were washed twice with Hank’s Balanced Salt Solution (HBSS) and then washed with M199 medium containing 10% FCS. Calcein acetoxymethyl ester (calcein AM) was used to label cancer cell lines (HT29, Caco2, HCT-116, and LS174T). Efficient fluorescence labeling of these colon cancer cells was achieved by incubating 250,000 cells per milliliter with 1 µM calcein AM as previously described (Lee et al. 2004) with modifications. Following loading of the cells with calcein AM for 20 min at 37°C, they were washed three times with HBSS and then with M199 medium containing 10% FCS. The calcein-AM-labeled cancer cells were added to the HUVEC monolayers and incubated for 20 min at 37°C. The nonadherent cells were removed from the monolayers after washing each well three times with HBSS. The number of adhered fluorescent cells was calculated using Image J Software.

Results

Production of glucans from fruiting bodies and from mycelium grown in submerged fermentation of P. pulmonarius

Polysaccharides were isolated from the edible mushroom P. pulmonarius grown under conventional conditions (i.e., fruiting bodies; FBE) or under biotechnological-oriented conditions (i.e., mycelia grown in submerged culture; ME) via hot-water extraction. These two extracts were further analyzed for their chemical properties as described in the following sections.

Chemical characterization of the FBE and ME fractions

Monosaccharide composition of FBE and ME glucans

The chemical composition and relative abundance of the monosaccharides of the FBE and ME glucans were analyzed by GC, following a procedure of hydrolysis, reduction, and acetylation. The monosaccharide composition was also confirmed by GC–MS. Data are summarized in Table 1;both FBE and ME fractions contained mainly glucose and galactose, as well as mannose, rhamnose, arabinose, and ribose. ME glucan had lower levels of glucose than FBE glucan but contained additional sugars, namely xylose and fucose.
Table 1

Monosaccharide composition of the fractions obtained from P. pulmonarius

Sugar

ME glucan (% of total)

FBE glucan (% of total)

Glucose

64.4

82.1

Galactose

8.3

8.3

Rhamnose

4.6

2.2

Fucose

2.7

nd

Ribose

4.1

2.6

Arabinose

4.6

2.5

Xylose

5.9

nd

Mannose

5.4

2.3

Monosaccharide composition of FBE glucan and ME glucan shown as percentage of total monosaccharides, analyzed by GC–MS and GC after successive hydrolysis, reduction, and acetylation

nd not detected

The content of polysaccharides following EtOH precipitation is 95 ± 1.5% of dry matter in FBE (n = 4) and 96 ± 0.8% of dry matter in ME (n = 4).

13C and 1H NMR analyses

Linkage within the polysaccharides was analyzed by 1H and 13C NMR. Since the main monosaccharide found in both polysaccharides was glucose, the main linkage analyzed in the FBE and ME polysaccharide was for this molecule. 1H NMR spectrum of the dissolved FBE polysaccharide showed anomeric carbon peaks at 5.10 and 4.51 ppm at a ratio of about 1:1, characteristic of α and β linkages, respectively. This was confirmed by the 13C-NMR spectrum which showed three anomeric carbon peaks: two at 91.98 and 93.11 ppm characteristic of the α-linked anomeric carbons and a single one at 95.79 ppm, characteristic of a β-linked carbon. Compared to the FBE polysaccharide, results with the ME-derived polysaccharide indicated only one α-glucan linkage showing one peak at 5.09 ppm. This was confirmed with a 13C-NMR spectrum showing a single signal at 93.19 ppm.

Molecular weight analysis of FBE and ME glucans

Three separate methods were used to characterize the molecular weight of the polysaccharide samples: SEC coupled with a multiangle light scattering detector and refractive index detector, MALDI-TOF MS, and HPLC coupled with a fluorescence detector. We compared the retention time of the samples detected by the refractive index detector to that of a standard dextran sample of 5,500 Da (Fig. 1a). Both of these polysaccharide samples shared the same retention time, indicating that both the FBE and ME samples’ molecular weights are lower than that of the dextran standard. The MALDI-TOF analysis revealed a main peak at 820.70 Da for both samples that could be assigned to a molecule containing four hexoses and one pentose. Additional peaks, all up to 3,000 Da, in both spectra, indicated the presence of a low molecular weight polymer. To further confirm the size of the polymer, we labeled the sample with 2-aminobenzamide, a sugar-specific fluorescent label that is attached to the reducing end of the sugar chain. This method allows the detection of oligosaccharides and polysaccharides of various sizes, while enabling the relative quantification of the various sugar structures in the sample. The samples are compared to a dextran partial hydrosylate that gives a ladder of “glucose units.” Results indicated that both samples contain a polymer that has up to about 18 monomers (up to 3,000 Da). The main peak was found to be at 3.4 glucose units, indicating that most of the polysaccharide is at a low molecular weight of approximately 500 Da (Fig. 1b, c). Cumulatively, all three methods gave an indication of a relatively low molecular weight polysaccharide in both the FBE and ME samples (between 500 and 3,000 Da).
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Fig. 1

Size characterization of FBE and ME polysaccharides. Size separation of polysaccharides using SEC (a) with a refractive index detector is compared to a standard dextran (5,500 Da). Results indicate that both FBE and ME polysaccharides are of similar molecular weights and both are smaller than the dextran standard. The normal-phase HPLC profiles of 2-aminobenzamide-labeled FBE (b) and ME (c) polysaccharides separated according to size show similar results. Both show a main peak at 3.4 glucose units (GU), indicating that the polysaccharide consists of mainly low-weight polymers (∼4 sugars), with additional minor structures containing higher-weight polysaccharides

Viscosity analysis of FBE and ME glucans

Viscosity of HWS and ME was determined to be 6.1 Cp/2.5 mg and 1.02 Cp/2.5 mg, respectively. These data are comparable to the glucan we have previously extracted from P. ostreatus (1.6 Cp/2.5 mg; Lavi et al. 2006) for which the molecular weight range detected was from 1,000 up to 10,000 Da. Generally, the relatively higher viscosity values reflect a relatively expanded chain of the polymer.

Expression of galectin-3 in colon cancer cell lines

The expression of the galactose binding molecule galectin-3 was tested in HT29, HCT-116, Caco2, LS174T, HM-7, and RSB colon cancer cell lines (Fig. 2). LS174T, HM-7, RSB, and HT29 cells expressed the highest concentration of galectin-3 while HCT-116 and Caco2 cells expressed the least (Fig. 1a, b).
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Fig. 2

a Expression of the 31-kDa protein galectin-3 in colon cancer cell lines with varying tumorigenic capacity. HT29, HCT-116, HM-7, LS174T, RSB, and Caco2 cells were analyzed for galectin-3 by SDS-PAGE as described in “Materials and methods.” b Densitometry analysis relative to beta actin. Results represent the average ± SD of the four independent experiments. Statistical analyses by Tukey–Kramer test, asterisks P < 0.01

Antiproliferative effect of low molecular weight FBE and ME glucans on colon cancer cell lines

FBE and ME preparations were analyzed for their antiproliferative activity on the human colorectal cancer cell lines HT29, HCT-116, Caco2, LS174T, HM-7, and RSB. The cell lines were exposed to low molecular weight FBE or ME glucans for 24, 36, 48, and 60 h at four different concentrations: 0.05%, 0.1%, 0.25%, and 0.5% (w/v). Both FBE and ME polysaccharide preparations exerted a significant and dose-dependent growth-inhibition effect in all cell lines except Caco2 cells (Figs. 3 and 4). The effects were already pronounced at 24 h of exposure in all colon cancer cells tested and for both FBE and ME preparations. As depicted in Figs. 3 and 4, the growth-inhibition effect of FBE in LS174T, HM-7, and RSB cells after 24 h was higher than in HT-29 and HCT-116 cells and much more than in Caco2 cells as compared to controls. At concentrations of 0.25% and 0.5%, a significant effect was seen in all cell lines.
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Fig. 3

Effect of FBE glucan on viability of a variety of colon cancer cell lines. HT29 (a), HCT-116 (b), HM-7 (c), LS174T (d), RSB (e), and Caco2 (f) cells were exposed to the indicated concentrations (w/v) of FBE glucan for 24, 36, 48, and 60 h. Cell viability was measured by the MTT cell viability assay, and the MTT reduction rate was calculated by setting each of the control survivals equal to 100%. Data are mean ± SD values of n = 6 experiments. Error bars, where not shown, are smaller than the symbol size. At the 48- and 60-h time points, all treatments were significantly different from the control (P < 0.01) as verified by Tukey–Kramer test

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Fig. 4

Effect of low molecular weight ME glucan on viability of a variety of colon cancer cell lines. HT29 (a), HCT-116 (b), HM-7 (c), LS174T (d), RSB (e), and Caco2 (f) cells were exposed to the indicated concentrations (w/v) of ME glucan for 24, 36, 48, and 60 h. Data are mean ± SD values of n = 6 replicates and are representative of three independent experiments. Error bars, where not shown, are smaller than the symbol size. At the 48- and 60-h time points, all treatments were significantly different from the control (P < 0.01) as verified by Tukey–Kramer test

The number of viable colon cancer cells was reduced significantly to approximately 40% to 45% of controls at a concentration of 0.5% but only after 36 h incubation with FBE glucan or ME glucan, as depicted in Figs. 3a–f and 4a–f, respectively (P < 0.01). We concluded that, in HT-29, Caco2, and HCT-116 cells, the effect of FBE and ME glucan treatment occurs after longer incubation periods (24 h) than in LS174T, HM-7, and RSB cells. Following 60-h incubation with ME and FBE glucan, a significant inhibition in cell viability (P < 0.01) was obtained in all cell lines, even at the low concentration of 0.05%. Growth inhibition was only 2% to 9% when the cells were exposed to the control polysaccharide neutral CMC for 60 h (not shown).

FBE glucan and ME glucan reduce the ability of colon cancer cell lines to adhere to FN

Adhesion of cancer cells to endothelial cells or to the extracellular matrix (ECM) is a key step in cancer progression and generation of tumor metastasis. Fluorescently labeled colon cancer cells were tested for their adhesion capability to FN, a key component of the ECM (Dallas et al. 2006). We measured the ability of the different colon cancer cell lines to adhere to FN following exposure to different concentrations of FBE, ME, or neutral CMC as a control. After incubation of the treated cancer cells for 24 and 48 h (not shown) on FN-precoated plates, the nonadhered cells were removed, and the adhered cells were incubated in DMEM containing MTT. Adhered cells were solubilized with DMSO, and viable cells were quantified using an ELISA reader as described in “Materials and methods.” Figure 5a–d shows that both low molecular weight FBE glucan and low molecular weight ME glucan reduced the ability of all cell lines to adhere to FN in a time- (not shown) and dose-dependent manner in all cell lines except Caco2 cells. High concentrations of both FBE glucan and ME glucan almost completely abolished the adhesion capability of the colon cancer cells to FN, while CMC was ineffective in this regard (as with its lack of antiproliferative effect).
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Fig. 5

Adhesion of human colon cancer cells to FN. Effect of FBE glucan or ME glucan on adhesion of HT29 (a), HCT-116 (b), LS174T (c), and Caco2 (d) cells to human FN. Data are mean ± SD of n = 3 replicates and are representative of three independent experiments. Error bars, where not shown, are smaller than the symbol size. At the 48-h time point, all treatments with 0.25% and 0.5% glucan were significantly different from the control (P < 0.05) as evidenced by Tukey–Kramer test

At all concentrations and times tested, we found that HT29, HCT-116, and LS174T cell lines were sensitive to FBE glucan and ME glucan treatment, whereas Caco2 cells, expressing low amounts of galectin-3, were to a large extent insensitive to FBE glucan and ME glucan treatment. Neutral CMC was used as a negative control and was indeed ineffective in inhibiting the cells’ adhesion capability, strengthening the importance of fungal glucans. CMC did not affect either cell viability or their adhesion capability.

FBE glucan and ME glucan reduce the ability of colon cancer cell lines to adhere to HUVECs

To explore the effect of FBE and ME glucans on cancer cell binding to vascular endothelial cells, HUVEC monolayers were preincubated with ME glucan or FBE glucans. HT29, HCT-116, Caco2, and LS174T cell lines were loaded with calcein AM, and their adhesion to HUVECs was determined by coculture experiments. Colon cancer cells were added 8 and 24 h after treatments. Both low molecular weight ME glucan and low molecular weight FBE glucan failed to influence HUVEC viability during the tested time periods (not shown). Representative micrographs of fluorescently labeled HT29 cells adhered to HUVECs are shown in Fig. 6a–h.
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Fig. 6

Effect of FBE and ME glucan on adhesion of calcein AM-labeled cancer cells to HUVECs. Representative pictures: calcein AM-labeled cancer cells were added to untreated (ad) and treated (eh) HUVEC monolayers. The adherent fluorescent cells are shown. Upper panel: calcein AM-labeled HT29 cells adhere to untreated HUVECs used as control. Magnification ×10 (a, b) and ×40 (c, d). Lower panel: calcein AM-labeled HT29 cells adhere to HUVECs treated with 0.5% FBE glucan for 24 h. Magnification ×10 (e, f) and ×40 (g, h). The adherent fluorescent cells were counted by Image J software (see Fig. 7)

As shown in Fig. 7a, several concentrations of FBE glucan similar to those used in the viability assay, i.e., 0.25% and 0.5%, significantly reduced tumor cell adhesion to the HUVEC monolayer relative to controls in all cell lines except Caco2 cells. As shown in Fig. 7b, several concentrations of MS glucan also significantly reduced tumor cell adhesion to the HUVEC monolayer relative to controls. At the concentrations and time points tested, it was apparent that Caco2 cells were much less sensitive to ME and FBE glucan treatment than the other cell lines.
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Fig. 7

Adhesion of human colon cancer cells to HUVECs. The adhered fluorescent cells were counted by Image J software. Data are mean ± SD values of n = 6 replicates and are representative of two independent experiments. Asterisks, significantly different from control (P < 0.05) as verified by Tukey–Kramer test

Discussion

Glucans from the edible mushroom P. pulmonarius are natural polysaccharides synthesized by the fungus when grown as a mycelium in liquid culture and by the fruiting bodies when grown under “natural” conditions, using straw as a substrate. These compounds are of importance due to their potential biological activities and medicinal properties (Wasser and Weis 1999). We compared the molecular weight and composition of the glycosyl residues, and the types of glycoside bonds, in polysaccharides harvested from P. pulmonarius under different growing conditions [glucan extracted from fruiting bodies (FBE) vs. glucan extracted from mycelia in submerged culture (ME)]. We found that the glucan extracted from FBE contained 84.6% glucose as compared to that extracted from ME contained only 64.4% glucose. Both FBE and ME glucans contained significant and equal amounts of galactose (8.3%). The ME polysaccharide also contained fucose and xylose, which were not found in the FBE glucan. The reason for the difference in carbohydrate content could be explained by the differences in sugar sources and growing conditions. Regarding the structural analyses of the glucan, 13C and 1H NMR analyses of the FBE preparation showed mixed α-linkages and β-anomeric carbon linkages, whereas the ME polysaccharide demonstrated only α-glucan linkages. With respect to size characterization, analyses performed by three independent methodologies demonstrated that polysaccharides extracted from P. pulmonarius grown under the two different conditions are relatively small.

Pleurotus species cell walls are composed of insoluble glucans, showing a structure of (1→3) linkage β-glucosyl residues (over 50%) together with (1→4) linkage α-glucosyl residues (Hadar and Cohen-Arazi 1986; Saito et al. 1976). Other researchers have isolated soluble β-glucans with biological activity not only from the fruiting bodies but also from the mycelium and from culture broth, e.g., Yoshioka et al. (1985) isolated β-glucan from P. ostreatus fruiting bodies, Sarangi et al. (2006) isolated β-glucan from P. ostreatus mycelia, and Zhang et al. (2001) and Tao et al. (2006) extracted β-glucan from Pleurotus tuber-regium sclerotia. Saito et al. (1976) also found α- and β-glucans in the aqueous extracts of P. ostreatus fruiting bodies. Gutierrez et al. (1996) described the structural characterization of extracellular polysaccharides produced by six fungi from the genus Pleurotus and compared with those extracted from the fruiting bodies of P. ostreatus. Glucans from mycelium showed mainly a β configuration of the main glucan linkages, in contrast to fruiting bodies that showed mixed α and β configurations. To the best of our knowledge, this is the only direct comparison of mycelium to fruiting bodies. However, in contrast to our study, those authors did not compare the biological activities of the isolated compounds. Data accrued to date agree with our results, showing that extracts from fruiting bodies contain a mixture of α and β configurations. This conformation differs from the glucans harvested from washed mycelium extracted from culture filtrate of Pleurotus ostreatoroseus (Rosado et al. 2003) and of glucans washed from mycelium of P. ostreatus (Lavi et al. 2006), where only the α configuration was detected. In the latter study, we demonstrated antiproliferation activity against the colon cancer cell line HT29. The biological and immunopharmacological effects of fungal glucans are mainly associated with those having a β-d-glucan structure, which is a major component of fungal cell walls, and among them, those with a high molecular weight, higher degree of branching, and higher water solubility (Borchers et al. 2004). In our study, we found a correlation between the bond types of the glucan and the level of cytotoxicity induced in the different neoplastic colon cell lines. Some studies have demonstrated that high molecular weight β-glucans are more effective than those of low molecular weight (Mizuno et al. 1999), whereas others show that α-glucans are active regardless of molecular size (Gao et al. 1996). In our study, the relatively low molecular weight glucan from ME (10,000 g/mol) already exerted a direct cytotoxic activity at 24 h in five from the six tested colon cancer cell lines, whereas similar but lower activity was observed after 60 h for the FBE glucan extracted from fruiting bodies. We conclude that α-glucans from P. pulmonarius are more cytotoxic, within a much shorter time. Several other fungal substances have also been demonstrated to inhibit the development of tumors and metastases by interfering with key cellular mechanisms (Petrova et al. 2007; Zaidman et al. 2005). A few reports have also demonstrated antitumor-related activities of α-glucans (Kiho et al. 1994; Yoshida et al. 1996).

It can be speculated that the binding ability of a relatively small molecule to a tumor cell is more efficient than that of a larger molecule, since the former is sterically more accessible and therefore its time to activity may be shorter, impinging on higher effectiveness. In addition to their significant antiproliferative effect, we investigated the possible effect of FBE and ME glucans on cell–cell interactions. We studied the interaction between the tumor cells and an extracellular matrix protein and between the tumor cells and cells from blood vessels. To this end, two different systems were selected to measure specific adhesion capabilities: one measuring adhesion to FN, a major component of the extracellular matrix and the other adhesion to endothelial (HUVEC) cells. In these systems, we examined four representative colon cancer cell lines. In addition, we took two approaches: in the first, the cancer cells received different concentrations of the isolated glucans, and then their adhesion to FN was examined; in the second, endothelial cells received the isolated glucans treatment and untreated cancer cells were added to the culture to test for adhesion. In both cases, treatment resulted in a significant reduction in the adhesiveness of three of the four cancer cells tested in a concentration- and time-dependent manner (not shown). We demonstrate herein that, in cells expressing high galectin-3 such as LS174T and HM-7 cells (see Fig. 5), FBE and ME glucans affect more significantly their viability than in Caco2 cells. The most striking differences in cell type response where in adhesion to FN and HUVEC. LS174T was the cell mostly affected followed by HT-29 and HCT-116 cell, and the cell line Caco2 was barely affected. The differential cell effect of the polysaccharides may be explained by their direct effect on the cell surface of colon cancer cells. The most feasible candidate for this cell type response is galectin-3, which is a carbohydrate-binding protein in charge of mediating the cell–cell interactions and demonstrates a high affinity to β-galactoside sugars. It has been previously shown that the expression of galectin-3 increases as the cancer stage advances and that blocking expression of galectin-3 in different carcinoma cells leads to a regression in the transformed phenotype and to a suppression of tumor development in nude mice (Honjo et al. 2001). In another study, Honjo et al. (2000) showed that obstruction of galectin-3 activity inhibits the adhesion and aggregation of cancer cells to one another and to normal cells and concomitantly to a delay in the formation of metastatic lesions. We show herein that FBE and ME in addition to containing glucose also contain significant amounts of galactose. As alluded to earlier, galactose sugars possess high affinity to galectin-3. We hypothesize that the low molecular weight glucans from FBE and ME contain significant amounts of galactose (see Table 1) and may bind more avidly to cancer cells expressing higher amounts of galectin-3 (HM7 and LS174T cells) than to cells expressing less galectin-3 (Caco2 cells) and thus affect their cell viability and adhesion capability to extracellular matrix proteins (FN) or to endothelial cells (HUVEC). In conclusion, our results show several distinct differences in the activities of low molecular weight glucans produced in two different ways, yielding slightly different chemical structures. Due to the wide variety of biological effects of fungal polysaccharides, the challenge is to determine the existence of glucan structure–function relationships that may be responsible for their medicinal properties, resulting from biological activities such as inhibition of proliferation and adhesion of tumor cells.

The effects of the glucans in vitro in colon cancer cells are indicative of putative effective effects of these molecules in in vivo models. Only recently, Rice et al. (2005) showed that fungal-derived soluble glucans translocate from the GI tract into the systemic circulation in normal animals. We have recently demonstrated that oral administration to dextran-sulfate-induced colitic mice of the different glucan preparations (HWS or ME) harvested from P. pulmonarius significantly attenuate the development of colonic inflammation, suggesting putative clinical utility of this extracts for the treatment of ulcerative colitis, and confirm the possibility that these glucan preparations reach the colonic cells (Lavi et al. 2009).

Cumulatively, in this study, we show for the first time that water-soluble glucans of the edible mushroom P. pulmonarius, from both fruiting bodies and washes of biomass grown in liquid medium, have a direct effect on the viability of colon cancer cells of different malignancies and on their adhesiveness to FN and endothelial cells. These steps are crucial in the formation of metastases. We therefore suggest that these fungal products may have an effect on different steps of tumor development.

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