An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells

Cancer Letters 244 (2006) 61–70 www.elsevier.com/locate/canlet

An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells Iris Lavi a, Dana Friesem b, Shimona Geresh c, Yitzhak Hadar b, Betty Schwartz a,* a

Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel b Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel c Department of Biotechnology, Ben-Gurion University of the Negev, Beer-Sheva, Israel Received 3 November 2005; accepted 4 December 2005

Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus were examined using HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight a-glucan. HT-29 cells were exposed to the different isolates and significant inhibition of proliferation was obtained in a dose-dependent manner. Proliferation inhibition was shown to be the result of apoptotic induction because the pro-apoptotic molecules Bax and cytosolic cytochrome-c were upregulated. Fluorescence-activated cell sorter analyses of polysaccharide-treated HT-29 cells showed a high percentage of Annexin-positive cells. Here, we describe a newly identified low-molecular-weight a-glucan with promising anti-tumorigenic properties, and demonstrate its direct effect on colon cancer cell proliferation via induction of programmed cell death. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Pleurotus ostreatus; Glucans; Colon cancer cells; Apoptosis

1. Introduction Edible mushrooms have beneficial effects on health and in the treatment of disease through their immunomodulatory, anti-neoplastic and lipid-reducing properties [1,2]. The Shiitake mushroom (Lentinus edodes), for example, has served as a model for investigating functional mushrooms and isolating pure compounds for pharmaceutical use [3]. Water extracts of the Shiitake fruiting bodies have been shown to prevent tumor growth in mice [4–6]. Mushroom’s polysaccharides, especially the high-molecular-weight b-Dglucan have been considered to have anti-cancer activity [7]. * Corresponding author. Tel.: C972 8 9489007; fax: C972 8 9363218. E-mail address: [email protected] (B. Schwartz).

The purpose of the present research was to evaluate the effect of a hot-water polysaccharide extract of Pleurotus ostreatus on the proliferation of human colon cancer cells. This included determining the culture conditions that would yield optimal production of this polysaccharide by the mushroom, identifying the polysaccharide and, ultimately, assessing the putative mechanism underlying the inhibition of colon cancer cell growth in vitro. 2. Materials and methods All chemicals and biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. P. ostreatus Florida (f6 ATCC #58053) was maintained on 2% agar basidiomycete synthetic medium

0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.12.007

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(BSM) as described [8]. Cultures were incubated on liquid BSM for 5 consecutive days. The resulting mycelia were homogenized which provided the basis for more homogeneous inoculums later incubated in liquid BSM for additional 2 days. Different BSMs were prepared with different carbon-to-nitrogen atomic ratios (16:1, 32:1, and 64:1) contained in glucose and asparagine. Varying the concentrations of glucose and asparagine changed the carbonto-nitrogen (C:N) ratios. Mycelia were grown for three different times (for BSM 16:1 and BSM 64:1; 5, 7, and 10 days) and six different times for BSM 32:1 (3, 5, 7, 9, 11, and 13 days). The extracellular medium and biomass were collected at the end of each incubation time. Biomass was dried and weighed or alternatively harvested from the fresh biomass and washed with distilled water at 80 8C. The resulting wash water was collected, frozen and lyophilized, and designated the Wash Water fraction—WW (WW 16:1, WW 32:1 and WW 64:1, depending on the C:N ratio. WW were extracted with 80% ethanol at 80 8C for 1 h, and the combined extracts were then evaporated. Two fractions were obtained: the precipitate (1) and the aqueous remaining supernatant (2), which were further proceed: (1) The precipitate was extracted with hot water and the filtrate lyophilized and designated the hot-water-soluble (HWS) fraction (see Fig. 1); (2) The aqueous remaining supernatant defatted with CH3Cl and the aqueous layer lyophilized and designated the defatted alcohol-soluble (DAS) fraction (see Fig. 1). Fig. 1 summarizes the isolation procedure in a schematic form. Protein concentration was determined by Bradford and glucose determined with Glucostat reagent (PGO enzymes, Sigma). HWS fractions were chemically analyzed in order to identify the nature of the polysaccharide secreted by the P. ostreatus mycelial cultures. Carbohydrates were determined colorimetrically by the phenol–sulfuric acid method

[9] with an LKB UV–VIS spectrophotometer (Biochrom Ltd, Cambridge, UK). A calibration curve was constructed with D-galactose as the standard. Sugar composition was determined by gas chromatography (GC) with a Hewlett Packard HP 19091C-133 GC, equipped with FID detector on a DB-1 capillary column (30 m, 0.25 mm id) from J&W Scientific (Folsom, CA, USA), with helium as the carrier gas at 220 8C. HWS derived polysaccharide samples (10 mg) harvested from the different C:N-ratio media were hydrolyzed acetylated, washed with water, dried, and redissolved in dry acetone. Particle size was determined by dynamic light scattering (DLS) measurements, using an ALV-HPPS (ALVGmbH, Langen, Germany). The light source was a He–Ne ion laser (632.8 nm). Spectra were collected at a scattering angle of 1738 and the correlograms were calculated using an ALV5000/EPP digital correlator operating in the pseudo-crosscorrelation mode. The time autocorrelation function of the scattered intensity was Laplace-inverted and analyzed by the Contin program [10,11]. The resultant diffusion coefficients (D) were converted to particle Stokes radii, Rh using the Stokes–Einstein relation, DZKT/phRh, where K is the Boltzmann constant, T is the temperature (K), h is the solvent viscosity and Rh is the hydrodynamic radius of the molecule. The apparent viscosity of the polysaccharide solutions (0.25%, w/v) was measured with a Brookfield programmable LVDVC2 viscometer (Brookfield Eng. Labs. Inc., Stoughton, MA) spindle 18, at 30 rpm. 13C-NMR spectra in DMSO-d6 were obtained with a 500 MHz Bruker NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). HT-29 human colon tumor cells (ATCC number HTB-38) were cultured as previously described [12]. Primary human normal fibroblasts were prepared and cultured as described [13]. Cells were incubated in a media containing different concentrations of solubilized WW, HWS or D-fraction

Wash Water (WW) 80% EtOH (80°C, 1h × 4 )

Precipitate water (80°C, 1h × 4 )

Supernatant Evaporation Remaining Aqueous Solution Extraction CH3Cl (× 3)

Aqueous Supernatant

Precipitate Aqueous Supernatant

Lyophilization HWS fraction

Lyophilization DAS fraction

Fig. 1. Scheme depicting the methodology used to obtain HWS and DAS fractions.

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(D-fraction extract from Maitake mushroom, containing 5.5 mg b-glucan/ml dissolved in glycerin was purchased from Maitake Products Inc, Paramus, NJ). Cell media was changed after 2 days; the solubilized treatment was replaced with fresh treatment and on the fifth day, cells were harvested by trypsinization and vital cells, excluding Trypan Blue, were counted in a Bright Line Neubauer counting chamber (SigmaAldrich Chemie, Steinheim, Germany). Apoptosis was assessed in living cultures using an Annexin V-FITC Kit (Bender MedSystems Diagnostics GmbH, Vienna, Austria) following the manufacturer’s specifications. Binding of fluorescein-conjugated Annexin V and PI was analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). Cytosolic fractions were prepared as previously described [14] and aliquots electrophoresed on 15% SDS polyacrylamide gels. Cytochrome c was detected with a monoclonal antibody (Pharmingen, BD Biosciences Clontech, Palo Alto, CA). Cell lysates were electrophoresed on 12% SDS polyacrylamide gel and Bax detected with rabbit anti-Bax, Santa Cruz Biotechnology, Santa Cruz, CA. All The blots were stripped and re-probed with anti-b-actin antibody (Sigma Chemical Co. St Louis, MO); which was used as a loading control. Western blots were scanned and quantified with densitometry software (Image J). The integration values for each band were taken and ratios to actin calculated. All values are expressed as meansGSEM. Statistical significance was calculated by Student’s t-test or was analyzed by one- or two-way ANOVA. Differences were considered significant at P!0.05.

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3. Results Table 1 indicates that the yield of biomass fraction obtained from P. ostreatus was very high in BSM 32:1, then lower in BSM 64:1 and very low yield in BSM 16:1. The polysaccharide starts to degrade after the seventh day in culture in BSM 32:1 as well as in BSM 64:1, and after the fifth day in BSM 16:1. We conclude that the optimal conditions for maximal polysaccharide yield from the P. ostreatus micela where a medium with carbon/nitrogen ratio of BSM 32:1 and 7 days of culture. Chemical characterization of all HWS fractions indicated that these fractions contained mainly a polysaccharide with a viscosity of 1.6 cP/2.5 mg HWS. Hydrolysis and GC analyses demonstrated glucose as the only carbohydrate present in the polysaccharide chain. The spectra obtained by 13C-NMR analyses (Fig. 2A) of the dissolved HWS polysaccharide proposes an a-glucan structure, rather than a b-glucan, molecules hitherto reported to be most commonly produced by this mushroom strain. The molecular mass of 80% of the polysaccharide contained in the HWS fraction was between 1000 and 10,000 Da (Fig. 2B), indicating that the polysaccharide has a low molecular weight, in contrast to the reported high molecular weights of polysaccharides bearing the b-glucan structure. The effect of various WW harvests from P. ostreatus grown

Table 1 Effect of different BSM media on average concentrations of protein, dry-weight polysaccharide and free glucose released to the medium, and dry-weight biomass C:N ratio

Day

Average protein concentration in the medium (mg/ml)

Average polysaccharide weight in the medium (g)

Average glucose concentration of in the medium (g/l)

Average biomass weight (g)

BSM 16:1

0 5 7 10 0 3 5 7 9 11 13 0 5 7 10

– 16.9G2.1 35.3aG3.3 35.3aG5.6 – 34.7G3.7 31.7G0.4 27.1bG2.8 15.6bG0.9 15.7bG0.4 14.9cG0.1 – 21.957G4.10 32.283aG3.03 51.819bG5.25

– 1.8G0.3 0.50cG0.017 0.214cG0.021 – 3.35G0.22 4.42bG0.10 6.03cG0.44 3.60G0.24 3.30G0.45 2.88G0.48 – 1.572G0.32 3.46bG0.073 1.15G0.24

5 0.46cG0.14 0.55cG0.07 0.61cG0.05 20 11.3G0.4 11.7G0.8 10.5bG0.96 9.2bG0.96 7.9bG0.959 7.9bG0.959 40 44.95G0.24 41.15G0.81 25.75bG0.96

– 0.45G0.01 0.44G0.01 0.38aG0.004 – 2.5G0.13 3.3bG0.11 4.5cG0.40 2.7G0.37 2.3G0.22 2.1G0.21 – 1.17G0.13 2.217bG0.10 2.238bG0.14

BSM 32:1

BSM 64:1

Data were obtained at three different times (for BSM 16:1 and BSM 64:1) and six different times (BSM 32:1) and at three different C:N ratios in the culture medium (BSM 16:1, BSM 32:1 and BSM 64:1). Values are meansGSEM and statistical analyses by Tukey–Kramer test. nZ5. Comparisons were performed for each measurement between consecutive days of incubation for each BSM condition. aP!0.05, bP!0.01, cP!0.005.

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in BSM with different C:N ratios was tested on the proliferation of HT-29 cells. All WW harvests from P. ostreatus grown in BSM 16:1, BSM 32:1 and BSM 64:1 affected colon cancer cell proliferation in a doseresponsive manner as depicted in Fig. 3. All of these treatments resulted in statistically significant inhibition (P!0.05). HT-29 cells were treated with two different fractions of the P. ostreatus WW: the HWS fraction and the DAS fraction (see Fig. 1 for fractionation details).

Treatment with 0.5% (w/v) DAS was not effective in downregulating colon cancer cell proliferation whereas treatment with 0.5% (w/v) HWS (from both WW 16:1 and WW 32:1) resulted in 67.75% and 93.75% inhibition, respectively (Fig. 4A). HWS extracts from P. ostreatus grown in different BSM containing different C:N ratios exerted similar inhibitory effects on HT-29 colon cancer cell proliferation (Fig. 4B) similar to WW extracts, supporting the view that the effect is due mainly

Fig. 2. A. Representative 13C-NMR analyses indicate that the spectrum obtained from the dissolved HWS polysaccharide suggests for a a-glucan structure. See signals obtained at 97.36 and 93.07 ppm indicative of the a-structure. B. Molecular weight detected by dynamic light scattering (DLS) technique: 80% of the polysaccharide contained in the HWS fraction has a molecular weight between 1000 and 10,000 Da. A representative analysis is shown.

I. Lavi et al. / Cancer Letters 244 (2006) 61–70

Proliferation (% of control)

A

160 140 120 100

a

80

a

60

b c

40 20 0

control 0.01% 0.025% 0.05% 0.1% 0.25% 0.5%

WW 16:1 Concentration (w/v)

Proliferation (% of control)

B 120 100 a

80

b

60

b 40 20 0 control 0.01% 0.025% 0.05% 0.1% 0.25% 0.5% WW 32:1 Concentration (w/v)

Proliferation (% of control)

C 120 100 80

b b

60 40

c

c

20 0 control

0.05% 0.1% 0.25% 0.5% WW 64:1 Concentration (w/v)

Fig. 3. Effect of Wash Water (WW) from Pleurotus ostreatus mushrooms grown in different C:N ratios media, on the proliferation of HT-29 cells. HT-29 cells were exposed to WW 16:1 (A), WW 32:1 (B) and WW 64:1 (C). On the 5th day, cells were harvested by trypsinization and vital cells, excluding Trypan Blue, were counted. Values are meansGSEM and statistical analyses by Tukey–Kramer test. nZ8. aZP!0.05, bZP!0.01, cZP!0.005. A. Number of cells in controlZ6.4!106. B. Number of cells in controlZ6.1!106. C. Number of cells in controlZ7.7!106.

to a common specific polysaccharide. We conclude that this polysaccharide characterized by its strong proliferation inhibition ability of colon cancer cells, is more concentrated in HWS preparations than in WW preparations. To test whether the effects exerted by the partially isolated polysaccharide is specific, or general and comparable to other polysaccharides, HT-29 colon cancer cells were exposed to two additional purified commercially available polysaccharides: carboxymethyl cellulose (CMC) and starch (both at 1% and

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0.05%, w/v). Starch at the concentrations used was not effective at inhibiting HT-29 colon cancer cell proliferation (not shown). CMC at the highest concentration used (1%) exerted only a borderline effect (less than 20%). At lower concentration (0.05%), no effect was detected. The ability of the D-fraction to inhibit proliferation of HT-29 cells was almost identical to that of the HWS fraction. The commercially available D-fraction, as well as our prepared HWS fraction, exerted a doseresponsive effect. Treatment with 0.5% (v/v) D-fraction resulted in 82.13% (P!0.005) inhibition (Fig. 4B). Treatment with 0.25%, 0.1% and 0.05% (v /v) D-fraction resulted in 53.33, 34.23 and 17.53% inhibition of cell proliferation, respectively (P!0.05). A significant difference existed not only between treated and non-treated (control) cells, but also between cells that were treated with D-fraction and those treated with glycerin-the solvent in which the D-fraction is dissolved, a finding proposing that the effect is also due to the solvent. The HWS fractions were not effective against primary fibroblast growth at concentrations at and above 0.05% (Fig. 5). We conclude that the HWS fraction more specifically inhibits the proliferation of cancer cells than the proliferation of normal cells. Fig. 6 shows representative dot plots of fluorescence intensities of HT-29 cells labeled with Annexin V/PI following no treatment (Fig. 6A), treatment with 0.5% HWS (Fig. 6B) and treatment with 0.5% D-fraction (Fig. 6C). Percent of surviving (Annexin V and PI negative, AnVK/PIK) and apoptotic (annexin V positive and PI negative, AnVC/PIK) cells are indicated in the quadrants. The percent of apoptosis in control HT-29 cells (2.57%) was significantly lower than in HWS-treated HT-29 cells (75.82%). D-fraction induced a significantly lower apoptotic effect (45.93%). Treatment with HWS and D-fraction resulted in a significant increase in the percent of AnVC/PIK HT-29 cells. The percent of early apoptotic cells (%AnVC/ PIK) were high in both HWS- and D-fraction-cultured cells (85.57 and 64.91%, respectively). The percent of surviving cells in the three groups were inversely related to the percentages of apoptotic cells. These results show a significant increase in apoptosis and decrease in survival in HT-29 cells exposed to polysaccharide extracted from edible mushrooms, with the HWS fraction exerting the most significant effect. Similar results were obtained in three additional independent experiments. Western blot analysis of cell lysates obtained from HT-29 cells indicated that proapoptotic proteins are overexpressed in cells treated

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Proliferation (% of control)

A 140 120 100 80 60 c

40

c

20 0

control WW 16:1 WW 16:1 WW 32:1 WW 32:1 DAS HWS DAS HWS

Treatment (0.5% w/v)

glycerin

Proliferation (% of control)

B D-fraction

120 100

a

HW S 16:1 a a

80

b b

60

HW S 32:1

b b

b

HW S 64:1 c c

40

c

c

20 0 control

0.05%

0.25%

0.5%

Concentration (w/v) Fig. 4. (A) Effect of HWS and DAS fractions prepared from WW from different C:N ratios (16:1 and 32:1). (B) Effect of HWS fractions from media with different C:N ratios as compared to D-fraction extract from Maitake mushroom on the proliferation of HT-29 cells. HT-29 cells were cultured in DMEM (control) or DMEM containing the solubilized D-fraction and glycerin (as a control for the solvent) at different concentrations. Cell treatment as described in Fig. 3. Values are meansGSEM, and statistical analyses by Tukey–Kramer test. nZ8. aZP!0.05, bZP!0.01, cZP!0.005. A. Number of cells in controlZ6.8!106. B. Number of cells in control D-fractionZ6.1!106, number of cells in control HWS 16: 1Z5.7!106, number of cells in control HWS 32:1Z6.6!106, number of cells in control HWS 32:1Z6.7!106.

proteins along with other mitochondrial proteins [16]. Defects in the cascade of apoptosis-related events during neoplastic development could well affect the execution of apoptotic death and disrupt homeostasis regulation of the colonic tissue. Therefore, a major

Proliferation (% of control)

with the HWS fractions from all C:N-ratio media. The HWS products caused a significant increase in Bax expression/corrected to equal loading with beta actin density (Fig. 7). No effect was seen on Bax in cells treated with D-fraction (not shown). The results indicate that HWS 16:1 exerts dose-dependent Bax up-regulation. The same pattern was observed for HWS 32:1 and for HWS 64:1. Cytochrome c levels also increased in response to the HWS 32:1 treatment (Fig. 8). Cytochrome c expression was upregulated following HWS 32:1 treatment. Maximal upregulation was achieved at 0.5% HWS 32:1 with milder effects at lower concentrations. The same pattern was observed for HWS 16:1 and HWS 64:1 (not shown).

120 100 80 60 40 20 0

4. Discussion It has been proposed that the transformation of normal colorectal epithelium to carcinomas involves progressive apoptotic inhibition [15]. Apoptosis entails the execution of specialized machinery, central components of which are the family of Bcl-2-related

control

0.05 %

0.1%.

0.25 %

0.5 %

HWS 32:1 Concentration (w/v) Fig. 5. Effect of HWS fraction from WW 32:1 C:N ratio, on the proliferation of normal primary human fibroblasts. Fibroblasts were cultured in DMEM or in DMEM containing the solubilized treatment at different concentrations. Values are meansGSEM, and statistical analyses by Tukey–Kramer test. No significant differences were found.

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Fig. 6. Flow cytometry analysis of HT-29 cells. HT-29 cells were treated with D-fraction or HWS fraction, or left untreated as controls. The cells were analyzed for apoptosis with an Annexin V-FITC Kit. Data are from one of two similar experiments. A. Untreated control cells. B. Cells treated with 0.5% HWS. C. Cells treated with 0.5% D-fraction.

strategy for colon cancer chemoprevention is the search for nutritional components directed at inducing apoptosis of cancer cells. Edible mushrooms have beneficial effects on health and in the treatment of disease through their immunomodulatory, anti-neoplastic, and lipid-reducing properties [17–19]. The present study evaluates a number of conditions used to produce polysaccharides from the edible A

Cont (%)

HWS 16:1 0.05 0.5

mushroom P. ostreatus. P. ostreatus is a legitimate candidate to look for chemopreventive elements since it has been shown to exhibit a wide variety of medicinal properties, including anti-tumor activity [19]. All polysaccharide partial isolates described in our study dose-dependently affected HT-29 cell proliferation to similar extents, indicating that regardless to the C:N ratio, a secreted molecule (s) was responsible for the growth inhibition and HSW preparations were

HW 32:1 0.05 0.25

0.5

0.05

HW 64:1 0.25

Bax 21 kDa Beta actin

Densitometry (bak relative to beta actin)

B

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 control

HWS (16:1) 0.05%

HWS (16:1) 0.5%

HWS (32:1) 0.05%

HWS (32:1) 0.25%

HWS (32:1) 0.5%

HWS (64:1) 0.05%

HWS (64:1) 0.25%

Fig. 7. Effect of HWS fraction from media containing different C:N ratios (16:1, 32:1, 64:1) on the expression of Bax in HT-29 cells. Blot was reprobed with beta actin antibody to test equal loading. Densitometry analysis relative to beta actin. Results represent the averageGSEM of three separate experiments. Statistical analyses by Tukey–Kramer test. aZP!0.05, bZP!0.01, cZP!0.005.

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A

HWS (32:1) Cont

0.05%

0.5%

0.1%

0.25% Cyt–c 15 kDa

Beta actin

Densitometry (cyt–c relative to beta actin)

B

b

5 4

a

a

3 2 1 0 control

HWS (32:1) 0.05%

HWS (32:1) 0.5%

HWS (32:1) 0.1%

HWS (32:1) 0.25%

Fig. 8. Effect of HWS fraction from media containing different C:N ratios on the expression of cytochrome c in HT-29 cells. Blot was reprobed with beta actin antibody to test equal loading. Densitometry analysis relative to beta actin. Results represent the averageGSEM of three separate experiments. Statistical analyses by Tukey–Kramer test. aZP!0.05, bZP!0.01.

significantly more effective in inhibiting HT-29 cell proliferation than their respective WW preparations. This phenomenon is indicative that in the purification procedure the concentration of the polysaccharide increases. We performed further isolation steps and found that the HWS fraction and not the DAS fraction, was responsible for the inhibition of HT-29 proliferation. We performed chemical analyses of the HWS fraction and identified the active component as a lowmolecular-weight a-glucan. 13C-NMR spectrum analysis of representative HWS fractions (Fig. 2A) yielded 19 resonance signals, indicating the a-anomer form of glucose from its chemical shifts. The signals at 97.36 and 93.07 ppm are assignable to a mixture of C6 carbon atoms in a-(1,4) and a-(1,6) linkages. Therefore, we conclude that the HWS fraction does not contain a b-configuration-glucan, which has its C1 peak at values above 100 ppm [20]. Moreover, the low molecular weight provides additional evidence that the HWS fraction indeed contains an a-glucan and not a b-glucan [21]. Mondal et al. [22] reported that a 1/6 a-glucan shows its C2, C3, C4, and C5 peaks at 72.87, 74.87, 71.02, and 71.65 ppm, respectively. Those values are similar to the HWS–NMR spectrum, where we also identified peaks located in the range of 70.97– 77.22 ppm. We assume that the HWS fraction does not contain a 1/3 a-glucan because the latter’s C3 peaks in DMSO-d6 appear at 83.2 ppm [23]. Based on these analyses, the structure of the HWS component

is essentially a mixture of 1/4 a- and 1/6 a-glucose units arranged into a soluble polysaccharide. Static light-scattering analyses indicated that HWS has a molecular mass between 1000 and 10,000 Da explaining the solubility of the molecule in water. The low molecular weight is also indicative of the polysaccharide not being a b-glucan. The inhibition of proliferation of HT-29 cells was the result of apoptosis induction, as evidenced by the FACS analyses of polysaccharide-treated HT-29 cells showing a high percent of Annexin-positive cells, in contrast to the negligible percentage in control cells. The finding that the HWS fraction affects mainly HT29 colon cancer cells and not normal human fibroblasts is encouraging because it shows specificity of the partially isolated polysaccharide towards neoplastic cells. The mechanism underlying the specific recognition remains to be determined. The expression levels of anti- and pro-apoptotic proteins of the Bcl-2 family such as Bax and Bak have previously been shown to directly engage the cell-death machinery, whereas Bcl-2 and Bcl-XL merely antagonize this interaction [24–26]. Bax, as well as its homologous protein Bak, promote cell death by competing with Bcl-2. Here we show that Bax protein expression and cytosolic cytochrome-c concentrations were strongly upregulated by the partially isolated a-glucan in the polysaccharidetreated HT-29 cells. The signaling events leading

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to apoptotic mitochondrion-related pathways lead to changes in mitochondrial membrane permeability and the subsequent release of pro-apoptotic factors involved in various aspects of apoptosis [26]. The released factors include cytochrome c, and cytosolic cytochrome c forms an essential part of the apoptosis complex ‘apoptosome,’ which is composed of cytochrome c, Apaf-1, and procaspase-9. Formation of the apoptosome leads to the activation of caspase9, which then processes and activates other caspases to orchestrate the biochemical execution of cells. Therefore, cytochrome-c release to the cytosol is indicative of mitochondrion-mediated apoptosis. In summary, we describe a newly identified lowmolecular-weight a-glucan with promising antitumorigenic properties, demonstrated to directly affect HT-29 colon cancer cell growth by regulating the expression of apoptosis-related proteins and inducing programmed cell death and inhibition of proliferation. This low-molecular-weight a-glucan preferentially exerts its anti-proliferative effects on HT-29 colon cancer cells and is significantly less effective towards normal human fibroblasts. Future work will focus on animal studies using timecourse and dose-response data, to test the effects of the whole extract and compare it to the putatively most potent purified components. The purified compound(s) may be very different from the whole mushroom in their inhibitory activity on proliferation. Future studies should answer some of these questions and provide guidelines for incorporating mushrooms and/or their extracts into the diet to take advantage of their ‘nutraceutical’ properties. Acknowledgments This study was supported in part by a grant from Yissum, Technology Transfer Company of the Hebrew University. References [1] S.P. Wasser, A.L. Weis, Therapeutic effects of substances occurring in higher basidiomycetes mushrooms: a modern perspective, Crit. Rev. Immunol. 19 (1999) 65–96. [2] T. Mizuno, H. Saito, T. Nishitoba, H. Kawagishi, Antitumor— active substances from mushrooms, Food Rev. Int. 11 (1995) 23–61. [3] S.C. Jong, J.M. Birmingham, Medicinal and therapeutic value of the shiitake mushroom, Adv. Appl. Microbiol. 39 (1993) 153–184. [4] R. Chang, Functional properties of edible mushrooms, Nutr. Rev. 54 (1996) S91–S93.

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