Food and Chemical Toxicology 48 (2010) 21152121

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Food and Chemical Toxicology

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Antitumor properties of a heteroglucan isolated from Astraeus hygrometricus

on Daltons lymphoma bearing mouse
S.K. Mallick a, S. Maiti a, S.K. Bhutia b, T.K. Maiti a,*
a
b

Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India
Department of Life Sciences, National Institute of Technology, Rourkela, India

a r t i c l e

i n f o

Article history:
Received 1 February 2010
Accepted 7 May 2010

Keywords:
Astraeus hygrometricus
Heteroglucan
Antitumor
Mushroom

a b s t r a c t
Polysaccharides such as glucans are the best known and most potent mushroom-derived substances with
antitumor and immunomodulating properties. However, a vast variety of mushroom species remain
unexplored. The present study aims at exploring the possible role of a heteroglucan designated as AE2,
isolated from Astraeus hygrometricus, on tumor bearing host. AE2 mediated tumor regression through
the reversal of tumor induced immunosupression. Tumor-associated macrophages from AE2 administered tumor bearing host have been demonstrated to possess augmented surface expression of co-stimulatory molecules and MHC-II, representing essentially elevated state of macrophage activation. NK cell
activity has also been found to be elevated with the AE2 treatment while the Th1 cytokine in the tumor
micro-environment increases with the treatment, the Th2 cytokine levels concomitantly decreases. Further, following the immunoactivation, Daltons lymphoma (DL) cells has undergone apoptosis, thereby
resulting in reduction of tumor growth and increased survival rate of the tumor bearing host. The present
study sheds light on the potent antitumor property of the heteroglucan AE2 isolated from A. hygrometricus, and can be extended further to develop therapeutic protocols for treatment of cancer or disease
resulting in immunosuppression.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Cancer is becoming the major concern for human health due to
the resulting deaths. Though all kinds of cancer follow similar
alteration of cell division, growth and their response to standard
therapeutic protocols (viz. chemotherapy, surgery, radiation therapy, cancer vaccine and antibody therapy), their degree of response
differs to a great extent due to the location, development and tumor induced immune system dysfunction. Existing cancer drugs
act as cell cycle inhibitors, apoptosis stimulators, signal transduction inhibitors, anti-inammatory compounds, anti-invasive
agents, anti-angiogenic compounds differentiating agents (Amin
et al., 2009). Till date there is no drug or therapeutic strategies that
do not harm normal cells of the host but prevent carcinogenesis
and inhibit growth of cancer cells only. Recently, attention has
been focused on the development of immunotherapy (active and
passive immunotherapy) to target and eliminate cancer cells. Such
compounds are classied as biological response modiers (BRMs).
BRMs possess a great potential not only in enhancing immune responses but also maintaining homeostasis and improving quality
of life, thereby mounting anti tumor immunity and alleviating
* Corresponding author. Tel.: +91 3222 283766; fax: +91 3222 255303.
E-mail address: tkmaiti@hijli.iitkgp.ernet.in (T.K. Maiti).
0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.05.013

the disease associated pathologies (Lull et al., 2005). In spite of

the great diversity of mushroom in nature, only six mushroom species have been investigated extensively till date (Wasser, 2002).
In a quest for such BRMs, studies are now undertaken to explore
and identify the mushroom species which has remained hitherto
neglected. We have been involved with these studies related to biological activity of mushroom derived bioactive molecules (Sarangi
et al., 2006; Shah et al., 2007) and A. hygrometricus is one of them.
A. hygrometricus is non cultivable, wild mushroom being used as
food for decades by the people of eastern part of India. However, except a few reports related to A. hygrometricus derived lectins (Yagi
et al., 2000) and immunomodulatory polysaccharides (Chakraborty
et al., 2004), the scientic studies pertinent to bioactive molecules
from this mushroom are grossly lacking. Recently, we have identied a bioactive protein fraction with anti-proliferative activity
towards cancer cells (Maiti et al., 2008). In addition, from the
above-mentioned mushroom, we have isolated a heteroglucan
using alkaline extraction, ethanol precipitation and chromatographic procedures. Correspondingly, we designate this group of
compound as AE2. AE2 features a high molecular weight and putative b-glucan structure and is composed of glucose, galactose, mannose and fucose. Further, AE2 has shown macrophage stimulatory
potential in vitro (Mallick et al., 2009). These preceding studies
have prompted us to explore the possible activities of these

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compounds. Towards this, the present study aims to understand the

role of this heteroglucan AE2 on the progression of cancer in a murine model.
2. Materials and methods
2.1. Chemicals
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) was purchased
from Loba chemicals, India), Ammonium chloride and BSA were procured from
SRL, India, Phytohaemagglutinin, Fluorescent labeled latex beads, Propidium iodide,
DNase free RNase, FITC-mannosylated BSA, and 3,30 -dioctadecyloxacarbocyanine
per chlorate (DiO) were purchased from Sigma, USA. Anti-CD80-PE, anti-CD86-PE,
anti-MHC II (I-A/I-E)-PE and cytometric bead array (CBA) were purched from BD,
USA. Fetal bovine serum (FBS) and RPMI-1640 media, trypsin EDTA, sodium azide
were obtained from Himedia, India and Limulus amebiocyte lysate (LAL) reagent
was purchased from Endosafe, USA.
2.2. Fruiting body and polysaccharide
Fruiting bodies of Astraeus hygrometricus (Pers.) Morgan (Gasteromycetideae)
were six collected from the markets of southern districts of West Bengal, India. A
heteroglucan was isolated from the fruit body of A. hygrometricus as described earlier (Mallick et al., 2009) and designated as AE2. Briey the residue of fruitbody after
an aquous extraction was subjected to alkaline extraction followed by ethanol precipitation and subsequent ion exchange chromatography. A yield of around 350 mg
of AE2 was obtained from 100 gm dry weight of mushroom. It contains galactose,
mannose, glucose, arabinose and fucose with the percent contribution of 16.0,
3.4, 69.0, 2.6 and 8.0, respectively, with a molecular weigh range of 500
1000 kDa as analyzed by gel ltration chromatography (Mallick et al., 2009). The
AE2 was subsequently passed through Polymyxin B Sepharose Matrix to remove
contaminating endotoxins, and no change was observed in the monosaccharide
composition and molecular weight of AE2 after passing through Polymyxin B Sepharose. An endotoxin level in AE2 to be used for biological assays was less than
1 ng/ml as determined by LAL assay.
2.3. Cell lines
YAC-1 cell line was obtained from National Centre for Cell Science (NCCS) Pune,
India and was maintained in RPMI-1640 media supplemented with 10% fetal bovine
serum.

ml lipopolysaccharide (LPS). All cultures were set up in triplicate for 72 h at 37 C

in a humidied atmosphere of 5% CO2. Proliferation of the cells was checked by
MTT assay method (Mosmann, 1983).

2.7. Isolation of tumor-associated macrophages (TAMs)

DL-bearing mice were sacriced by cervical dislocation and peritoneal exudates
cells (PEC) were harvested by peritoneal lavage (Singh et al., 2004). The PEC was
incubated in plastic tissue culture asks at 37 C in a CO2 incubator for 2 h. The cultures were then washed thrice with warm serum-free medium with gentle ushing
to ensure that all the DL and/or other non-adherent cells were removed. Approximately 95% of the adherent cell populations were macrophages as determined by
morphology. The TAMs were detached from the tissue culture ask with a cell scraper and counted.

2.8. Assay for nitrite production

After harvest, TAMs (106 cells/ml) were cultured in complete RPMI media in
96-well plate with LPS or IFN-c for 24 h at 37 C in humidied CO2 incubator. Production of nitric oxide was estimated by measuring nitrite levels in cell supernatant
with Griess reagent (1% sulfanilamide in 2.5% phosphoric acid, 0.1% napthylethyldiamine dihydrochloride in 2.5% phosphoric acid) and absorbance was read at 550 nm
(Bhutia et al., 2008).

2.9. Phagocytosis of latex beads by tumor-associated macrophages

TAMs were seeded to six well plates at 2  105 cells per well were treated with
LPS or IFN-c for 24 h followed by incubation with latex beads (amine-modied
polystyrene, uorescent red, 1.0 lm mean particle size, Sigma, USA) in fresh medium for 2 h. They were then harvested with trypsin EDTA and ow cytometry was
performed on a BD FACSCalibur ow cytometer using CellQuest pro software. The
phagocytosis was reported as mean uorescence intensity (Ozaki et al., 1995).

2.10. Macrophage mannose receptor (MMR) assays

TAMs were seeded to six well plates at 2  105 cells per well were treated with
IFN-c for 24 h followed by assay for surface display of MMR. After treatment, TAMs
were harvested and incubated with saturating concentrations of FITC-mannosylated BSA (250 ng/ml ligand/5  105) for 1 h at 4 C. Subsequently, cells were analyzed
on a BD FACSCalibur ow cytometer by using CellQuest pro software. The MMR
expression was reported as mean uorescence intensity (Stein et al., 1992).

2.4. Mice and tumor system

Swiss albino mice (20 2 g, 68 weeks old) were used for immunomodulation
studies. Mice were housed in open top cages and maintained on food and water
ad labium. Room temperature was maintained at 22 2 C with 14/10 h light and
dark cycle. All animal experiments were performed according to the rules of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Environment and Forests, Government of India and Institutional
Animal Ethics Committee, Indian Institute of Technology, Kharagpur, India. DL
(1  106/mouse) was maintained in ascetic form by serial transplantation in Swiss
albino mice or in an in vitro cell culture system by serial passage. Irrespective of
whether the cells were obtained from in vitro culture or from ascetic uid they
exhibited typical phenotypic features (Bhutia et al., 2008).

2.11. Immunophenotyping by ow cytometry

Tumor-associated macrophages were Seeded to six well plates, at 106 cells per
well in 10% FBS containing RPMI media. MHC II, CD80 and CD86 levels were measured using anti- MHC-II, anti-CD80-PE and anti-CD86-PE (BD, San Diego, CA),
respectively. Surface staining was performed with proper combinations of antibodies for 30 min on ice followed by washing in PBS containing 0.1% BSA and 0.01% sodium azide before data acquisition. Flow cytometry data was acquired on a BD
FACSCalibur ow cytometer and analyzed by CellQuest pro software (Caruso
et al., 1997).

2.12. Estimation of cytokines from peritoneal uid of DL-bearing mice

2.5. Tumor transplantation, AE2 treatment and survival study of DL-bearing mice
For survival study, mice were injected i.p. with 5  105 DL cells on day zero of
the experiment. On day 2 the mice were divided into three groups of ten mice each
and administered with PBS or AE2 on alternate days. The doses of 10 and 20 mg/kg
body weight of AE2 were selected based on our previous ndings on in vivo immunomodulatory studies (Mallick et al., 2010). After 5th dose of AE2, mice were observed till they succumb to tumor growth and the day is recorded. For all other
studies mice with treatment on similar schedule were sacriced on day 9 of tumor
transplantation.

Mice bearing DL were sacriced on day nine of tumor transplantation and peritoneal lavage was collected with the injection of two ml PBS. The peritoneal lavage
was centrifuged at 500 g for 10 min and the supernatant collected was used for
cytokine quantication by Cytometric bead array (CBA) from BD (San Diego, CA,
USA) for mouse IFN-c, IL-2, IL-4, IL-5, IL-10, and TNF-a and were performed by ow
cytometer. Standard curves for each cytokine, from each kit, were generated by
using the reference cytokine concentrations supplied by the manufacturers. The
quantication and analysis was done by ow cytometric analysis program (FCAP)
array software (Young et al., 2008).

2.6. Ex vivo proliferation of cells obtained from DL-bearing mice

2.13. Cell cycle analysis of DL cells

A single cell suspension of spleen, thymus and Bone marrow cells (BMC) were
prepared from DL-bearing mice treated with or with out AE2 under aseptic conditions by homogenization in Hanks balanced salt solution (HBSS) (Maiti et al., 2008).
A group of mice with out tumor were used as normal control. The contaminating
RBC from cell preparations was removed by hypotonic ammonium chloride treatment. After two washes in HBSS the cells were resuspended in complete RPMI medium. About 2  106 cells/well were plated in 96 well at bottom tissue culture
plates and incubated with or without 5 lg/ml phytohaemagglutinin (PHA), 2 lg/

The DL cells were washed PBS and xed in 70% ethanol for 4 h at 20 C. Then,
the cells were washed with ice cold PBS (10 mM, pH 7.4) and resuspended in 200 ll
of PBS followed by incubation with 20 ll DNase free RNase (10 mg/ml) and 20 ll of
DNA intercalating dye propidium iodide (PI) (1 mg/ml) at 37 C for 1 h in dark.
Apoptotic cells were determined by their hypochromic sub-diploid staining proles. The distribution of cells in the different cell cycle phases was analyzed from
the DNA histogram using BectonDickinson FACSCalibur ow cytometer and Cell
Quest pro software (Maiti et al., 2008).

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2.14. Ex vivo NK cell assay
A single cell suspension of spleen was prepared as mentioned before. The cells
were plated on plastic tissue culture asks at 37 C in a CO2 incubator for 2 h.
Non-adherent cells were taken as a source of NK (effecter) cells these cells were
incubated with different concentrations of YAC-1 cells (Target cells), labeled with
lipophilic membrane binding dye DiO (50 lg/ml) for 2 h. Effecter cells and target
cells at the ratio of 80:1, 40:1 and 20:1 were taken in 5 ml poly propylene (PP)
tubes. The tubes were then centrifuged at 250 g for 4 min to ensure effecter and target cell contact. The cells were then incubated for 4 h in a humidied 37 C, 5% CO2
incubator after the incubation period PI was added to the cells and ow cytometry
was performed. The equation given by Kane et al. (1996) was used to calculate the
percentage of YAC-1 cells killed.

Table 1
Cell cycle analysis of DL cells.
DNA cell cycle analysis

Sub G0/G1 (apoptosis) (%)

G0/G1 (%)
S phase (%)
G2/M (%)

Control

10 mg/kg AE2

20 mg/kg AE2

13.04 3
57.67
28.42
13.91

28.68 8.5*
47.39
44.13
8.48

29.79 7.2*
60.07
30.10
9.82

Daltons lymphoma cells obtained from mice administered with PBS (Control) or
AE2 were xed in ethanol and stained with DNA intercalating Dye PI. Cell cycle
analysis was done with ModFit software, data of sub G0/G1 (population) was represented as mean SD percent of population (N = 5). Students t-test was performed
and *indicates statistical signicance as compared to control group.

2.15. Statistical analysis

All data were given as the mean S.D. Experimental results were analyzed by
Students t-test. P < 0.05 was considered as the level of signicance for values obtained for treated compound to control.

3. Results
3.1. AE2 prolongs the survival of tumor bearing mice
The DL-bearing mice administered with PBS or AE2 (10 or
20 mg/kg of body weight) on alternate days for 14 days were observed and the days of survival were recorded. With AE2 treatment, the survival of DL-bearing mice increased (Fig. 1), the
medial survival times of 10 and 20 mg/kg AE2 treated groups were
found to be 27 and 29 days, respectively, where as the median survival times of control DL bearing group remained 23 days, as obtained by Kaplan Meirers survival analysis.

3.2. Morphological and DNA analysis of apoptotic DL cells

For a better understanding of the regression of DL growth, we
studied the DL apoptosis by DNA cell cycle analysis and the apoptotic body visualization by hematoxylene staining. There was an
increase in sub G0/G1 hypodiploid population in DL cells obtained
from mice treated with AE2 as compared to those isolated from
control mice (Table 1). The morphological studies of the DL smear,
after staining with hematoxylene showed increased occurrences of
cells with distinguished membrane associated blebs (a hallmark of
apoptosis) in the AE2 treated group as compared to the control
ones (Fig. 2).

Fig. 2. Morphological analysis of DL smear: A thin smear of DL cells from tumor

bearing mice peritoneal uid, 9 day post DL transplant was stained with hematoxylene and photographed. Three groups of mice received four dose of (A) sterile PBS
(B) 10 mg/kg of AE2 and (C) 20 mg/kg of AE2 on alternate days from 2 days post DL
transplant. Arrows point to the prominent membrane blebs on apoptotic cells.

3.3. Effect of AE2 on the ex vivo proliferation of immune cells from

treated mouse

Fig. 1. Effect of AE2 on survival rate of DL-bearing mice. Mice (10 per group) were
treated with PBS alone or 10, 20 mg/kg body weight of AE2, three days after tumor
transplantation for 12 days and left till death. Group1 received PBS while group 2
received 10 mg and group 3 received 20 mg/kg body weight of AE2. Survival of DLbearing mice was measured by monitoring the life span of DL-bearing mice and
data were analyzed using KaplanMeier method. Survival time plotted as days post
tumor transplant from three independent experiments. *Designates the signicance
of the log rank test of survival advantage of group 2 and group 3 as compared to
group 1 with p 6 0.05.

Proliferation of splenocytes, thymocytes, and bone marrow cells

obtained from DL-bearing mice was studied as a representative
measure of immune status of the tumor bearing host. As delineated
in Fig. 3A, the splenocytes from DL-bearing mice treated with AE2
at both 10 and 20 mg/kg body weight had signicantly higher proliferative potential than those of the control DL-bearing mice.
Again, the Splenocyte from AE2 treated group showed higher
responsiveness to PHA (added to culture medium) whereas those
from DL-bearing mice were observed to be less responsive to

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Ex Vivo proliferation splenocytes from DL bearing Mice

1.8
1.6
1.4

SPI

1.2

*
PBS
PHA

1
0.8
0.6
0.4
0.2
0
PBS

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

BPI

PBS
PHA

PBS

20mg/kg

Ex Vivo Proliferation of Thymocytes from DL bearing mice

TPI

10mg/kg

10mg/kg

20mg/kg

Ex Vivo proliferation BMCs from DL bearing Mice

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

PBS
PHA

PBS

10mg/kg

20mg/kg

In Vivo AE2 Treatments

Fig. 3. Effect of AE2 treatment on the ex vivo proliferation immune cells obtained from DL-bearing mice treated with or without AE2 (10 mg or 20 mg per kg body weight).
About 2  106/ml of cells obtained from mice were plated and cultured for 72 h and proliferation scored with MTT assay. (A) Ex vivo proliferation of splenocytes with/ without
PHA 5 lg/ml, (B) ex vivo proliferation of thymocytes with or without PHA 5 lg/ml and (C) BMC proliferation. Data are reported as the mean S.D. of three different
observations (ve animals per treatment group) and compared against PBS control by using Students t-test. P values < 0.05 are considered signicant compared with PBS
control (*signicant compared to PBS control).

PHA. Further, it was demonstrated (Fig. 3B) that treatment with

AE2 at both 10 and 20 mg/ kg body weight reversed the tumor
mediated suppression of proliferation and was approximately
equivalent to that of normal mice. Consequently, the reversal
was identied to be optimum at 10 mg/kg treatment of AE2. Also,
a reversal in the tumor induced suppression of bone marrow cells
of AE2 treated mice was observed to occur in a dose dependent
manner (Fig. 3C).
3.4. Ex vivo studies of TAMs obtained from DL-bearing mice
Tumor-associated macrophages are known to support the tumor growth; hence we studied the TAMs obtained from DL-bearing
mice for the ability of NO production and the surface expression of
MHC II along with other co-stimulatory markers. The NO production by TAMs from the AE2 treated and non-treated tumor group
was observed to almost similar with insignicant difference. How-

ever, TAMs from AE2 treated group in presence LPS or IFN-c,

showed a signicant increase of NO in culture supernatant
whereas those from control group did not show any appreciable
change in NO. The IFN-c responsiveness observed with TAMs from
mice treated with 20 mg/kg of AE2 was the highest of all the
groups indicating the reversal of the tumor induced macrophage
suppression as shown in Fig. 4. For a better comprehension of
the TAM Status, the surface expression levels of MHC II and mannose receptor were studied (Table 2a). The MHC-II expression on
TAMs obtained from mice treated with AE2 was signicantly higher as compare to the control ones, with 10 mg/kg AE2 treated
group producing the optimum results. We also examined the alteration in phagocytic potential of TAMs using uorescent labeled latex beads. Again, a reversal in tumor induced macrophage
suppression was observed as the TAMs from AE2 treated groups
showed signicantly higher phagocytic potential than the control
ones.

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S.K. Mallick et al. / Food and Chemical Toxicology 48 (2010) 21152121

Ex Vivo NO production by TAM from DL bearing Mice

120

NO (uM)

100
80

PBS
LPS
IFN

60
40
20
0
PBS

10mg/kg

20mg/kg

In Vivo AE2Treatments
Fig. 4. Ex vivo NO production By TAM: TAM isolated from DL bearing mouse peritoneum with or with out AE2, and were treated ex vivo with LPS or IFN-c. NO secreted to
culture medium was estimated as nitrates by Griess reagent, and represented as lM of NO produced and reported as the mean S.D. of three different experiments and ve
animals were taken per treatment group (*signicant compared to PBS control).

Table 2
Expression cell surface molecules and phagocytosis by TAM.

4. Discussion

Control

AE2 Treatment

PBS

10 mg /kg

20 mg/kg

MHC II
PBS
IFN-c

37.62 3.27
81.27 4.22

81.00 7.52
156.2 2.1*

35.71 2.14
167.4 24.22*

Phagocytosis
PBS
LPS
IFN-c

19.19 1.26
43.86 3.69
27.95 3.2

41.95 2.2
81.47 7.2*
34.85 3.4*

45.58 8.2
58.66 10.2
39.19 11.3*

Mannose receptor
PBS
1397.7 127
IFN-c
1347.9 40

1077.9 59
572.5 21*

1162.7 71
722.6 43*

Activation marker (MFI)

CD 80
25.26 4.9
CD 86
36.19 6.4

44.39 3.8*
72.39 11.8*

47.77 7.2*
85.27 9.4*

Tumor-associated macrophages isolated from DL-bearing mice treated with or

without AE2 were investigated for expression of surface molecules and phagocytosis by ow cytometry and data represented as mean uorescence intensity (MFI).
Students t-test was performed for statistical signicance (*) as compared to PBS
treated controls. P value of <0.05 was considered signicant.

3.5. Ex vivo activity of NK cells obtained from DL-bearing mice

Having noted a reversal of immune suppression both on macrophages and lymphocytes, we, further, investigated the NK cell
activity, which was perceived to play a major role in checking tumor growth. While assaying NK cell activity by YAC-1 cell killing,
a signicantly higher response was observed with the cells obtained from AE2 treated groups with respect to those from the control DL-bearing mice (Fig. 5).
3.6. Cytokine levels in the tumor micro-environment
In order to observe the role played by AE2 in modulating the tumor micro-environment with respect to the soluble mediators of
immune system; we quantied the cytokine levels in the peritoneal uid of DL-bearing mice. With the treatment, there was an
elevated level of Th1 cytokines namely IFN-c, TNF and IL-12 in
AE2 treated group as compared to the control group while IL2 levels in both treaded and non-treated group did not exhibit any signicant difference. With respect to Th2 cytokines, IL-4 and IL-10
levels decreased signicantly, where as IL-5 level remained comparable among the control and treatment groups (Fig. 6).

The important role played by polysaccharides in anti-cancer

therapy was rst recognized more than 50 years ago when it was
discovered that the remission of cancer could be effectively mediated by this class of compounds (Nauts et al., 1946). Mushroom
polysaccharides were realized to exert their antitumor action
predominantly via activation of the immune response of the host
organism (immuno-enhancing activity) or, alternatively, by antiproliferative effect towards tumor, changing the expression of signals within tumor cells. However, the underlying mechanism has
remained largely unclear. In this respect, while, till date, researchers have isolated several structural diversied polysaccharides
with biological activities (Moradali et al., 2007; Borchers et al.,
2008) large varieties of available mushrooms have remained
unexploited.
In the present investigation, we have endeavored to elucidate
the possible antitumor activity of a heteroglucan isolated from A.
hygrometricus having in vitro immunomodulatory properties (Mallick et al., 2009). Here, it is pertinent to mention that Astraeus
hygrometricus has been not explored much with respect to the biological properties. Previously, Chakraborty et al. (2004) has reported a polysaccharide possessing splenocyte stimulating
activity while we have reported an anti-proliferative protein (Maiti
et al., 2008) as well as a heteroglucan with immunomodulatory
property, designated as AE2. The heteroglucan, AE2 with high
molecular weight and putative b-glucan structure, has noted to
activate macrophages (Mallick et al., 2009) in vitro with no auxiliary toxic activity towards the accounted cells. The promising macrophage stimulatory property of AE2 has subsequently prompted
us to scrutinize any plausibility of polysaccharide mediated antitumor activity.
A general observation in the regression of tumor growth following therapeutic protocol is the prolonged survival of the tumor
bearing host (Bhutia et al., 2009; Ghosh and Maiti, 2007). In tune
with this, under the present study, the administration of AE2 markedly increased the median survival time of DL-bearing mice (Fig. 1)
from 23 days in case of control to 29 days in 20 mg/kg AE2 treated
group. Successively, to appreciate the mode of tumor regression,
we examined the occurrence of apoptosis in DL from mice treated
with AE2, with respect to morphological and DNA changes. The
morphological studies of the peritoneal DL smear illustrated prominent membrane blebs indicating the occurrence of higher apoptosis in DL from AE2 treated mice. For a further understanding of the
apoptosis, cell cycle was studied employing propidium iodide, a

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NK Cell activity
16

% Target Killed

14
12

10

PBS
10mg/kg
20mg/kg

8
6
4
2
0
80 : 01

40 :01

20:01

Effector to arget cell ratio

Fig. 5. Effect of AE2 administration on the NK cell activity. Non-adherent splenocytes were taken as effector cells from normal or DL-bearing mice treated with or with out
AE2. DiO labeled YAC1 cells were incubated in presence of effector cells at different ratios for 4 h and killing was observed by PI staining followed by ow cytometry. Data
reported as the mean S.D. and compared against PBS control by using a Students t-test (*signicant compared to PBS control).

Cytokines in tumor micro-environment

Conc. (pg/ml)

160
140

Control

120

10 mg/kg

100
80
60

20 mg/kg

*
*

40
20
0
IL-2

IL-4

TNF

IL-10

IL-12
p70

IL-5

Cytokines
Fig. 6. Effect of AE2 administration to DL-bearing mice on the cytokine level in peritoneal uid. Peritoneal uid from DL bearing mouse was collected after centrifugation and
used for estimation of IFN-c, IL-2, IL-4, IL-5, IL-10, IL12 p70 and TNF-a by using CBA method on a BD FACSCalibur. All data were repeated in triplicates and reported as the
mean S.D. of three different observations and compared against PBS control by using Students t-test and ve animals were taken per treatment group. P values < 0.05 were
considered signicant (*signicant compared to control).

ratio metric DNA intercalating dye. The results revealed a signicant hypodiploid fraction of cells in AE2 treated group, conrming
the tumor regression due to induction of apoptosis in DL cells.
Again, it has been reported previously by us (Sarangi et al., 2006)
and others (Wang et al., 1997; Wasser, 2002) that polysaccharides
mediate the tumor cell apoptosis by the activation of host immune
system to induce tumor immunity. Also, polysaccharides with antitumor properties have been demonstrated to exhibit the said effect
by the restoration of homeostasis (Leung et al., 2006) and reversal
if the immune suppression by inducing Th1 cytokine productions
by the immune cells (Harnack et al., 2009; Murata et al., 2002).
In order to verify whether the tumor regression is through the
reversal of the DL induced immune suppression, we studied the
ex vivo expansion of splenocytes, thymocytes and BMC with or
without a mitogenic agent like PHA. The splenocytes, thymocytes
and BMC from DL-bearing mice treated with AE2 showed higher
proliferation in culture as compared to the non-treated control
DL-bearing mice. Moreover, the cells instantiated higher mitogenic
response to PHA, essentially indicating an activated status of immune system of AE2 treated mice as compared to control ones.
Pertinently, it is believed that even if the overall immune status
of the host may be modulated by a BRM, the role played by the
molecule in modulating the cells present in tumor environment
is more crucial. The tumor micro-environment containing various
Th2 cytokines that induce tolerance and anergy to tumor, facilitate
the process of tumor escaping the immune surveillance system. In

this respect, macrophages at tumor sites, generally known as tumor-associated macrophages, are often associated with secretion
of suppressor cytokines like TGF and IL-10. Concomitantly, they exhibit reduced phagocytic potential as well as lower level of MHC-II
expression. Appreciating profound inuences of TAMs in growth
and priming of tumor environment, we attempted to probe the
characteristic behavior of tumor-associated macrophages with relevance to NO production, phagocytosis and cytokine production. In
subsequence, the TAMs obtained from DL-bearing mice treated
with AE2 were observed to elicit augmented level of both NO production and phagocytosis of latex beads ex vivo in response to LPS
or IFN. Similarly, enhanced surface expressions of MHCII and CD86
were observed in the AE2 treated groups. While mannose receptor
expression on TAM was studied, after 24 h ex vivo culture with or
without IFN-c, IFN-c was to found mediate the down regulation
of MR on TAMs obtained from AE 2 treated mice exclusively.
Preceding studies related to MMR expression on macrophage in
presence of cytokines have delineated an increased expression level with IL4 (Stein et al., 1992) and IL10 (Martinez-Pomares
et al., 2003) treatment while the expression has been shown to decrease with IFN (Harris et al., 1992). In this study, it was divulged
that IFN-c, though, failed to decimate the MR expression in TAMs
from control mice, it effectively down regulated MR level in TAMs
of the AE2 treated mice. In addition, cytokines in tumor microenvironment playing important role in the immunosuppession
and tumor progression (Mocellin et al., 2005; Kim et al., 2006)

S.K. Mallick et al. / Food and Chemical Toxicology 48 (2010) 21152121

we opted to study Th1 and Th2 cytokines from the tumor milieu.
An enhanced level of Th1 cytokines like IFN- c, IL-12 and lower
production of Th2 cytokines like IL-4 and IL-10 were observed in
the peritoneal uid. However there was no signicant change in
IL-2 and IL-5 levels in peritoneal uid. These observations, in correlation with the previous ndings involving polysaccharides as
anti tumor agents, indicate that the polysaccharide AE2 isolated
from Astraeus hygrometricus possibly possess indirect antitumor
properties via the activation of immune system of the host, which
results into the induction of tumor regression by enhanced apoptosis of the tumor cells. This is, therefore, a novel effort towards the
exploration of potent anti tumor and immunomodulatory properties of A. hygrometricus, and is expected to facilitate a deeper revelation the biologically active fraction of this largely unexplored
species. Further studies are required to decipher the mechanism
of action lying behind these observations.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgments
The authors would like to acknowledge Central research facility
IIT Kharagpur and Sanjaya K. Mallick acknowledges CSIR, New Delhi for providing the fellowship. This study was funded by Department of Science and Technology (DST), Govt. of India (Grant No.
Sr/So/HS-71/2006). The language editing of the manuscript by Tamal Das is also acknowledged.
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