Effect of Agaricus blazei Murrill Extract on HT-29 Human Colon Cancer Cells in SCID Mice In Vivo

Abstract

. Agaricus blazei Murrill (ABM) popularly known

as ‘Cogumelo do Sol’ in Brazil, or ‘Himematsutake’ in

Japan, is a mushroom native to Brazil and widely cultivated

in Japan for its medicinal uses and is now considered one of

the most important edible and culinary-medicinal

biotechnological species. This study is the first tumor growth

model to evaluate the amelioratory effect of ABM extract

using HT-29 human colon cancer cells in severe combined

immunodeficiency (SCID) mice. Forty SCID mice were

inoculated with HT-29 cells to induce tumor formation and

were then divided into four groups. All the four groups

(control, low, medium and high concentration treatment) of

mice were separately orally administered 0 mg, 1.125 mg,

4.5 mg or 45 mg ABM extract daily. After six weeks of

treatment, 8 out of the 40 mice had not survived including

one mouse which scored +++ (tumor up to 15 mm diameter)

and four mice which scored ++++ (tumor over 15 mm

diameter) in the control group and three mice which scored

++++ on the low-dose ABM treatment. After high- or

medium-dose treatment, all ten mice in each group survived.

The oral administration of ABM does not prevent tumor

growth, as shown by increased tumor mass, but compared

with the control group, the tumor mass seems to grow more

slowly depending on the ABM dose.

For males and females in Taiwan, the colon/rectum is the third

leading primary cancer site because of the poor prognostic

outcome of colorectal cancer due to its resistance to current

therapies, it is the leading cause of cancer-related death.

A number of phytochemicals present in medicinal plants

are known to possess substantial anticarcinogenic and

anti-mutagenic activities (1-8). Successful treatment with

chemotherapeutic agents is largely dependent on their ability

to trigger cell death in tumor cells; therefore, novel inducers

of apoptosis provide a new anticancer therapeutic approach,

and certain phytochemicals present in medicinal herbs have

demonstrated apoptosis induction in cancer cells (9-10).

Mushrooms and primarily basidiomycetous fungi are a

popular and valuable food, low in calories and high in

minerals, essential amino acids, vitamins and fiber (11);

some ‘medicinal’ mushrooms produce substances having

potential medical effects. Among the mushroom species of

Basidiomicetes, Agaricus blazei Murrill (ABM), a species

native to Brazil, where it is generally known as ‘sun

Correspondence to: Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 404, Taiwan, R.O.C. Tel: +886 422053366 ext. 2161, Fax: +886 422053764. e-mail: [email protected] and Hsu-Feng Lu, Department of Clinical Pathology, Cheng-Hsin General Hospital, No.45, Cheng Hsin St., Taipei 112, Taiwan, R.O.C. Tel: +886 228264400 ext 5850, Fax: +886 228264517, e-mail: [email protected]

Key Words: Agaricus blazei Murrill, antitumor effects, HT-29 colon cancer cells, SCID mice.

Effect of Agaricus blazei Murrill Extract on HT-29

Human Colon Cancer Cells in SCID Mice In Vivo

MING-FANG WU

, YUNG-LIANG CHEN

, MEI-HUI LEE

, YUNG-LUEN SHIH

,

YU-MING HSU

, MING-CHU TANG

, HSU-FENG LU

, NOU-YING TANG

,

SU-TSO YANG

, FU-SHIN CHUEH

and JING-GUNG CHUNG

1

_{Animal Medicine Center, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC;}

_{Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan, ROC;}

3

_{Department of Genetic Counseling Center, Changhua Christian Hospital, Changhua Taiwan, ROC;}

_{Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei Taiwan, ROC;}

5

_{Department of Clinical Pathology, Cheng-Hsin General Hospital, Taipei Taiwan, ROC;}

_{Department of Restaurant, Hotel and Institutional Management, Fu-Jen Catholic University, New Taipei;}

7

_{Department of Radiology, China Medical University Hospital, Taichung Taiwan, ROC;}

_{School of Chinese Medicine,}

_{Department of Biological Science and Technology,}

China Medical University, Taichung Taiwan, ROC;

Departments of

Health and Nutrition and Biotechnology and

Biotechnology,

Asia University, Taichung, Taiwan, R.O.C.

mushroom’, is often consumed as food and tea in different

parts of the world recently (12-13). It has traditionally been

used for the prevention of a range of diseases, including

cancer, hepatitis, atherosclerosis, hypercholesterolemia,

diabetes and dermatitis (14-19).

ABM antitumor activity has been shown in some reports

(20-22), but there are few reports regarding HT-29 human

colon cancer in severe combined immunodeficiency (SCID)

mice. The purpose of this research was to examine whether

ABM extract was effective against HT-29 tumor-bearing

SCID mice and to determine whether the treatment effect

was dependent on the concentration of ABM extract.

Materials and Methods

Experimental animals and housing conditions. SCID male mice, specific pathogen-free and 5 weeks old, were obtained from the Animal Medicine Center, College of Medicine, National Taiwan University (our own breeding colony). The animals were kept in polypropylene cages (five animals/cage) covered with metallic grids in a room maintained under constant environmental conditions, with air filter tops in a filtered laminar air flow-controlled room, with an ambient temperature of 20±2˚C, relative humidity 75±15%, and with a 12-h light-dark cycle. The mice were raised and cared for on laboratory pellet chow, given autoclaved water and fed ad libitum following the animal procedures approved up by the National Science Council of the Republic of China. The experiments were performed according to the law, regulations and guidelines for animal experiments in Taiwan, which are in agreement with the Helsinki declaration.

ABM extract and administration. Powdered ABM was obtained from S. Canaan Biotechnology Development Co. (Taipei, Taiwan, ROC) and 22.5 and 90 or 900 mg were separately suspended in 6 mL distilled water at 60˚C for 10 min, then cooled to room temperature and left for 5 h with stirring at 200 rpm to form the low, medium and high concentration aqueous extracts.

Colon cancer formation and experimental treatment. Forty mice in this study were inoculated with HT-29 human colon cancer cells in the dorsal area (2×107_{cells/mouse). Around 2-5 weeks after inoculation,}

the mice with carcinomas of 3 mm in diameter as measured by using caliper were divided into 4 groups of 10 mice each. The mice were fed with regular diet and double-distilled water. All the four groups (control, low, medium and high concentration treatments) of mice were separately orally administered 0 mg, 1.125 mg, 4.5 mg or 45 mg ABM extract daily. After 6 weeks of treatment, all the survivors were sacrificed under anesthesia by CO₂. The colon tumor size was scored under gross examination. The tumor status was categorized into: +, tumor diameter up to 5 mm; ++ and +++, tumor diameter up to 10 mm and 15 mm respectively, and over 15 mm diameter scored ++++.

Results

Effect of ABM extract on colon cancer formation. HT-29

cells inoculation induced 3 mm diameter tumors after around

2-5 weeks in all forty mice. After 6 weeks’ treatment, 8 out

of the 40 mice had not survived, including one mouse scored

+++ and four mice scored ++++ in the control group and

three mice scored ++++ in the low-dose ABM treatment.

After the high- or medium-dose treatments, all ten mice in

each group survived.

By naked eye, the 40 mice had obvious tumor masses in

the colon without full dissection (Figure 1). All ten mice of

the control group were scored +++/++++, showing that the

size of the tumor mass increased sharply to around 10 mm

to over 15 mm during the 6 weeks of incubation. After

low-dose ABM treatment, five mice still scored ++++, showing

that low-dose treatment was not very effective. After the

high- or medium-dose treatments, no mouse was scored

++++ (Table I). The oral administration of ABM did not

prevent tumor growth since the tumor masses increased, but

compared with the control group, the tumor mass seemed to

grow more slowly with ABM treatment. Tumor growth

inhibition was dependent on the ABM dose.

Discussion

Whole-mushroom extracts contain compounds including an

α-1,6- and α-1,4-glucan complex and α glucomannan with

a main chain of β-1,2-linked D-mannopyranosyl residues

that may modulate tumorigenesis and carcinogenesis at

different stages and/or may act at the same stage through

different mechanisms (23, 24). A combination of distinct

downstream responses involving different cell subsets could

conceivably provide greater tumor inhibition than could be

induced by a single polysaccharide. However, the wide

number of different and only partially homogeneous ABM

extracts used represents a difficult challenge for establishing

the best extract and active substances. Moreover additive, or

even synergistic, effects may occur while increasing

fractionations of one ABM extract enhanced some biological

activities, but abolished others (25).

Whilst to have anticlastogenic properties with a 100%

reduction of chromatid and 144.4% reduction of

isochromatid breaks, is apparently really important for

cancer prevention in humans since it is usually consumed in

its natural form as tea or as food (26). A aqueous extracts of

ABM have variously demonstrated no clastogenic activity.

While an n-butanolic extract was both anticlastogenic and

clastogenic (26) and at different concentrations, hexane

extracts were genotoxic, cytotoxic and anticlastogenic,

suggesting further studies are needed (27). A strong

protective effect (28-30), activation, of apoptosis (31) and no

protective effect have been shown in some cell types (32-34).

Additionally, differences in the cultivation, storage and

extract preparation might influence the effectiveness of

aqueous extracts of ABM (34).

Thus lineages and pre-treatment influence the

pharmacological anticancer activity of ABM extracts and as

confirmed by Manzi and Pizzoferrato (35), β-glucans,

apparently the most important constituent, are distributed

variably in mushrooms, both in the soluble and in the

insoluble fraction. Luiz et al. (33) also suggested that fatty

acids (especially linoleic and eicosapentanoic acid) in

ethanol and chloroform/ methanol extracts could have a role

in the antimutagenic activity.

In the present study, while ABM treatment did not stop the

growth of HT-29 tumor masses, it did inhibit rapid growth.

Aqueous ABM extracts given in the drinking water have

demonstrated protection at the initiation step of liver

carcinogenesis in rats and mice, but were ineffective when

administered in the post-induction period (32, 36-37). Our

next study may investigate ABM treatment prior to cancer

induction. ABM fed in dry powdered form to Wistar rats

exhibited significant chemopreventive influence on the

promoting phase of chemical hepatocarcinogenesis (38).

Among nearly 40 articles directly associated with ABM in

animal studies, this study was the first to evaluate the effect

of ABM extract on HT-29 cell colon cancer growth in SCID

mice, and this tumor growth model performed well because

colon cancer was successfully induced in all forty mice.

Although the results point to effective treatment by ABM,

one should be very cautious about applying these data to

human clinical studies, because this study was performed on

a specific strain of mice using a HT-29 colon cancer cell line.

Which substance in ABM inhibits the cells needs to be

established. Moreover, a more detailed study of its specificity

to organs or any other cell inoculation may be valuable in

terms of clarifying its mode of action, and its antitumor

effectiveness should be studied in different tumor inoculation

Table I. Tumor size score at death or after 6-week treatment of SCID mice with or without ABM. Tumor status categorized as: +: up to 5 mm; ++: up to 10 mm, +++: up to 15 mm; ++++: over 15 mm. Low: 1.125 mg; medium: 4.5 mg; high: 45 mg ABM orally daily.

Treatment + ++ +++ ++++

Survived Died Survived Died Survived Died Survived Died

Control 5 1 4

Low 1 4 2 3

Medium 1 4 5

models. Careful clinical studies comparing the activity of

isolated compounds, whole-mushroom extracts and

epidemiological data are still necessary to determine whether

ABM can provide real clinical benefits. Dose–response

studies and isolation, as well as chemical identification and

quantification of specific compounds responsible for the

potential benefit from ABM should be fully developed.

In conclusion, ABM extracted by hot water led to tumor

growth rate decline in the tumor growth model, in vivo. The

treatment effect and survivor rate are dependent on the ABM

dose used.

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Received March 11, 2011

Revised April 19, 2011

Accepted April 21, 2011