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Anti-Tumor Activities of the Antlered Form of Ganoderma lucidum in Allogeneic and Syngeneic Tumor-Bearing Mice

Yuji N ONAKA,1; y Hiroshi S HIBATA,1 Masaaki N AKAI,1 Hiroshi K URIHARA,1; * Hiroko I SHIBASHI,2 Yoshinobu K ISO,1 Takaharu T ANAKA,1 Hideyo YAMAGUCHI,2 and Shigeru A BE2
Institute for Health Care Science, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan 2 Institute of Medical Mycology, Teikyo University, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan
Received September 21, 2005; Accepted May 22, 2006; Online Publication, September 7, 2006 [doi:10.1271/bbb.50509]
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We investigated the anti-tumor eects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF signicantly countered the depression of splenic CD8 cells and protected the decrease in interferon-gamma (IFN- ) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer. Key words: antlered form of Ganoderma lucidum; antitumor activity; syngeneic tumor; cytokine production; immunodepression

Ganoderma lucidum (Fr.) Karst (reishi in Japanese), an Oriental fungus, is a famous traditional Chinese medicine used in China, Japan, and other Asian countries.1) It has been prescribed for the prevention and treatment of various diseases, such as hypertension,2) hyperlipidemia,3,4) diabetes,5) hepatitis,6,7) allergy,8,9) and cancer.1012) Recently, active components, such as -D-glucans and triterpenoids, which exhibited anti-tumor eects, have been isolated from G. lucidum, and many experimental data in vitro and in vivo have been reported.1019) Anti-tumor activities of edible and medical mushy

rooms in vivo have been reported,20) but Sarcoma 180 tumor-bearing mice have been used in many studies. Sarcoma 180 tumor is an allogeneic tumor, and so it cannot be denied that the anti-tumor mechanism in this model depends on the allograft rejection of the host animal. Therefore, it is indispensable to use a syngeneic tumor to elucidate the anti-tumor activity and its immunological mechanism of active compounds. The antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese) is a variant type of G. lucidum rarely found in nature. Recently, a cultivation technology was developed, and G. lucidum AF is articially cultivated in China, Korea, and Japan. It has been reported that G. lucidum AF contained large amounts of -D-glucans,21) and that the triterpenoids in G. lucidum AF were much larger than those in the normal type of G. lucidum.17) It is expected that the antitumor activity of G. lucidum AF might be much stronger than that of the normal type of G. lucidum. Kohguchi et al.21) found that G. lucidum AF can activate macrophages and induce Th1-associatied immune activities in vivo, but no information has been reported as to anti-tumor activity and the immunological mechanism of G. lucidum AF in a syngeneic tumormice model. In the present study, we investigated the anti-tumor eects of G. lucidum AF by oral administration (p.o. and free-feeding) not only in allogeneic Sarcoma 180 tumor-bearing ddY mice, but also in syngeneic MM 46 mammary carcinoma-bearing C3H/He mice, and then elucidated the immuno-stimulating eect of G. lucidum AF in syngeneic MM 46 tumor-bearing mice using splenocytes and lymphocytes from regional lymph nodes.

To whom correspondence should be addressed. Fax: +81-75-962-1690; E-mail: Yuji Nonaka@suntory.co.jp Present address: Jinan University, Shinpai, Guangzhou, 510632, China Abbreviations: G. lucidum AF, antlered form of Ganoderma lucidum; p.o., post orally; i.p., intraperitoneally; s.c., subcutaneously; IFN- , interferon-gamma; IL-4, interleukin-4; FBS, fetal bovine serum; PBS, phosphate-buered saline; Con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; CD, cluster of dierentiation; BRM, biological response modiers
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Materials and Methods


Materials. We used G. lucidum AF in this study, grown commercially in China, Korea, and Japan. All G. lucidum AF samples (Chinese 4, Japanese 3, and Korean 1) were purchased from Shinwa Bussan (Osaka, Japan). They were crushed to a 200-mesh path equivalent. A G. lucidum AF-containing diet was prepared from AIN-93M chow (Oriental Yeast, Tokyo). Sarcoma 180 cells were kindly donated by Dr. M. Hayashi (Kitasato Institute for Life Sciences and School of Pharmaceutical Sciences, Kitasato University, Tokyo). MM 46 cells were from the collection of the Institute of Medical Mycology, Teikyo University. Animals. Male ddY and C3H/He mice (56 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and Charles River Japan (Yokohama, Japan) respectively. These animals were housed and put on a commercial diet (AIN-93M) and tap water ad libitum for 1 week before experimentation at 25 1  C and 60 5% humidity under a 12 h light-dark cycle. Experiments were performed according to the Guidelines for the Care and Use of Experimental Animals of the Japanese Association for Laboratory Animals. Anti-tumor tests. Allogeneic tumor model: Sarcoma 180 cells (1 106 cells) were inoculated intraperitoneally (i.p.) into ddY mice for passage culture. Tumor cells were collected from the peritoneal cavity 7 d after inoculation and then suspended in MEM medium (Nacalai Tesque, Kyoto, Japan). The cells were subcutaneously (s.c.) inoculated into the left chest of ddY mice (1 106 cells/0.1 ml/mouse). G. lucidum AF (0.005, 0.05, or 0.5 g/kg/d) was suspended in 0.6 ml of distilled water and post orally (p.o.) administered to mice for 19 consecutive d starting from the day before inoculation. Water was administered to the control mice. The tumor diameter was monitored and the tumor weight was determined 18 d after inoculation. An AIN93M diet with or without 2.5% G. lucidum AF was also administered to the mice by free feeding from the day before until 100 d after inoculation. The tumor diameter and the survival rate were monitored during the experiment. Syngeneic tumor model:22) MM 46 mammary carcinoma (1 106 cells) was inoculated i.p. into C3H/He mice. Tumor cells for the experiment were prepared similarly to Sarcoma 180 cells. The cells were inoculated s.c. into the inguinal region of C3H/He mice (2 106 cells/0.2 ml/mouse). The mice were equivalently divided into two groups according to tumor size 7 d after inoculation. Then each group of mice was administered an AIN-93M diet with or without 2.5% G. lucidum AF, and the survival rate was monitored until 100 d after inoculation. Half of the mice in each group were killed at 28 d after inoculation, and the tumor weight was determined.

The average diameter was calculated using a slide caliper as follows: Tumor diameter (mm) p the largest diameter the smallest diameter The anti-tumor test was performed at least twice, and representative results are shown. Preparation of splenocytes and lymphocytes. Splenocytes and lymphocytes were prepared from spleens and regional (inguinal) lymph nodes from MM 46 tumorbearing mice killed 28 d after tumor inoculation, as described above. After removing the spleen or the regional (inguinal) lymph node from a mouse, each single-cell suspension of splenocytes or lymphocytes was prepared by passing it through a 70 mm nylon mesh. The cells were washed with a hemolytic buer (155 mM NH4 Cl, 10 mM KHCO3 , 0.1 mM EDTA) and phosphatebuered saline (PBS) in that order. The cells were suspended in RPMI 1640 medium containing 10% FBS and a 1% antibiotic-antimycotic mix cocktail (Nacalai Tesque). Flow cytometric analysis. CD4 and CD8 cells were stained for 45 min using CY-CHROME conjugated anti-mouse CD4 monoclonal antibody (BD Pharmingen, San Diego, CA) and FITC-conjugated anti-mouse CD8 monoclonal antibody (Immunotech, Marseille, France) respectively. The cells were washed with PBS, and CD4 and CD8 expression in lymphocytes was analyzed using Epics XL (Beckman Coulter, Fullerton, CA). The analysis was based on 15,000 cells gated as lymphocytes, and calculated using EXPO32 software. Assay for cytokine production. Splenocytes and lymphocytes (5 106 cells/ml each) were stimulated with 2.5 mg/ml of concanavalin A (Con A) (Wako Pure Chemical Industries, Osaka, Japan). After incubation for 24 h at 37  C, the culture supernatant was collected and the levels of IFN- and IL-4 production were determined using an OptEIA kit (BD PharMingen). Statistical analysis. The statistically signicant differences for each value were tested by one-way ANOVA, followed by Fishers PLSD as a post-hoc test. Cumulative survival curves were constructed using KaplanMeier methods, and signicant dierences were tested by the log-rank test.

Results
Anti-tumor activity of G. lucidum AF in Sarcoma 180 tumor-bearing mice The anti-tumor activities of G. lucidum AF from eight dierent growing-districts (Chinese 4, Japanese 3, and Korean 1) were tested using Sarcoma 180 tumor-bearing mice. Each sample was orally administered to mice daily by gastric gavage at a dose of 0.5 g/kg/d from one

day before tumor inoculation. There was no correlation between the anti-tumor activity and the total -glucan content of G. lucidum AF in this experiment, although the total -D-glucan contents were more than 40% in all of the eight dierent district-derived G. lucidum AF samples (data not shown). A kind of Chinese G. lucidum AF (from Sang Tong) exhibited the highest activity, inhibiting tumor growth by 48% 18 d after inoculation. Hence we used this Chinese G. lucidum AF in all the experiments described below. As shown in Fig. 1, G. lucidum AF signicantly inhibited the growth of Sarcoma 180 tumor at a dose of 0.05 or 0.5 g/kg/d when administered daily by gastric gavage. An ordinary type of commercially available G. lucidum did not show anti-tumor activity at either 0.05 g/kg/d (data not shown) or 0.5 g/kg/d (Fig. 1A).

The tumor weights of mice administered 0.05 and 0.5 g/ kg/d of G. lucidum AF were signicantly smaller on day 18 (Fig. 1B). In the preliminary experiments, the anti-tumor activity at 1 g/kg/d G. lucidum AF was weaker than that at 0.5 g/kg/d. No adverse eects of G. lucidum AF was observed up to 1 g/kg/d (data not shown). Anti-tumor and life span-elongation eects of G. lucidum AF in Sarcoma 180 tumor-bearing mice We examined the anti-tumor eect of G. lucidum AF by free feeding in Sarcoma 180 tumor-bearing mice, since oral administration by gastric gavage can induce experimental stress in mice. A control diet (AIN-93M) or the G. lucidum AF diet (0.156, 0.625, or 2.5%) was given. There was no dierence between the groups in food intake. A mouse took about 75 mg/d (2.5 g/kg/d) in the 2.5% G. lucidum AF-fed group. In the 2.5% G. lucidum AF-fed group, tumor growth was signicantly inhibited from 15 d after tumor inoculation. The lower amounts of G. lucidum AF groups also showed a repression tendency against tumor growth as compared with the control group (Fig. 2A). In the G. lucidum AFfed groups, body weights were slightly lighter than in the control group, but were not signicantly dierent. No adverse eect of G. lucidum AF was observed up to 10% (data not shown). Survivals of Sarcoma 180 tumor-bearing mice, with or without a 2.5% G. lucidum AF-containing diet, were monitored up to 100 d after tumor inoculation. There was no dierence between the groups in food intake, but food intake reduction was observed starting about 30 d after tumor inoculation. The life span of mice fed the G. lucidum AF diet was signicantly prolonged as compared with the control group. The survival periods giving 50% lethality were 54 and 64 d for the control and G. lucidum AF-fed mice respectively (Fig. 2B). Anti-tumor and life span-elongation eects of G. lucidum AF in MM 46 tumor-bearing mice G. lucidum AF also exhibited anti-tumor activity against a syngeneic MM 46 tumor in C3H/He mice in spite of post-feeding from 7 d after tumor inoculation. The control diet (AIN-93M) or G. lucidum AF diet (0.156, 0.625, 2.5%) was given as described in Materials and Methods, and there was no dierence between the groups in food intake. As shown in Fig. 3A, G. lucidum AF signicantly reduced tumor weight 28 d after inoculation when orally administered at a dose of 2.5% G. lucidum AF-containing diet. Survivals of MM 46 tumor-bearing mice, with or without the 2.5% G. lucidum AF diet, were monitored up to 100 d after tumor inoculation. The life span of the mice fed G. lucidum AF diet was signicantly prolonged as compared with the control group. The survival periods giving 50% lethality were 49 and 62 d for the control and the G. lucidum AF-fed mice respectively (Fig. 3B). They were not signicantly dierent in body

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A
Tumor diameter (mm) 10 ** ** ** 5 ** 0 0 5 10 15 D after inoculation 20 **
Control G. lucidum AF (0.5 g/kg)

1.5 Tumor weight (g)

1.0

**
0.5

**

0 Control 0.005 0.05 0.5 G. Lucidum AF (g/kg)


Fig. 1. Anti-Tumor Eects of G. lucidum AF in Sarcoma 180 Tumor-Bearing Mice. Sarcoma 180 (1 106 cells) was inoculated s.c. into ddY mice. G. lucidum AF was p.o. administered to mice for 19 consecutive d starting from 1 d before inoculation. The tumor diameter (A) was monitored, and tumor weight (B) was determined 18 d after inoculation. Determination of the tumor diameter is described in Materials and Methods. Data are the means S.E. (n 13{15). Asterisks indicate signicant dierences from the control group ( , p < 0:05; , p < 0:01; one-way ANOVA followed by Fishers PLSD).

25 Tumor diameter (mm) 20 15 10 5

8 Tumor weight (g)


Control G. lucidum AF 0.156 % 0.625 % 2.5 %

A *

6 4 2 0

* *

0 0 5 10 15 20 25 D after inoculation

Control 0.156 0.625

2.5

G. Lucidum AF (%)

G. lucidum AF

Survival rate (%)

100 Survival rate (%) 80 60 40 20 0 0

100 80 60 40 20 0 0

B
G. lucidum AF p < 0.05

p < 0.05

Control

Control

20 40 60 80 100 D after inoculation

20 40 60 80 D after inoculation

100

Fig. 2. Anti-Tumor and Life Span-Elongation Eects of G. lucidum AF in Sarcoma 180 Tumor-Bearing Mice. Sarcoma 180 (1 106 cells) was inoculated s.c. into ddY mice. The mice were fed an AIN-93M diet or G. lucidum AF (0.156, 0.625, and 2.5%) from 1 d before inoculation. A, The tumor diameter was determined as described in Materials and Methods. The data are the means S.E. (n 10). Asterisks indicate signicant dierences from the control group ( , p < 0:05; one-way ANOVA followed by Fishers PLSD). B, Survival rate was monitored (n 29{30). Cumulative survival curves were constructed using the KaplanMeier methods, and signicant dierences were tested with the log-rank test.

weight, although food intake reduction was observed starting about 42 d after tumor inoculation. Immunological function of G. lucidum AF in MM 46 tumor-bearing mice We checked the activity of immunocompetent cells isolated from MM 46 tumor-bearing mice fed the G. lucidum AF diet. Splenocytes and lymphocytes were prepared from spleens and regional (inguinal) lymph nodes from MM 46 tumor-bearing mice killed 28 d after tumor inoculation, as described in Fig. 3A. Splenocytes and lymphocytes from the normal mice were similarly prepared, as a control. The populations of CD4 and CD8 cells in both splenocytes were decreased by MM 46 inoculation. G. lucidum AF signicantly recovered the reduction of CD8 cells in splenocytes. No changes in cell populations were detected in any of the groups of regional lymph nodes (Fig. 4).

Fig. 3. Anti-Tumor and Life Span-Elongation Eects of G. lucidum AF in MM 46 Tumor-Bearing Mice. MM 46 mammary carcinoma was inoculated s.c. into the inguinal region of C3H/He mice (2 106 cells/0.2 ml/mouse). The mice were divided equivalently into two groups according to the tumor size 7 d after inoculation. Then each group of mice was administered an AIN-93M diet or G. lucidum AF (0.156, 0.625, 2.5%). A, Ten arbitrarily-chosen mice in each group were killed 28 d after inoculation, and the tumor weight was determined. Determination of the survival rate is described in Materials and Methods. The data are the means S.E. Asterisks indicate signicant dierences from the control group ( , p < 0:05; one-way ANOVA followed by Fishers PLSD). B, The survival rate was monitored (n 29{30). Cumulative survival curves were constructed using KaplanMeier methods, and signicant dierences were tested with the log-rank test.

IFN- and IL-4 production by Con A-stimulated splenocytes or lymphocytes were also measured. G. lucidum AF signicantly protected the decrease in IFN- production in splenocytes and lymphocytes in regional lymph nodes, respectively. The IL-4 level in regional lymph nodes was also recovered by feeding the G. lucidum AF-containing diet (Fig. 5).

Discussion
We demonstrated, for the rst time, the anti-tumor activities of G. lucidum AF, a variant type of G. lucidum, by oral administration not only in allogeneic Sarcoma 180 tumor-bearing mice, but also in syngeneic MM 46 mammary carcinoma tumor-bearing mice (Figs. 1, 2 and 3). G. lucidum AF inhibited the increase

Splenocytes 30
CD4+ cells (%)

Lymphocytes 80 60
IFN- (ng/ml)

Splenocytes 40 30 20 10 0 40
IL-4 (pg/ml)

Lymphocytes 1.0 0.8 0.6 0.4 0.2 0 4 3 2

20 10 0 6 ** **

40 20 0 20 15 10 5

** **

CD8+ cells (%)

4 2 0 **

**

30 20 10 0 ** **

1 0

G. lucidum AF

G. lucidum AF

MM 46 tumor

MM 46 tumor

MM 46 tumor

MM 46 tumor

Fig. 4. Changes in CD4 and CD8 Cell Populations in Spleens and Regional Lymph Nodes from MM 46-Tumor Bearing Mice. Splenocytes and lymphocytes were prepared as described in Materials and Methods. CD8 and CD4 cells were analyzed using Epics XL. Open bars indicate normal mice (without tumor inoculation), hatched bars indicate tumor bearing mice fed a control diet, and closed bars indicate tumor bearing mice fed the G. lucidum AF-containing diet. The data are the means S.E. (n 10). , p < 0:01 vs. open bar; y, p < 0:05 vs. hatched bar; one-way ANOVA followed by Fishers PLSD.

Fig. 5. Cytokine Production from Spleens and Regional Lymph Nodes from MM 46-Tumor Bearing Mice. Splenocytes and lymphocytes were prepared as described in Materials and Methods. The cells were stimulated with 2.5 mg/ml of concanavalin A for 24 h. The levels of IFN- and IL-4 in the culture supernatant were measured using an OptEIA kit. Open bars indicate normal mice (without tumor inoculation), hatched bars indicate tumor bearing mice fed a control diet, and closed bars indicate tumor bearing mice fed the G. lucidum AF-containing diet. , p < 0:05 vs. open bar; , p < 0:01 vs. open bar; y, p < 0:05 vs. hatched bar; one-way ANOVA followed by Fishers PLSD.

in tumor weight and elongated the life span of the tumor-bearing mice when orally administered by free feeding (Figs. 2, 3). Furthermore, we found that G. lucidum AF protected the tumor-bearing mice from immunodepression due to tumor growth (Fig. 4, 5). It has been expected that G. lucidum AF has strong anti-tumor activity because of the high content of -Dglucan in its fruiting body. -D-glucan, a major component of G. lucidum AF, exhibited immuno-stimulating activities in vivo and in vitro.1016) Kohguchi et al. found that the -D-glucan content in G. lucidum AF was 40.1%, and that total percentage of -D-(1,3)glucan in the -D-glucan was 85.3%.21) They also indicated that the oral administration of G. lucidum AF resulted in Th1-associated immuno-potentiating activities in BALB/c mice, but there was no correlation between the anti-tumor activity and -D-glucan content of G. lucidum AF in this study, although all the total D-glucan contents were more than 40%. We should take notice that the anti-tumor activity of G. lucidum AF could not be predicted by the total -D-glucan content. We used Chinese G. lucidum AF in all experiments, which exhibited the highest activity of eight samples. Recently, various structures and bioactivities of -Dglucans derived from G. lucidum have been reported, e.g., protein-bound -glucan,23) -D-(1!3)-glucan,12,24) -D-(1!6)-glucan with -(1!3) or -(1!4) branches,15) and -D-(1!3)-glucan with -(1!6) branches.13,16) The cytotoxic actions of triterpenes, such as ganoderic acid, lucidenic acid, and ganoderiol, in G. lucidum AF have been also studied vigorously.18,19)

Further study should be done to determine the active component responsible for the anti-tumor activity with oral administration of G. lucidum AF. We examined the anti-tumor activity of G. lucidum by oral administration with diet, in order to avoid physical and mental stress by gastric gavage. G. lucidum AF showed anti-tumor activity in Sarcoma 180 tumorbearing mice by free feeding from 1 d before tumor inoculation. G. lucidum AF also exhibited anti-tumor activity against a syngeneic MM 46 tumor in C3H/He mice and elongated the life span of tumor-bearing mice, in spite of post-feeding from 7 d after tumor inoculation. These results suggest that ingestion of G. lucidum AF is useful not only for prevention, but also for curing cancer. The mechanism of the anti-tumor eect of G. lucidum AF remains to be claried. At this point, we consider as follows: The immunological mechanism may be related to inhibition of MM 46-tumor growth, because MM 46 carcinoma in C3H/He mice is moderately antigenic,25) and its growth can be inhibited without cytotoxicity by immuno-stimulating agents, including mushroom D-glucans such as lentinan22,26) and PSK.2729) In this study, G. lucidum AF signicantly countered the depression of splenic CD8 cells and protected against the decrease in IFN- and IL-4 production in regional lymph nodes in MM 46 tumor-bearing mice. In addition, in a recent study, in vivo depletion of IFN- abrogated the anti-tumor vaccination eect,30) indicating that IFN might play an important role in immunomodulating

anti-tumor action. These ndings suggest that one of the anti-tumor mechanisms of G. lucidum AF might be due to its IFN- -inducible activity and CTL activation under the cancer condition. In summary, we conrmed the anti-tumor activity of G. lucidum AF not only against allogeneic tumors, but also against syngeneic tumors. We also suggest that the anti-tumor eects of G. lucidum AF might be explained by the relieving activity from tumor-induced immunodepression. BRMs derived from mushrooms are generally treated with chemotherapeutic agents to counteract their side eects, such as myelosuppression or leukocytopenia.31,32) G. lucidum AF can clinically alleviate chemotherapeutic agent-induced immunodepression. Currently, the anti-tumor activities of concomitant administration of G. lucidum AF and chemotherapeutic agents are under investigation in our laboratory. Finally, the present study suggests that G. lucidum AF might be useful for the prevention of relapse and as a co-adjuvant in the medical treatment of cancer.

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