Effects of D-Fraction, a Polysaccharide from Grifola frondosa on Tumor Growth Involve Activation of NK Cells

Noriko Kodama,*a Kiyoshi Komuta,* Norio Sakai,c and Hiroaki NANBAa

a
Department of Microbial Chemistry, Kobe Pharmaceutical University; 4-19-1 Motoyama-kitamachi, Higashinada-ku,


Kobe 658-8558, Japan: b International Department, Osaka Police Hospital; 10-31 Kitayamacho, Tennoji, Osaka 543-0035, Japan: and c
Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University; 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8551, Japan.

Received July 12, 2002; accepted August 16, 2002


Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell
cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic
activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we
examined tumor volume and levels of IFN-

y and TNF-a

in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases
in TNF-

a and IFN-y released from spleen cells and a significant increase in TNF-a

expressed in NK cells. This sug­gests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction in­creased
macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early
effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from
macrophages.

Key words IL-12 production; macrophage; NK cell

tients for long periods of time, exceeding one year (Table 1). To investigate maintenance of NK cell activation by D-Frac­tion, we examined NK cell
activation in MM-46 carcinoma- bearing mice treated with D-Fraction for 19 d. Our data in this paper may support a critical role for NK cell activation in
immunotherapy with D-Fraction for cancer patients.

MATERIALS AND methods

Materials
For the detection of human NK activity, chromium-51 (Daiichi Kagaku Yakuhin Co., Tokyo) and lymphopacel (d= 1.077) (IBL Co., U.S.A.) were prepared.

Animals
Male C3H/HeN mice (4-weeks-old) were ob­tained (Japan Crea Co., Osaka) and were raised for one week before being used for experiments. Food and water were
given freely to these mice until used for experiments.

Cells
MM-46 carcinoma cells were kindly donated by Dr. Kanki Komiyama. 51Cr labeled K-562 cells were used as target cells of human NK.

Preparation of D-Fraction
D-Fraction was prepared from the dried powder of the fruit body of maitake mush­rooms (Grifola frondosa) (Yukiguni Maitake Co., Niigata),
according to the method described in our previous paper.6) The level of LPS contained in D-Fraction was determined by using Endospecy ES-20S Set
(Seikagaku Industry Co., Tokyo), and the ratio (%) of LPS in D-Fraction was less than

We previously reported that the (1®3)-branched (1®6)-^- glucan termed D-Fraction, extracted from the fruit body of the maitake mushroom

(Grifola frondosa)

(Fig. 1), is a bio­logical response modifier (BRM) like lentinan, which is found in

Letinus

edodes.

1—3)

These polysaccharides enhance the activity of immunocompetent cells such as macrophages, helper T cells, and cytotoxic T cells, which attack tumor cells.
These effects of BRMs led us to hypothesize that oral administration of D-Fraction would be an effective treatment for cancer patients. Orally administered
D-Fraction was shown to reduce tumor size in mice, without causing un­wanted side effects.

2

-

1

Therefore, D-Fraction has been com­mercialized as a health material, the safety of which has been confirmed by the Consumer Product Testing Co. (New
Jer­sey, U.S.A.) We have already tried a non-randomized clinical trial of D-Fraction therapy for patients with lung and breast cancer in stages II—IV

4)

In this trial, we found that IL-2 pro­duction was enhanced in D-Fraction-treated patients, sug­gesting that macrophages and T cells were activated by D-
Fraction. In addition, D-Fraction is revealed to be effective for human immunodeficiency virus (HIV) infection

5)

and lis­teriosis,

6

-

1

by reinforcing the immune system. These results indicate that one network of the immuno-response pathway by which D-Fraction enhances anti-tumor effects is
depen­dent on an adaptive immunity associated with cytotoxic T cell activation.

Fig. 1. Chemical structure of D-Fraction

© 2002 Pharmaceutical Society of Japan

* To whom correspondence should be addressed. e-mail: n-kodama@kobepharma-u.ac.jp


Natural killer (NK) cells rapidly recognize and lyse a large variety of tumor or virus-infected cells, without the need for either prior sensitization or
MHC-dependent recognition, which is different from cytotoxic T cell.

7)

Pham-Nguyen

et al.

have reported that the early NK cell response alone is not sufficient for tumor clearance, and that both T cells and NK cells are required in the
development of the long-term sur­vival in the hepatic tumor model by IL-12-mediated gene therapy.

8)

In our clinical study, we also found that D-Fraction activated NK cells in lung, breast and liver carcinoma pa­

0.000007%. In the experiment using macrophage cell line RAW 264.7 cells, D-Fraction was pretreated with polymixin B (30 mg/ml) for 2h.

Administration of D-Fraction to Cancer Patients
Dr. Kiyoshi Komuta of the Osaka Police Hospital gave informed consent to 8 cancer patients. Eight stage II—IV cancer pa­tients between 43- and 74-years-old
were orally administered 100 mg of D-Fraction on consecutive days for 34 months and their NK activity was examined.

Administration of D-Fraction to MM46 Carcinoma- Bearing C3H/HeN Mice
MM-46 carcinoma cells (2X106) were implanted in the right axillary region of 5-week-old male C3H/HeN mice. After 24 h, D-Fraction (5 mg/kg/d)
was administered to MM-46 carcinoma-bearing mice intraperi- toneally (i.p.) for 19 consecutive days. Tumor inhibition ratio (T.I.R.) was calculated as
follows: [1-(weight of tumor mass from mice treated with D-Fraction)/(weight of tumor mass from mice treated with PBS)]X 100.

Detection of Human NK Cell Activity
NK cells (1 X 106) obtained from blood using the Conray-Ficoll method9) were mixed with 2X107 of 51Cr-labelled
K-562 (target cell of NK cell) cells in a tube. After incubation for 1h, release of 51Cr into the culture supernatant was detected using a g-
counter. NK activity was calculated as: NK activity (%) = [(experimental release-spontaneous release) cpm/(max-re- lease-spontaneous release) cpm (by 1n
HCl treatment)] X 100.

RESULTS AND DISCUSSION


Effects of D-Fraction on MM46-Carcinoma Cell Growth

On the 20th day of D-Fraction administration to MM-46 tumor-bearing mice, D-Fraction significantly decreased tumor growth as compared with mice
administered phosphate bufferent saline (PBS), when the T.I.R. was 82% (Fig. 2). The effect of D-Fraction was also investigated in C3H/HeJ mice that are
non-response to LPS; the T.I.R. was 78%. These results indicated that D-Fraction inhibited the growth of tumor.

Effects of D-Fraction on NK Cell Activation
D-Frac- tion has already been reported to enhance cellular immunity by stimulating cells such as macrophages, helper T cells and cytotoxic T cells. 2) To detect NK cell activation, we exam­ined IFN-g and TNF-a release from whole spleen cells on the 20th day of D-Fraction administration by
mouse IFN-g and TNF-a ELISA kits (Genzyme Co., Minneapolis, U.S.A.). As shown in Figs. 3A and 3B, levels of IFN-g and TNF-a were significantly increased by
1.5- and 2.5-fold, re­spectively, compared to control mice. Intracellular expression of TNF-a in splenic NK cells under D-Fraction administra­tion was also
investigated using flow cytometric analysis. To detect NK cells, 2X106 cells were stained with R-PE-conju- gated PanNK and Cy-Chrom™-conjugated
CD3e antibodies (PharMingen Co., San Diego, CA, U.S.A.) and CD3e nega­tive and PanNK positive was determined to represent the NK cell. Fluorescein
isothiocyanate (FITC)-conjugated TNF-a antibody (PharMingen Co.) was used for detecting TNF-a expression in NK cells. After staining, NK cells expressing
TNF-a were analyzed using a FACSCalibur analyzer (Beck- ton Dickinson Co., Grenoble, France) and histograms were calculated with CellQuest software (Becton
Dickinson,

Fig. 2. Effect of D-Fraction on MM-46 Carcinoma Cell Growth


D-Fraction (5 mg/kg/d) was administered to MM-46 carcinoma-bearing mice i.p. for 19 consecutive days, and then tumor volume (mm3=longest
diameterXshortest diame- ter2/2) was measured on day 20.16) Data are expressed as mean±S.E.M. of 8 experi­ments. **,
/><0.01; ***, _p<0.001 compared to control mice (Student’s Mest).






Figs. 3A and 3B. Effects of D-Fraction on Release of IFN-g or TNF-a from Whole Spleen Cells in MM-46 Carcinoma-Bearing Mice

On day 20 in mice administered D-Fraction (5 mg/kg/d) or PBS (—) i.p., splenocytes were collected according to previously described methods. 6) Splenocytes were plated at 1X106 cells/well in a 96-well plate and cultured with Con A (10 mg/ml) for 24h in 5% CO

2

at 37 °C. After activation, levels of INF-g and TNF-a in culture supernatant were assessed using ELISA. Data are expressed as mean±S.E.M. of 4—8
experiments. **, /><0.01; ***, _p<0.001 compared to control mice (Student’s Mest).




Fig. 3C. Effect of D-Fraction on Intracellular TNF-a Expression in NK Cells of MM-46 Carcinoma-Bearing Mice


On day 20 in mice administered D-Fraction (5mg/kg/d) or PBS (—), intracellular TNF-a expression in splenic NK cells was investigated using flow
cytometric analysis. Shown are representative histograms of triple-color flow cytometric analysis of spleno­cytes stained with R-PE-conjugated PanNK,
Cy-ChromTM-conjugated CD3£, and FITC-conjugated TNF

-a
antibodies.

Mountain View, CA, U.S.A.). As shown in Fig. 3C, D-Frac- tion administration resulted in a 1.3-fold increase in intracel­lular TNF-aexpression in
splenic NK cells compared to con­trol mice. TNF-a is a cytokine released from activated NK cells in the same way as IFN-g,10,11) and
has various func­tions relating to inflammatory and cytotoxic reactions, in ad­dition to cytotoxicity and the ability to directly cause hemorrhagic tumor necrosis.

Fig. 4. IL-12 Release from RAW 264.7 Cells Stimulated with D-Fraction RAW 264.7 cells (1X
106 cells/well in a 24-well plate) were stimulated for 18h with D-Fraction. To remove any possible LPS contamination, D-Fraction was treated
with polymyxin B (30 mg/ml) for 2h before use. At the indicated time of incubation, IL-12 in the culture supernatants was measured using ELISA. Data
are expressed as mean ±S.E.M. of 3 experiments. *, p
<0.05 as compared with the basal (0 mg/ml of D- Fraction) (Scheffe’s F-test).

Show more details – table 1 >>>

12) Consequently, significant increases in release of IFN-g and TNF-a suggest activation of NK cells by D-Fraction.

Effects of D-Fraction on IL-12 Release from Macro­phages
IL-12 is a cytokine released by monocytes and macrophages, and is critical to the functions of NK and T cells. It induces IFN-g release from both NK and T
cells.12—14) NK cells can lyse a variety of different tumor cells by exocytosis of perforin-containing granules, and sub­sequent formation of
lytic pores by perforin on the target cell membrane. Activation of NK cells with IL-2 and IL-12 has recently been reported to increase the binding of
perforin to the target cell membrane and subsequent lysis of tumor cells.15) IL-12 is released from monocytes and macrophages. To investigate
the effect of D-Fraction on IL-12 release, macrophage cell line RAW 264.7 cells were incubated with various concentrations of D-Fraction and secretion of
IL-12 was assessed. When cells were treated with D-Fraction (500—1000 mg/ml) for 24 h, the amount of IL-12 released significantly exceeded basal levels (0 mg/ml D-Fraction) (Fig. 4). Also, the effect of D-Fraction on IL-12 production was investigated in vitro using peritoneal macrophages
from nor­mal C3H/HeJ mice. When the normal peritoneal macro­phages were stimulated with D-Fraction (500 mg/ml) for 24 h, IL-12 production showed
an increase compared with that of the control (data not shown). These results suggested that the long-term anti-tumor effects of D-Fraction are
attrib­utable to activation of NK cells via macrophage-derived IL- 12, and to T cell activation. NK cells may be further acti­vated by INF-g released from
not only themselves but also activated T cells.

Effect of D-Fraction on the Activation of NK Cells in Cancer Patients
We concluded that D-Fraction was safe with no toxicity according to data confirmed by the Con­sumer Product Testing Co. Then, a non-randomized clinical
trial of D-Fraction was conducted to check the effect in NK cells of 8 cancer patients, who agreed to the trial. As shown in Table 1, the NK cell
activities of these patients were en­hanced 1.2—2.7 times by treatment with D-Fraction. Even though these tests were limited and in a non-controlled trial,
these results indicated that D-Fraction was effective for acti­vation of NK in cancer patients.

In conclusion, D-Fraction represents an important BRM for NK cells by enhancing IL-12 release from macrophages. In immunotherapy using D-Faction for cancer
patients, NK cells are responsible for early anti-tumor responses, while both NK and T cells are responsible for long-term anti-tumor responses. Although
the dependence of the immune system on NK cells requires further study, our results also indicate the possibility of immunotherapy by various agents, which
enhance the activity of NK cells for cancer patients.

Acknowledgements
We wish to thank Dr. Kanki Komi- yama of Kitasato Institute for the tumor cells utilized in our study, and also Mr. Yasuo Ohdaira, Manager of the
Develop­ment Division of Yukiguni Maitake Co. who contributed maitake to our studies.

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