Isolation of an Antitumor Compound from Agaricus blazei Murill and Its Mechanism of Action

Takeshi Takaku*, Yoshiyuki Kimura2 and Hiromichi Okuda

(Journal of Nutrition. 2001;131:1409-1413.) © 2001 The American Society for Nutritional Sciences

Second Department of Medical Biochemistry and *Central Research Laboratory, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. In the present study, we examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei. Tumor growth was retarded by the oral administration of the lipid fraction extracted from A. blazei with a chloroform/methanol mixture in sarcoma 180-bearing mice. The substance with the antitumor activity in the lipid fraction was isolated via silica gel column chromatography, eluted with an acetonitrile/methanol (3:2) mixture and identified as ergosterol by direct comparison of the 1H NMR and mass spectrometry spectral data of an authentic sample. The oral administration of ergosterol to sarcoma 180-bearing mice significantly reduced tumor growth at doses of 400 and 800 mg/kg administered for 20 d without side effects, such as the decreases in body, epididymal adipose tissue, thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs. Ergosterol had no cytotoxicity against tumor cells. To clarify the antitumor activity of ergosterol, we examined the effects of ergosterol on tumor-induced angiogenesis using two in vivo models. Intraperitoneal administration of ergosterol at doses of 5, 10 and 20 mg/kg for 5 consecutive d inhibited the neovascularization induced by Lewis lung carcinoma cell-packed chambers, suggesting that either ergosterol or its metabolites may be involved in the inhibition of tumor-induced neovascularization. Therefore, we further examined the inhibitory effects of ergosterol on Matrigel-induced neovascularization. Female C57BL/6 mice were subcutaneously inoculated with Matrigel containing acidic fibroblast growth factor and heparin with or without ergosterol. Ergosterol inhibited the Matrigel-induced neovascularization, suggesting that ergosterol directly inhibits Matrigel-induced neovascularization. From these results, it seems likely that the antitumor activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumors. This is the first report of ergosterol as an antiangiogenic substance.

1. Antitumor activity and some properties of water-soluble polysaccharides from “Himematsutake,” the fruiting body of Agaricus blazei Murill.
Mizuno T., Inagaki R., Kanno T., Hagiwara T., Nakamura T., Itoh T., Shimura K., Sumiya T., Asakura A.
Agric. Biol. Chem. 1990;54:2889-2896

2. Inhibitory action of (1-6)-ß-glucan-protein complex (FIII 020b) isolated from Agaricus blazei Murill (“Himematsutake”) on Meth A fibrosarcoma-bearing mice and its antitumor mechanism. Jpn.
Itoh H., Ito H., Amano H., Noda H.
J. Pharmacol. 1994;66:265-271[Medline]

3. Formolysis of a potent antitumor (1-6)-ß-glucan-protein complex from Agaricus blazei fruiting bodies and antitumor activity of the resulting products.
Kawagishi H., Kanao T., Inagaki R., Mituno T., Shimura K., Itoh H., Hagiwara T., Nakamura T.
Carbohydr. Polym. 1990;12:393-403

4. Selective tumoricidal effect of soluble proteoglucan extracted from the Basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apotosis.
Fujimiya Y., Suzuki T., Oshiman K., Kobori H., Moriguchi K., Nakashima H., Matumoto Y., Takahara S., Ebina T., Katakura R.
Cancer Immunol. Immunother. 1998;46:147-159[Medline]

5. Antitumor promoting effect of an active component of polyporus ergosterol and related compounds on rat urinary bladder carcinogenesis in a short-term test with concanavalin
Yazawa Y., Yokota M., Sugiyama K.
A. Biol. Pharm. Bull. 2000;28:1298-1302

6. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor.
Passaniti A., Taylor R. M., Pili R., Guo Y., Long P. V., Haney J. A., Pauly R. R., Grant D. S., Martin G. R.
Lab. Invest. 1992;67:519-528[Abstract]

7. Current status and possible future of antitumor or drugs: with special reference to pharmacology of oriental medicine.
Chihara G.
Yakugaku Zasshi 1988;108:171-186[Medline]

8. Antitumor effects of a new polysaccharide-protein complex (ATOM) prepared from Agaricus blazei (Iwade strain 101) “Himematsutake” and its mechanisms in tumor-bearing mice.
Ito H., Shimura K., Itoh H., Kawase M.
Anticancer Res 1997;17:277-284[Medline]

The antitumor activity of the i.p. or p.o. administration of polysaccharide-protein complex, ATOM (antitumor organic substance Mie) prepared from cultured mycelia of Agaricus blazei (Iwade strain 101) “Himematsutake” examined against four kinds of established mouse tumors. ATOM was highly effective at the doses of 10 and 20 mg/kg/day x 10 on subcutaneously implanted Sarcoma 180 in mice, and was also active against Ehrlich ascites carcinoma, Shionogi carcinoma 42 and Meth A fibrosarcoma at doses of 50 and 100 mg/kg/day x 10. ATOM has no direct cytotoxic action on tumor cells in vitro. Thus the tumor growth-inhibitory effect of ATOM is apparently due to immunological host-mediated mechanisms. The number of peritoneal macrophages, the phagocytosis of polystyrene latex beads and the proportion of the third component of complement (C3)-positive fluorescent cells were increased in the mice treated with ATOM. These results suggest that the macrophage activa-tion and alterations of the C3 are necessary for the induction of an antitumor effect of ATOM.

PMID: 9066665 [PubMed - indexed for MEDLINE]

9. Inhibition of tumor growth and angiogenesis by vitamin D3 agents in murine renal cell carcinoma.
Fujioka T., Hasegawa M., Ishikura K., Matsushita Y., Sato M., Tanji S.
J. Urol. 1998;160:247-251[Medline]

10. Inhibition of angiogenesis as a mechanism for inhibition by 1-hydroxyvitamine D3 and 1.25-dihydroxyvitamine D3 of colon carcinogenesis induced azomethane in Wistar rats.
Iseki K., Tatsuta M., Uehara H., Iishi H., Yano H., Sakai N., Ishiguro S.
Int. J. Cancer 1999;81:730-733[Medline]

11. Antitumor effects of 1, 25-dihydroxyvitamine D3 and vitamin D analogs.
Van Den Bend G. J., Pols H. A., Van Leeuwen J. P.
Curr. Pharm. Des. 2000;6:717-732[Medline]

12. Antitumor beta glucan from the cultured fruit body of Agaricus blazei.

Ohno N, Furukawa M, Miura NN, Adachi Y, Motoi M, Yadomae T.

Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy & Life Science, Hachioji, Japan. ohnonao@ps.toyaku.ac.jp

Agaricus blazei is a medically important mushroom widely eaten and prescribed in Japan. Polysaccharide fractions were prepared from cultured A. blazei by repeated extraction with hot water (AgHWE), cold NaOH (AgCA), and then hot NaOH (AgHA). By chemical, enzymic, and NMR analyses, the primary structures of AgHWE, AgCA, and AgHA were mainly composed of 1,6-beta-glucan. Among these fractions, the NaOH extracts showed antitumor activity against the solid form of Sarcoma 180 in ICR mice. To demonstrate the active component in these fractions, several chemical and enzymic treatments were applied. These fractions were found to be i) neutral beta-glucan passing DEAE-Sephadex A-25, ii) resistant to periodate oxidation (I/B) and subsequent partial acid hydrolysis (I/B/H), iii) resistant to a 1,3-beta-glucanase, zymolyase, before I/B, but sensitive after I/B/H. In addition, after I/B/H treatment of the neutral fraction of AgCAE, a signal around 86 ppm attributable to 1,3-beta glucosidic linkage was detectable in the 13C-NMR spectrum. These facts strongly suggest that a highly branched 1,3-beta-glucan segment forms the active center of the antitumor activity.

PMID: 11456124 [PubMed - indexed for MEDLINE]

[Antitumor effects of intratumoral injection of Basidiomycetes preparations]

[Article in Japanese]

Ebina T.

Division of Immunology, Miyagi Cancer Center Research Institute.

The antitumor effects of Basidiomycetes preparations in an experimental mouse model, the “double grafted tumor system” were analyzed. Some BRMs prevented metastases by utilizing the anti-tumor immunological cascade reactions, which activates macrophages in the body. The following Basidiomycetes preparations were analyzed: PSK was a hot water extract of cultured mycelia from Coliolus versicolor and a protein bound beta-glucan. Matsumax was extracted from mycelia of Tricholoma matsutake and was a protein bound (38%) a-glucan. The Agaricus preparation was extracted from fruit bodies of Agaricus blazei and a protein-bound (17%) a-glucan, beta-glucan. Himematsutake preparation was extracted from fruit body of Agaricus blazei (Himematsutake) and a protein bound (5%) glucan. Lentinan was purified from fruit bodies of Lentinus edodes and is a purified beta-glucan. PSK cured both primary and metastatic tumors in the double grafted tumor system. Lentinan inhibited the growth of neither primary nor metastatic tumors. Matsumax and Agaricus preparation cured primary tumor and inhibited the growth of metastatic tumor. Himematsutake preparation inhibited the growth of primary tumor. Immunosuppresive acidic protein (IAP) is produced by activated mactrophates. The PSK, Matsumax, Agaricus preparation and Himematsutake preparation induced IAP but Lentinan did not.

PMID: 16315899 [PubMed - indexed for MEDLINE]

Effect of a medicinal extract from Agaricus blazei Murill on gene expression in a human monocyte cell line as examined by microarrays and immuno assays.

Ellertsen LK, Hetland G, Johnson E, Grinde B.

Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway.

Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).

Effects on gene expression and viral load of a medicinal extract from Agaricus blazei in patients with chronic hepatitis C infection.

Grinde B, Hetland G, Johnson E.

Division of Infectious Disease Control, Norwegian Institute of Public Health, Ulleval University Hospital, Oslo, Norway.

Extracts from the mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer and infections, including hepatitis. We previously demonstrated a potent immunomodulating effect of a particular preparation on monocytes in vitro, and a protective effect on bacterial infections in mice. Here we report the effect on gene expression in peripheral blood cells from four chronic hepatitis C patients, using global (29 k) oligo-based, single channel microarrays. The viral load was slightly, but not significantly, decreased after 1 week of AbM treatment. The cytokine genes most strongly induced in vitro were not induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled receptor signalling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the beta-glucans of the extract, which presumably are responsible for cytokine induction, did not readily enter the blood, while other components, such as substances proposed to have anticancer effects, were active in the blood.

STUDY SHOWS EXTRACTS SUPPERIOR TO CAPSULES

In vitro augmentation of natural killer activity and interferon-gamma production in murine spleen cells with Agaricus blazei fruiting body fractions.

Zhong M, Tai A, Yamamoto I.

Bizen Chemical Co., Ltd., Okayama, Japan.

Aqueous extracts of the Agaricus blazei fruiting body prepared at different temperatures were fractionated by ethanol precipitation with various ethanol concentrations. The original aqueous extracts of A. blazei failed to stimulate natural killer (NK) cell activity in murine spleen cells in vitro, but the strongest effect was observed in a 30% ethanol-soluble-50% ethanol-insoluble fraction prepared from the extract at 40 degrees C (fraction A-50). Fraction A-50 also showed the strongest augmenting effect on interferon (IFN)-gamma production. This augmentation of NK activity and IFN-gamma production by fraction A-50 was completely abrogated by a heat treatment

Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells.

Kim GY, Lee MY, Lee HJ, Moon DO, Lee CM, Jin CY, Choi YH, Jeong YK, Chung KT, Lee JY, Choi IH, Park YM.

Department of Microbiology and Medical Research Institute, Pusan National University, College of Medicine, Ami-dong 1-10, Seo-gu, Pusan 602-739, Republic of Korea.

Water-soluble proteoglycan (WSPG) of Agaricus blazei Murill has been known to stimulate the non-specific complements and humoral immune functions to act as polyclonal activators of B cells and to inhibit tumor growth and metastasis. However, little is known about its immunomodulating effects on murine bone marrow (BM)-derived dendritic cells (DC). In the present study, we examined the maturation process of murine BM-DC. BM cells were cultured in the presence of IL-4 and GM-CSF and the generated immature DC were stimulated with WSPG or LPS (WSPG-DC and LPS-DC, respectively) for 24 h. WSPG significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) II, as did LPS. IL-12p70 production in WSPG-DC was also identical to that in LPS-DC. The antigen-uptake capacity of WSPG-DC was determined by FITC-labeled dextran uptake. WSPG-DC lost dextran uptake capacity comparable to LPS-DC. The antigen-presenting capacity of WSPG-DC as analyzed by allogeneic T cell proliferation was significantly increased in comparison with immature DC, was identical to LPS-DC, and induced higher levels of IL-2 in responding T cells. These results indicate the immunomodulatory properties of WSPG, which might be therapeutically useful in the control of cancers and immunodeficient diseases through the up-regulation of DC maturation.

PMID: 16023604 [PubMed - indexed for MEDLINE]

Effect of supplementation of Agaricus mushroom meal extracts on enzyme activities in peripheral leukocytes of calves.

Kimura N, Fujino E, Urabe S, Mizutani H, Sako T, Imai S, Toyoda Y, Arai T.

Division of Animal Nutrition, Department of Animal Science, Nippon Veterinary and Animals Science University, 1-7-1 Kyonancho, Musashino, Tokyo 180-8602, Japan.

To investigate the effect of Agaricus mushroom meal on the energy metabolism in animal tissues; plasma glucose, triglyceride, cholesterol and immunoreactive insulin (IRI) concentrations and activities of enzymes related to energy metabolism in plasma and peripheral leukocytes were measured in Japanese Black WagyuxHolstein F1 calves supplemented with Agaricus blazei Murill (A. blazei) extract in milk-replacer at the dose of 60g/head/day for 4 weeks. Activities of malate dehydrogenase and aspartate aminotransferase in cytosol and glutamate dehydrogenase in mitochondria, and the malate dehydrogenase/lactate dehydrogenase ratio in cytosol in peripheral leukocytes of calves with A. blazei were significantly higher than those in control calves without A. blazei. It was concluded that supplementation of Agaricus mushroom meal extract was effective in activation of enzymes related to energy metabolism in peripheral leukocytes of calves.

An extract of the mushroom agaricus blazei Murill protects against lethal septicemia in a mouse model of fecal peritonitis.

Bernardshaw S, Hetland G, Grinde B, Johnson E.

Department of Gastroenterological Surgery, Ulleval University Hospital, Oslo, Norway. sorn@ulleval.no

Bacterial septicemia is frequently occurring during gastroenterological surgery. Because of increasing problems in hospitals with bacteria developing multiresistance against antibiotics, prophylactic treatment using immunomodulators is interesting. We have examined the putatively anti-infective immunomodulatory action of the edible mushroom, Agaricus blazei Murill (AbM), in an experimental peritonitis model in BALB/c mice. The mice were orally given an extract of AbM or phosphate-buffered saline 1 day before the induction of peritonitis with various concentrations of feces from the mice. The state of septicemia, as measured by the number of colony-forming units of bacteria in blood, and the survival rate of the animals were compared between the groups. Mice that were orally treated with AbM extract before bacterial challenge showed significantly lower levels of septicemia and improved survival rates. Our findings suggest that the AbM extract, when given prophylactically, may improve health. Further studies are needed on humans when considering whether AbM could be used as an alternative treatment modality for patients at risk of contracting serious bacterial peritonitis.

An extract of the mushroom Agaricus blazei Murill administered orally protects against systemic Streptococcus pneumoniae infection in mice.

Bernardshaw S, Johnson E, Hetland G.

Department of Gastroenterological Surgery, Ulleval University Hospital, Oslo, Norway.

The aim was to investigate the antibacterial effect of the biologically active and edible mushroom Agaricus blazei Murill (AbM). A water extract of AbM or PBS control was administered orally before or with challenge to NIH/OlaHsd mice, experimentally infected intraperitoneally with the moderately virulent Streptococcus pneumoniae serotype 6B. End points were bacteraemia and survival rate. The AbM extract, protected against systemic S. pneumoniae 6B infection in the mice. It was most effective when given 24 h before inoculation but did also have protective effects when given together with challenge compared with control. The lack of antibiotic effect on pneumococci in vitro and increased levels of cytokines MIP-2 and TNF-alpha in the serum of mice receiving AbM extract, indicated that the protective effect of AbM was due to the involvement of the native immune system. This is the first report of anti-infection effects of AbM in vivo. Our results suggest that AbM extract may be useful as additional prophylactic and possibly therapeutic treatment against bacterial and possibly other infections in humans.

PMID: 16253127 [PubMed - indexed for MEDLINE]