We previously found that ergosterol isolated from Agaricus blazei inhibited tumor growth through the inhibition of tumor-induced neovascularization. In the present study, we isolated further anti-angiogenic substances (A-1 and A-2) from this fungus using an assay system of angiogenesis induced by Matrigel supplemented with vascular endothelial growth factor, and A-1 was identified as sodium pyroglutamate. Next, we examined the antitumor and antimetastatic actions of A-1 using Lewis lung carcinoma ( he Basiodiomycete fungus Agaricus blazei Murill (Japanese name: Himematsutake or Agarikusutake) has been traditionally used as a health food in Brazil for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. It has been reported that 200,000 to 400,000 kg of the dried body of A. blazei is produced every year in Japan.1, 2) A. blazei is used by 300,000 to 500,000 persons for the prevention of cancer and/or as an adjuvant with cancer chemotherapy drugs after the removal of a malignant tumor.1, 2) The water extract of A. blazei is generally administered at 3 to 5 g three times daily. The hot water extract of A. blazei has potent antitumor activity in sarcoma 180-bearing mice, [3][4][5][6] and the antitumor activity was postulated to reside in the β-(1-6) glucan fraction.
3-6)Previously we examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei and identified ergosterol as an active substance. 7) We found that ergosterol inhibited tumor-induced neovascularization.7) Furthermore, we found that the acetone-soluble fractions of ethanol extracts inhibited tumor growth and lung metastasis in Lewis lung carcinoma (LLC)-bearing mice, and inhibited Matrigel-induced angiogenesis (unpublished data). In a preliminary experiment, we found a large amount of D-mannitol in the acetonesoluble fraction of the ethanol extracts. However, D-mannitol had no effect on Matrigel-induced angiogenesis in vivo (data not shown). Therefore, we prepared the methanol-soluble fraction of the chloroform-methanol (1:1, v/v) extracts in order to remove D-mannitol. We then attempted to isolate antiangiogenic substances from the methanol-soluble fraction, which showed antitumor and antiangiogenic activities. We isolated two active substances (A-1 and A-2) from A. blazei with the aid of a Matrigel-induced angiogenesis assay system. This report describes the determination of the structure of A-1 and the effects of A-1 on tumor growth and lung metastasis in LLC-bearing mice. In addition, to clarify the mechanisms of the antitumor and antimetastatic activities of A-1, we examined the effects of A-1 on splenic immune function, tumor apoptosis and tumorinduced neovascularization.
Materials and MethodsGeneral experimental procedures. The 1 H-NMR (499.83 MHz) and 13 C-NMR (125.68 Hz) spectra were recorded in D 2 O with a Varian Unity Inova 500 spectrometer (Tosoh, Tokyo). Mass spectra were measured with a Hitachi M-4000 H spectrometer (Tokyo). Thin layer chromatography (TLC), prepar...
