| The medicinal fungi are famous of their medicinal value in oriental countries for over two thousands years, and the first medicinal book "Shennong Bencao Jing" in the world has recorded the clinical effect of medicinal fungi Lingzhi, Fuling, Leiwan and Zhuling etc., including the activity of antitumor, antivirus, hypocholesterolemic, reducing blood pressure and antiplatelet-aggregating, and the antitumor activity are the most conspicuous. In the fifties of the twentieth century, Lucas etc. had found that the extracts from the fruiting bodies of several basidiomycetes could inhibit the growth of S180 cells, and then the antitumor activity of medicinal fungi had been attracting the attention of the scientists from western countries. Polysaccharide has been generally regarded to be the main antitumor component in the medicinal fungi. However, the polysaccharide is usually used as adjuvant in the clinical tumor therapy. Are there any other important components with antitumor activity from medicinal fungi?A kind of active protein component Yt, extracted from medicinal mushroom Agrocybe aegerita, showed high antitumor activity both in vitro and in vivo. We also extracted the protein components from another three medicinal fungi Cordyceps sinensis, Lentinula edodes, Ganoderma Lucidum through the same approach, and found that they showed similar tumor rejection effect as Yt on H22 bearing mouse. All these data indicated that besides polysaccharides, active protein components from medicinal fungi possessed high antitumor activity and needed further elucidation.The component of Yt was determined by Schiffs and phenol-sulfuric acid method, which showed that there was little carbohydrate in Yt. Yt was separated using synthetic adsorbent sepabead HP20 to Yp (mainly contains proteins) and Ys (mainly contains small molecule compounds), which accounted for 59% and 41% of Yt. The 2-D gel result showed that there were about 20 kinds of proteins in Yt and MS results showed that Yt contained proteins such as lectin, and serine proteinase, AAL and other unknown proteins.In vitro, Yt could induce high cytotoxicity on tumor cells H22, HepG2, HeLa and Hep3B etc. and remarkably inhibit the growth of S180 tumor tissue, but had low or no cytotoxicity on fibroblast NIH3T3 and muscle tissues. In tumor H22-bearing mouse model, Yt could also remarkable inhibit H22 growth and prolong the life span of the BALB/c; Yt induced the antitumor memory effect. Moreover, the toxicological studies of Yt showed that intratumoral injection of Yt could not induce the production of a large amount of antibody, it did not affect the increasing of body weight, it did not induce tissue pathological change, it changed some of blood values and inhibit the percentage of live sperms of tumor-bearing male mouse. Yt was not effective against the growth of H22 in the nude mouse hosts but could prolong the lifespan. The immunoregulation effect of Yt was investigated, including spleen cell proliferation stimulated by Con-A was promoted ex vivo by Yt treatment; when the mouse was treated with Yt, splenocyte cytotoxicity activity was enhanced; the mRNA level of cytokines in spleen was changed significantly, such as up-regulation of the mRNA of TNF-α, IL-2, and IFN-γand down-regulation of TGF-β; the serum cytokine IL-2 expression increased after Yt treated.Furthermore, Yp and Ys both showed antitumor effect on H22 bearing mouse model, and Yp was better than Ys in the tumor growth inhibition and life span prolong effect. The results of body weight, blood value and serum antibody expression indicated that Yp and Ys had no obvious toxicity to tumor bearing mouse. The antitumor effect of Yp was reduced when Yp was treated with proteinase K, which indicated that the main active component of Yp was protein. In vivo, Yp could induce up-regulation of cytokine IL-10 and TGF-β, Ys could induce up regulation of GM-SCF, and neither of them could change the serum cytokine IL-2 and TNF-αexpression.Yt exerted the antitumor activity via direct cytotoxicity and immunoregulation, and we speculated that intratumor injection of Yt killed the tumor cells and induced strong immune response, resulting in tumor rejection. Hence, we simulated the treatment of Yt that was injected intratumorly, which was to mix Yt and whole tumor cell (UV-treated cells) vaccine and determined the adjuvant effect of Yt in the whole tumor cell vaccine. Both of the whole tumor cell vaccine and the mixed vaccine (Yt+whole tumor cell) showed 100% protect effect and memorial protection. The whole tumor cell vaccine could not induce 100% cross reactive protection, but the mixed vaccine could. We used different mouse models, and found that the protection effect was completely lost on nude mouse, partial lost on CD4+, CD8+T cell depletion mouse that was depleted with monoclonal antibody, and partial lost on macrophage depletion mouse model that was depleted with liposome-clodronate (liposomeciodr). Moreover, the protection effect of the whole tumor cell vaccine depended on the frequency of vaccination, the cell surface glycosylation and nucleic acid, and the intact of tumor cells. |
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