Studies in humans
Polysaccharide fractions of Ganoderma lucidum have been shown to have potent immunomodulating effects in pre-clinical trials. A clinical study of healthy volunteers demonstrated that G. lucidum did not affect their immune functions. Subsequently, an open-labeled study (i.e. not double-blind or placebo controlled) has evaluated the effects of water-soluble G. lucidum polysaccharides (Ganopoly) in patients with advanced colorectal cancer. Forty-seven patients were enrolled and treated with Ganopoly at 5.4 g/day for 12 weeks. In 41 assessable cancer patients, treatment with Ganopoly tended to increase mitogenic reactivity to phytohemagglutinin. The results indicated that Ganopoly may have beneficial immunomodulating effects in patients with advanced colorectal cancer, although further studies are needed to determine the mechanism of action, efficacy, and safety of the water- soluble G. lucidum polysaccharides in cancer patients (Gao et al., 2005).
A randomized, placebo-controlled, clinical trial has evaluated the effects of dietary supplementation with Agaricus sylvaticus on hematological, immunological, and glycemia levels of postsurgical patients with colorectal cancer. The study used 56 patients with colorectal cancer with the data suggesting that supplementation with A.sylvaticus produces benefits in hematological and immunological parameters and can reduce glycemia levels in patients with colorectal cancer (Fortes et al., 2009).
In vitro studies (human cell lines)
An Agaricus bisporus lectin (ABL) has been shown to inhibit incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87%, Caco-2 colon cancer cells by 16%, MCF-7 breast cancer cells by 50%, and Rama-27 rat mammary fibroblasts by 55% when the cells were grown for 24h in serum-free medium. Similar inhibition of proliferation of HT29 cells by ABL was found. ABL caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells, and inhibition of proliferation in HT29 cells was reversible after removal of the lectin (Yu et al., 1993).
The reversibility of the anti-proliferative effect of Agaricus bisporus lectin was associated with its release from cancer cells after internalization. The internalization and subsequent slow release, with little degradation of the lectin, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells (Yu et al., 2000).
An extract from Ganoderma lucidum has been reported to have apoptotic and anti-inflammatory functions in HT-29 human colonic carcinoma cells. Ling Zhi extract (LZE) is an herbal mushroom preparation that has been shown to induce apoptosis, anti-inflammatory action and differential cytokine expression during induced inflammation in the human colonic carcinoma cell line, HT 29. The extract caused no cytotoxicity in HT-29 cells at doses less than 10,000µg/ml. Increasing concentrations reduced prostaglandin E2 production, but increased nitric oxide production. LZE treatment induced apoptosis by increasing the activity of caspase-3. LZE at a concentration of 5,000µg/ml decreased the expression of cyclooxygenase-2 mRNA. Among 42 cytokines tested by protein array in this study, supplementation of LZE at doses of 500 and 5,000 µg/ml to HT-29 cells reduced the expression of interleukin-8, macrophage inflammatory protein 1-delta, vascular epithelial growth factor, and platelet-derived growth factor. These results suggest that LZE has pro-apoptotic and anti-inflammatory functions, as well as inhibitory effects on cytokine expression during early inflammation in colonic carcinoma cells (Hong et al., 2004). Similar anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus have been reported in HT-29 colon cancer cells in vitro (Lavi et al., 2006).
Similar effects in the same cell line (HT-29 human colon cancer cells) have been reported for an aqueous extract of Inonotus obliquus. The extract inhibited cell growth in a dose-dependent manner, and this inhibition was accompanied by apoptotic cell death. In addition, the apoptotic cell percentage was closely associated with down-regulation of Bcl-2 and up-regulation of Bax and caspase-3. The results suggest that the extract would be useful as an antitumor agent via the induction of apoptosis and inhibition of the growth of cancer cells through up-regulation of the expression of proapoptotic proteins and down-regulation of antiapoptotic proteins (Lee et al., 2009c).
A polysaccharide extract of Ganoderma lucidum has been shown to inhibit DNA synthesis in SW 480 human colorectal cancer cells and reduce the formation of DPPH radicals indicating that G. lucidum extracts inhibit proliferation of human colorectal cancer cells and possesses antioxidant activity (Xie et al., 2006).
Ergosterol peroxide from mushrooms has been shown to suppress inflammatory responses in RAW264.7 macrophages and the growth of HT29 human colon adenocarcinoma cells. Ergosterol peroxide appeared to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells (Kobori et al., 2007).
A study on the action of lentinan (extracted from Shiitake mushrooms (Lentinus edodes) has been conducted using murine lymphoma (K36) cells in an AKR mouse model. Further investigation on the effectiveness of the extracted lentinan was then performed using human colon-carcinoma cell lines in mice. Six established human colon-carcinoma cell lines segregated into three groups of different degrees of differentiation were used inthis study. One group was not fed (control) and the second group was prefed with lentinan for 7 days prior to inoculations with the cancer cells. The size of the tumours that developed was rated after 1 month. Significant regression in tumour formation was observed in prefed mice compared to control (unfed) mice when K36 or human colon-carcinoma cells were used. Significant reductions in the size of the tumours were observed in mice prefed with lentinan. Follow-up investigation proceeded with the use of nude mice (athymic). Lymphocytes extracted from AKR mice prefed with lentinan for 7 days were inoculated into the nude mice. This was followed by inoculation of the human colon-carcinoma cell lines into these mice. Much smaller tumours were formed in nude mice inoculated with lymphocytes, in contrast to the larger tumours formed in nude mice without lymphocyte inoculation. The study concluded that the anti-tumour property of lentinan was maintained with oral administration. In addition, "primed" lymphocytes, when given passively to immuno-deficient mice, were able to retard the development of tumours in these mice (Ng and Yap, 2002).
Splenic-sympathetic nerve activity (SNA) was suppressed by an intraduodenal Lentinus edodes injection in urethane-anesthetized rats, which significantly inhibited increases in the tumour volume of human colon and breast cancer cells implanted in athymic nude mice. The findings suggested that Lentinus edodes has an inhibitory effect on tumour proliferation, possibly via a reduction in NK cytotoxicity through the suppression of splenic-SNA (Shen et al., 2009).
Animal model (mouse) studies
There is epidemiological evidence that populations with high faecal beta-glucuronidase activity have greater risk of colon cancer than populations with low faecal beta-glucuronidase. This relationship has been investigated using the mouse-dimethylhydrazine colon carcinogenesis model and a fraction of Ganoderma lucidum which is a beta-glucuronidase inhibitor. Mice with low faecal beta-glucuronidase activity induced by consumption of an ether fraction of G. lucidum had significantly fewer aberrant crypts after injections of 1,2-dimethylhydrazine (DMH) than mice treated with DMH alone, supporting the hypothesis that the ether fraction of G.lucidum can provide some protection (in this animal model) against the induction of colon cancer (Kim et al., 1995).
4,7-Dimethoxy-5-methyl-l,3-benzodioxole (SY-1) has been shown to decrease the proliferation of human colon cancer cells (COLO 205) through G0/G1 cell-cycle arrest and induction of apoptosis. In contrast, SY-1 treatment did not induce significant changes in G0/G1 phase cell-cycle regulatory proteins in normal human colonic epithelial cells (FHC). The findings demonstrated that SY-1 inhibited human colon cancer cell proliferation through inhibition of cell growth and anchorage-independent colony formation (Lien et al., 2009).
A 23 kDa polysaccharide isolated from Grifola frondosa, while not affecting the proliferation of colon-26 cells in vitro, did significantly inhibit tumour growth in BALB/cA mice inoculated with colon-26 cancer cells, via activation of cell-mediated immunity (Masuda et al., 2009).
Animal model (rat) studies
Intake of dry powdered Lentinula edodes (Shiitake) has been reported to have no effect on the relative incidence of tumours in the colon or small intestine (duodenum) in azoxymethane-treated Sprague-Dawley rats. Consumption of 1% Shiitake stimulated growth of invasive adenocarcinomas in the mid colon and favoured a non significant increase in median frequency of aberrant crypt foci in this same region. In contrast, Shiitake at 4% intake elicited a reduction in colon tumour multiplicity. The authors suggested a stimulatory action of 1% Shiitake on rat colon tumourigenesis which is puzzling as the data were not statistically significant. However, the inhibitory actions of 4% Shiitake mushroom on the indices of rat colon tumourigenesis were statistically validated (Frank et al., 2006).
The effect of pleuran (beta-1,3-D-glucan isolated from the Oyster mushroom Pleurotus ostreatus) on the antioxidant status of the organism and on the development of precancerous aberrant crypt foci (ACF) lesions in the colon have been studied in the male Wistar rat. A diet containing either 10% pleuran or 10% cellulose was compared with a cellulose-free diet and both were found to significantly reduce conjugated diene content in erythrocytes and in liver. Particularly significant was the reduction of conjugated dienes in the colon following pleuran administration. ACF lesions developed in the colon of all animals fed a cellulose-free diet; however, the incidence was reduced to 64% and 60% following the cellulose and pleuran diets, respectively. The highest average count of the most frequent small ACF lesions, and highest total count, was seen in animals fed a cellulose-free diet. Although ACF lesions were reduced by the cellulose diet, the more significant reduction statistically (>50%) was achieved with the pleuran diet (Bobek and Galbavy, 2001).
Chemopreventive and immunomodulatory potential of methanolic (MET) and dichloromethanic (DCl) extracts of Agaricus blazei were investigated in the postinitiation stage of colon carcinogenesis in male Wistar rats. Administration of DCl extracts did not suppress 1,2dimethylhydrazine-induced colonic aberrant crypt foci nor did it affect the crypt multiplicity, but the highest dose of MET significantly reduced the development of preneoplastic lesions in the colon and liver. Lymphoproliferative response was slightly decreased in the initiated control group, which was restored by treatment with MET. No toxicity from DCl and MET extracts was observed (Ribeiro-Santos et al., 2008).
The potential blocking effect of Agaricus blazei (Ab) intake on the initiation stage of colon carcinogenesis has been investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as a biomarker in male Wistar rats. All 1,2-dimethylhydrazine (DMH)-treated rats developed ACF mainly in the middle and distal colon. Agaricus blazei intake at 5% did not alter the number of ACF induced by DMH or the proliferating cell nuclear antigen indices in the colonic mucosa. The results did not confirm a chemopreventive activity of Ab on the initiation stage of rat colon carcinogenesis (Ziliotto et al. 2008) (Ziliotto et al. 2009).(Ziliotto et al., 2008, Ziliotto et al., 2009).