Anti Cancer Properties of Mushrooms

Effects on Carcinogenesis

Summary of Anti-Cancer Properties

Anti-tumor effects, primarily in human cell lines, have been reported for polysaccharides extracted from various mushrooms.

The polysaccharides generally belong to the beta-glucan family of compounds and appear to exert their anti-tumorigenic effects via enhancement of cellular immunity. Apoptosis and/or anti-proliferative effects on carcinomas and cell lines is also a mechanism shared by several mushrooms and their extracts in studies of anti-cancer effects.

Anti-tumor effects of proteoglycan fractions from a variety of mushrooms, including Agaricus bisporus, involve the elevation of natural killer (NK) cell numbers and the stimulation of inducible nitric oxide (NO) synthase gene expression, which is then followed by NO production in macrophages via activation of the transcription factor, NF-kappaB.

Activation of NK cells is likely via interferon-gamma and interleukin mediated pathways. In addition to the apoptotic and anti-proliferative effects, the anti-inflammatory and anti-microbial / viral effects described in this report may also contribute to the anti-carcinogenic effects of mushrooms and their extracts, although such direct links have not been established to date. While studies in human cell lines provide supporting evidence, well-designed human clinical trials are required before the anti-cancer mechanisms and health outcomes in humans can be validated. In recent years, a number of human trials have been undertaken. See section on Studies in Humans.

Bladder Cancer & Mushrooms

In vitro studies (human cell lines)

Maitake mushroom D-fraction in combination of interferon (IFN)-alpha has been shown to potentiate the anticancer activity of IFN-alpha in bladder cancer T24 cells in vitro. The combination of IFN-alpha 2b (10 000 IU/mL) and Maitake mushroom D-fraction (200 g/mL) reduced growth in T24 cells by ~75% by an induction of G1 arrest with DNA-dependent protein kinase activation (Louie et al., 2010). Anti-tumour potential of the medicinal mushrooms Pleurotus florida and Calocybe indica against T24 bladder cancer cell lines have also been demonstrated (Selvi et al., 2011).

Cordycepin (3′-deoxyadenosine), from Cordyceps militaris, has been shown to have anti-tumor effects in two different bladder cancer cell lines, 5637 and T-24 cells. Cordycepin treatment, at a dose of 200 mM (IC50) during cell-cycle progression resulted in a significant and dose-dependent growth inhibition, which was largely due to G2/M-phase arrest (Lee et al., 2009d).

Breast Cancer & Mushrooms

Animal model studies

Extracts from Agaricus bisporous mushrooms have been suggested as potential breast cancer chemopreventive agents, as they suppress aromatase activity and estrogen biosynthesis. A study has evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo and found the major active compounds to be unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of

linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibited aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicated that they are non-competitive inhibitors with respect to androstenedione. As only conjugated linoleic acid was found to inhibit the testosterone-dependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that the mushroom extract decreased both tumor cell proliferation and tumour weight with no effect on the rate of apoptosis (Chen et al., 2006).

study of the effects of Ganoderma lucidum (Basidiomycetes) polysaccharide (GL-PS) extract on tumor volume and T(CD4+/CD8+) ratio of tumour infiltrating lymphocytes (TILs) in breast cancer bearing mice has indicated that GL-PS (100mg/kg/day) could effectively increase the delayed type hypersensitivity response against sRBC in BALB/c mice. Furthermore, intraperitoneal injection of this extract in breast cancer bearing mice could increase T-cell infiltration into the tumour, suggesting a potent immunomodulatory effect (Mojadadi et al., 2006).

In vitro studies (human cell lines)

Estrogen is a major factor in the development of breast cancer and in situ estrogen production by aromatase/estrogen synthetase in breast cancer plays a significant role in tumour proliferation. An aqueous extract of Agaricus bisporus has been shown to suppress aromatase activity in a dose-dependent manner. In situ aromatase activity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phytochemicals in the mushroom aqueous extract inhibited aromatase activity and proliferation of MCF-7aro cells. These results suggest that diets high in mushrooms may modulate the aromatase activity and function in chemoprevention in post-menopausal women by reducing the in situ production of estrogen (Grube et al., 2001).

2 Aqueous extracts Maitake (Grifola frondosa), Crimini (Agaricus bisporus), Portabella

(Agaricus bisporus), Oyster (Pleurotus ostreatus) and White Button (Agaricus bisporus) mushrooms significantly reduced cellular proliferation in MCF-7 human breast cancer cells by up to 33%, with Maitake and Oyster mushrooms being the most effective. Maitake also significantly induced apoptosis and cytotoxicity in these human breast cancer cells (Martin and Brophy, 2010) with the activation of apoptosis possibly being mediated via BAK-1 gene activation (Soares et al., 2011). Another study in MCF-7 breast cancer cells has shown that Coprinus comatus (OFMull.: Fr.) Pers. (Agaricomycetideae), also called the Shaggy Inc Cap Medicinal Mushroom, contains potent compounds capable of inhibiting NF-kappa B function that may also act as an antitumor agent (Asatiani et al., 2011).

Screening of 38 species of edible mushrooms on human estrogen-receptor positive (ER+) (MCF-7) and estrogen-receptor negative (ER-) (MDA-MB-231, BT-20) breast cancer cells showed that water-based extracts of three mushroom species, Coprinellus sp., Coprinus comatus, Flammulina velutipes (CME, CCE and FVE, respectively), haveanti-tumour activities including marked growth inhibition of both ER+ and ER- breast cancer cells, induction of rapid apoptosis (cell death) on both ER+ and ER- cells, and significant inhibition of MCF-7 tumour colony formation in vitro. The anti-proliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ER- breast cancer cells was very high. Mushroom extracts CME and FVE induced a rapid (within 5 hours) apoptosis on MCF-7 and MDA-MB-231 cells. MCF-7 tumour colony formation rate was reduced by 60% in CCE- and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that the mushroom species Coprinus comatus, Coprinellus sp. and Flammulina velutipes contain potent anti-tumour compounds for breast cancer (Gu and Leonard, 2006). The cultivated mycelium of Cordyceps sinensis (Cs), has also been shown to have a significant and dose-dependent inhibitory effect on the proliferation of MCF-7 breast cancer cells (Wu et al., 2007b).

Ganoderma lucidum (Reishi) selectively inhibits cancer cell viability in inflammatorybreast cancer although it does not affect the viability of noncancerous mammary epithelial cells. Reishi has been shown to inhibit cell invasion and disrupt the cell spheroids that are characteristic of inflammatory breast cancer. Reishi decreased the expression of genes involved in cancer cell survival, proliferation, invasion and metastasis, whereas it increases the expression of IL8 (Martinez-Montemayor et al., 2011).

An array of low molecular weight compounds (including phenolic acids, flavonoids, tocopherols, carotenoids, sugars and fatty acids) from wild mushrooms have been used in molecular docking experiments against three known protein targets involved in breast cancer (aromatase, estrone sulfatase and 17beta-HSD-1) using docking software. The estimated inhibition constants for the low molecular weight compounds, and the potential structure-activity relationships for the compounds were determined. The compounds 4-O-caffeoylquinic, naringin and lycopene were the top-ranked potential inhibitors for aromatase, estrone sulfatase and 17beta-HSD1 which are protein targets in breast cancer (Froufe et al., 2011, Froufe et al., 2012).

Ganodermanontriol (GDNT), a Ganoderma alcohol is able to suppress proliferation (anchorage-dependent growth) and colony formation (anchorage-independent growth) of human breast cancer cells MDA-MB-231. GDNT suppressed expression of the cell cycle regulatory protein CDC20, which is over-expressed in pre-cancerous and breast cancer cells compared to normal mammary epithelial cells, and also over-expressed in tumors when compared to the tissue surrounding the tumor in specimens from breast cancer patients. GDNT also inhibited invasive behavior (cell adhesion, cell migration, and cell invasion) through the suppression of secretion of urokinase-plasminogen activator (uPA) and inhibited expression of uPA receptor (Jiang et al., 2011a).

Ganoderic acid DM (GADM), a triterpenoid isolated from Ganoderma lucidum, inhibits cell proliferation and colony formation in MCF-7 human breast cancer cells. The mechanisms involved G1 cell cycle arrest, and apoptosis induced by GADM may be partially due to GADM-induced DNA damage of the breast cancer cells (Wu et al., 2012).

Antrodia camphorata has been shown to promote cell cycle arrest and apoptosis ofhuman estrogen-nonresponsive MDA-MB-231 cells and to markedly inhibit the invasion/migration of highly metastatic MDA-MB-231 human breast cancer cells. A. camphorata suppressed the phosphorylation of ERK1/2, p38, and JNK1/2, and inhibited NF-kappaB binding and activation in a dose-dependent manner. These data suggest that the anti-metastatic activities of Antrodia camphorata against human breast cancer cells are mediated through suppression of the MAPK signaling pathway (Yang et al., 2011).

Ganoderma lucidum suppresses the invasive behaviour of breast cancer cells byinhibiting the transcription factor NF-kappaB. It has been shown that Ganoderma lucidum inhibits proliferation of breast cancer MDA-MB-231 cells by downregulatingAkt/NF-kappaB signaling. Ganoderma lucidum has been shown to suppress phosphorylation of Akt on Ser473 and downregulate the expression of Akt, which results in the inhibition of NF-kappaB activity in MDA-MB-231 cells. The biological effect of Ganoderma lucidum was demonstrated by cell cycle arrest at G0/G1, which was theresult of the downregulation of expression of NF-kappaB-regulated cyclin D1, followed by the inhibition of cdk4. These results suggest that Ganoderma lucidum inhibits the growth of MDA-MB-231 breast cancer cells by modulating Akt/NF-kappaB signaling and could have potential therapeutic use for the treatment of breast cancer (Jiang et al., 2004a).

A subsequent study by the same group on the proliferation of human estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cells has reported that G. lucidum inhibits proliferation of human breast cancer cells and contains biologically active compounds with specificity against the estrogen receptor and NF-kappaB (transcription factor) signalling (Jiang et al., 2006). More recently, the same group reported the effects of the structurally related lanostane-type triterpenes, ganoderic acid A, F and H from Ganoderma lucidum on highly invasive human breast cancer cells. The activity of ganoderic acids is linked to the hydroxylation in the triterpene lanostane structure. Hydroxylated triterpenes from G. lucidum could be promising natural agents for further study of invasive breast cancers (Jiang et al., 2008).

An aqueous extract of Cordyceps militaris (AECM) has been shown to induce apoptosis via the inhibition of Akt activation in a time-dependent manner. The data suggested that the apoptopic effect may relate to the activation of caspase-3 and mitochondria dysfunctions that correlate with the inactivation of Akt (Jin et al., 2008).

The effect of G. lucidum on oxidative stress-induced metastatic behaviour of poorly-invasive MCF-7 breast cancer cells has also been studied and it has been shown that G. lucidum inhibited oxidative stress-induced migration of MCF-7 cells by the down-regulation of mitogen activated protein kinase (MAPK) signalling, which is involved in hormonal signalling cascades. G. lucidum suppressed oxidative stress stimulated phosphorylation of extracellular signal-regulated protein kinases (Erk1/2), which resulted in the down-regulation of expression of c-fos, and in the inhibition of transcription factors AP-1 and NF-kappaB. The biological effect of G. lucidum on cell migration was mediated by the suppression of secretion of interleukin-8 from MCF-7 cells exposed to oxidative stress. These results suggest that G. lucidum inhibited the oxidative stress-induced invasive behaviour of breast cancer cells by modulating Erk1/2 signaling and could possibly be considered as an antioxidant in adjuvant cancer therapy (Thyagarajan et al., 2006).

A further study by the same group has also shown that an extract from green tea (GTE) increased the anti-cancer effect of G. lucidum extract (GLE) on cell proliferation (anchorage-dependent growth) as well as colony formation (anchorage-independent growth) of breast cancer cells. The effect was mediated by the down-regulation of expression of the oncogene c-myc in MDA-MB-231 cells. (Thyagarajan et al., 2007).

While Ganoderma lucidum has shown significant inhibitory effects on NF- kappa B activity in breast cancer cells, other mushrooms which have also been reported to produce biologically active substances and have demonstrated in vitro and in vivo breast cancer inhibitory activity are Agaricus bisporus, A. brasiliemis, Trametes versicolor, Grifola frondosa, Inonotus obliquus, Lentinus edodes, Leucoagaricus americanus, Pleurotus ostreatus and Sparassis crispa (Petrova et al., 2005). Phellinus linteus hasalso been shown to suppress growth, angiogenesis and invasive behaviour of breast cancer cells (Sliva et al., 2008). An apoptopic effect in the human breast cancer cell line ZR-75-30 of a polysaccharopeptide from Coriolus versicolor has also been reported (Wan et al., 2008).

An alcohol extract from the spore of Ganoderma lucidum has been shown to inhibit the in vitro proliferation of human umbilical vein endothelial cells and MDA-MB-231 humanbreast cancer cells. Further fractionation of the alcohol extract revealed that the ethyl acetate fraction inhibited both cell lines in a dose-dependent manner from 2 to 40mg/ml (Lu et al., 2004).

An ethyl acetate fraction from Shiitake (Lentinus edodes) mushrooms has been investigated using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human non-malignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent anti-proliferative effects of the fraction were observed in all cell lines. Approximately 50mg/L of the fraction induced apoptosis in 50% of the population of four human tumour cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by significant decrease of the S phase, which was associated with the induction of cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumour cells, non-malignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% anti-proliferative effect occurred at the highest concentration of the fraction (800mg/L). The data suggest that inhibition of growth in tumour cells by the Shiitake mushroom extract may result from an induction of apoptosis (Fang et al., 2006).

Extracts from Lentinula edodes (Shiitake) have been widely reported to have anti-tumour activity. However, this activity has been shown to be host-mediated and not by direct cytotoxic activity to cancer cells. A study (Israilides et al., 2008) has demonstrated cytotoxic and cell growth inhibitory (cytostatic) effects of aqueous extracts of the mushroom on the MCF-7 human breast adenocarcinoma cell line. The effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore, mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC50 values and the simultaneous higher respective values on normal fibroblast cells. In addition to the direct inhibition of the proliferation of human breast cancer cells in vitro, the Lentinula edodes extract had immuno-stimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

Pleurotus ostreatus (Oyster mushroom) has been shown to suppress proliferation ofbreast cancer (MCF-7, MDA-MB-231) and colon cancer (HT-29, HCT-116) cells, without affecting proliferation of epithelial mammary MCF-10A and normal colon FHC cells. Flow cytometry revealed that the inhibition of cell proliferation by P.ostreatus was associated with the cell cycle arrest at G0/G1 phase in MCF-7 and HT-29 cells. P.ostreatus also induced expression of the tumour suppressor p53 and cyclin-dependent kinase inhibitor p21(CIP1/WAF1). It appears that P. ostreatus suppresses the proliferation of breast and colon cancer cells via a p53-dependent as well as a p53-independent pathway (Jedinak and Sliva, 2008).

Hispolon extracted from Phellinus linteus has also been shown to have antiproliferative effects in breast and bladder cancer cells (Lu et al., 2009).

Ovarian, Endometrial Cancer & Mushrooms

Animal model studies

Protein extracts of Lentinus edodes C91-3 fermentation broth administered to mice with cervical cancer prolonged the lifespan of tumour-bearing mice significantly and killed cervical cancer U14 cells in vitro (Liu et al., 2009).

In vitro studies (human cell lines)

Polysaccharides from the Lingzhi (Ganoderma Lucidum) have been shown to decrease CyclinB1 mRNA expression in

cervical cancer CaSki cells and inhibit CaSki and HeLa cell proliferation (Chen et al., 2010d). Ganoderma lucidum has also been shown to inhibit cell growth and disruption of cell cycle progression via down regulation of cyclin D1 in the ovarian cancer cell line OVCAR-3. Chemopreventive activities were demonstrated by suppression of oxidative stress via the induction of antioxidant SOD and catalase as well as the phase II detoxification enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase PI (GSTP1) via the Nrf2 mediated signaling pathway known to provide chemoprotection against carcinogenicity (Hsieh and Wu, 2011).

Ethanol and hot water extracts of Pleurotus ferulae have been shown to have anti-tumourigenic properties in human cervical cancer cell lines and in human lung cancer cell lines. When A549, SiHa and HeLa cells were incubated with different concentrations of ethanol and hot water extracts, the ethanol extracts showed strong cytotoxicity against A549 cells at concentrations over 10mg/mL and against SiHa and HeLa cells at over 40mg/mL (Choi et al., 2004a). Ganoderma lucidum (Lingzhi) polysaccharides have also been shown to have an inhibitory effect on cervical cancer cells (CA Ski and HeLa cells) (Chen et al., 2010d).

An anti-proliferative effect of clitocine from the mushroom Leucopaxillus giganteus on human cervical cancer HeLa cells has also been reported. The anti-proliferative effect was via an induction of apoptosis (Ren et al., 2008a). An anti-proliferative effect of ethanol and water extracts of Pleurotus tuberregium against HeLa cervical cancer cells has also recently been reported (Maness et al., 2011).

Colon / Colorectal Cancer & Mushrooms

Animal model (mouse) studies

Treatment of mice with Pleurotus ostreatus at 100 and 500 mg/kg has suggested that P. ostreatus may prevent inflammation-associated colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by dextran sodium sulfate (DSS), via combined modulatory mechanisms of inflammation and tumor growth via suppression of COX-2, F4/80, Ki-67 and cyclin D1 expression in mice. However, incidence of colon tumors and high grade dysplasia was reduced by 50 and 63% only in the 500 mg/kg dose (Jedinak et al., 2010). A further study by the same group also showed that the anti-inflammatory activity of Pleurotus ostreatus is mediated through the inhibition of NF-

kappaB and AP-1 signaling (Jedinak et al., 2011a). Induction of natural killer (NK) activity, activation of macrophages, and inhibition of angiogenesis have all been reported to be involved in the mechanisms affecting colon cancer cells and reduction of tumor size in mice (Kim et al., 2011b).

Dietary administration of the fruiting body extract or mycelia extract of the edible mushroom Pleurotus pulmonarius to mice reduced the formation of aberrant crypt foci, which precedes colorectal cancer, and of microadenomas. The treatments significantly lowered the expression of proliferating cell nuclear antigen and increased the number of cells undergoing apoptosis in the colon as well as inhibiting the expression of the proinflammatory cytokine TNF-alpha in colonic tissue. The extracts inhibited colitis-associated colon carcinogenesis induced in mice through the modulation of cell proliferation, induction of apoptosis, and inhibition of inflammation (Lavi et al., 2011).

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 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 ofprecancerous 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 1,2-dimethylhydrazine-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).

In vitro studies (human cell lines)

Ganodermanontriol, a lanostanoid triterpene from Ganoderma lucidum has been shown to be able to modulate of the beta-catenin pathway, which is involved in the progression of colorectal cancer. Ganoderma lucidum inhibited proliferation of HCT-116 and HT-29 colon cancer cells without a significant effect on cell viability, while ganodermanontriol inhibited transcriptional activity of beta-catenin and protein expression of its target gene cyclin D1 in a dose-dependent manner. A marked inhibitory effect was also seen on Cdk-4 and PCNA expression, whereas expression of Cdk-2, p21 and cyclin E were not affected. Ganodermanontriol also caused a dose-dependent increase in protein expression of E-cadherin and beta-catenin in HT-29 cells, and suppressed tumor growth in a xenograft model of human colon adenocarcinoma cells HT-29 implanted in nude mice without any side-effects and inhibited expression of cyclin D1 in tumors (Jedinak et al., 2011b). In HT-29 human colon cancer cells, triterpenes from Ganoderma lucidum have also been shown to induce programmed cell death (Type II-autophagy) via a mechanism involving inhibition of p38 mitogen-activated kinase (p38 F) (Thyagarajan et al., 2010). A polysaccharide extract of Ganoderma lucidum has also 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).

Ganoderic acid T (GA-T) of Ganoderma lucidum inhibited proliferation of HCT-116 cells, a human colon carcinoma cell line. Cell aggregation and adhesion assays showed that GA-T promoted homotypic aggregation and simultaneously inhibited the adhesion of HCT-116 cells to the extracellular matrix (ECM) in a dose-dependent manner (Chen et al., 2010c). Wound healing assays indicated that GA-T also inhibited the migration of HCT-116 cells in a dose-dependent manner, and suppressed the migration of 95-D cells, highly metastatic human lung tumor cell line, in a dose- and time-dependent manner. In addition, GA-T inhibited the nuclear translocation of nuclear factor-kappa B (NF-kappa B) and the degradation of inhibitor of kappa B-alpha (I kappa B alpha), which leads to down-regulated expression of matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Animal and Lewis Lung Carcinoma (LLC) model experiments demonstrated that GA-T suppressed tumor growth and LLC metastasis and down-regulates MMP-2 and MMP-9 mRNA expression in vivo. These results indicate that GA-T effectively inhibits cancer cell invasion in vitro and metastasis in vivo (Chen et al., 2010c). An anti-proliferative effect of ethanol and water extracts of Pleurotus tuberregium against HCT-116 colon cancer cells has also recently been reported (Maness et al., 2011).

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 a 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).

The effects of an extract of Agaricus blazei Murrill (ABM) has been evaluated on HT-29 human colon cancer cells in severe combined immunodeficiency (SCID) mice. Oral administration of ABM (up to 45 mg ABM daily for 6 weeks) did not prevent tumor growth, but compared with the control group (0 mg ABM), the tumor mass appeared to grow more slowly following ABM doses of 4.5 and 45 mg) (Wu et al., 2011b).

Phellinus linteus has been reported to inhibit tumor growth, invasion, and angiogenesisvia the inhibition of Wnt/beta-catenin signaling in SW480 human colon cancer cells (Song et al., 2011).

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).

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).

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 a 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 in this 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 inthe 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).

Gastric Cancer & Mushrooms

Animal model studies

The effects of natural polysaccharides isolated from Phellinus gilvus (PG) in vitro and in vivo againstgastric cancer have been evaluated. PG decreased cell proliferation and increased cell apoptosis in a dose-dependent manner in vitro and also led to a marked inhibition of tumour growth and significant decrease in the incidence of peritoneal carcinomatosis. Histological analysis of the tumour

confirmed a significant increase in tumour cell apoptosis by PG, indicating reduced tumour cell proliferation. The data showed that polysaccharides isolated from PG significantly inhibited tumour growth and metastasis in an orthotopic model of human gastric adenocarcinoma, without detectable adverse effects (Bae et al., 2006).

Toth and co-workers have reported the inhibition of intestinal cancer by a hot water extract of the Coriolus versicolor (Turkey Tail) mushroom in C57bl/6j-Apc(Min) mice (Toth et al., 2007).

In vitro studies (human cell lines)

The induction of apoptosis by extracts of Ganoderma lucidum has previously been reported, however, more recent data have proposed that the mechanisms involved (at least in human gastric carcinoma cells) involve caspase pathways which are associated with inactivation of the Akt signalling pathway (Jang et al., 2010b).

Recombinant Lz-8 (rLz-8), a protein from Ganoderma lucidum induced endoplasmic reticulum stress-mediated autophagic cell death in the human gastric cancer cell line SGC-7901, but caspase inhibitors did not prevent rLz-8-induced cell death, and therefore the autophagic response induced by rLz-8 is independent of caspase activation (Liang et al., 2012).

Induction of apoptosis in human gastric epithelial AGS cancer cells by an aqueous extract of Agaricus blazei has been demonstrated. It was found that an Agaricus blazei extract could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death via caspase-3 activation (Jin et al., 2006).

An exopolysaccharide produced from the medicinal mushroom Fomes fomentarius, has been reported to have a direct anti-proliferative effect in vitro on SGC-7901 human gastric cancer cells in a dose- and time-dependent manner (Chen et al., 2008).

A water-soluble extract of Grifola frondosa has been shown to inhibit the proliferation of four human gastric cancer cell lines (TMK-1, MKN28, MKN45 and MKN74) in a time-dependent manner. The inhibition was most pronounced in TMK-1 cells, which exhibited up to 90% inhibition after treatment with 10% extract for 3 days. Induction of apoptosis was confirmed by fluorescence-activated cell sorting analyses, while Western blot analyses of TMK-1 cells after treatment with the extract revealed increases in intracytoplasmic cytochrome c and cleavage of caspase-3 and poly(ADP-ribose) polymerase, but no expression of p21 or Bax. The caspase-3 protease activities in lysates of TMK-1 cells treated with 1% or 10% of the extract were approximately 3-fold higher than in control cells. The data suggest that this extract from Grifola frondosa produces potential antitumour effects on gastric cancer via an induction of apoptosis of TMK-1 cells by caspase-3-dependent and -independent pathways (Shomori et al., 2009).

Leukemia & Mushrooms

Animal model studies

The effects of a beta-glucan supplement (Lentinan) from Lentula edodes (Shiitake) on BN rats have been studied and in a preclinical model of acute myeloid leukemia. BN rats supplemented daily with lentinan exhibited weight gains, increased white blood cells, monocytes and circulating cytotoxic T-cells, and had reduction in anti-inflammatory cytokines IL-4, IL-10, and IL-6. A combination of lentinan with standards of care in acute myeloid leukemia, idarubicin, and

cytarabine increased average survival compared with monotherapy and reduced cachexia suggesting that nutritional supplementation of cancer patients with lentinan would warrant investigation (McCormack et al., 2010).

In vitro studies (human cell lines)

A study demonstrated that agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells in vitro (Endo et al., 2010b). Agaritine inhibited the proliferation of leukemic cell lines U937, MOLT4, HL60 and K562, but showed no significant effect on normal lymphatic cells. The data also showed that this activity was distinct from that of beta-glucan, which indirectly suppresses proliferation of tumor cells. This conclusion of direct anti-tumor activity by agaritine against leukemic tumor cells in vitro contrasts to the carcinogenic activity previously ascribed to it in animal studiescarried out around 20-30 years ago. A more recent study by the same group has now shown that the mechanism by which agaritine may act in U937 leukemic cells is via the moderate induction of apoptosis via caspase activation through cytochrome C release from mitochondria (Akiyama et al., 2011). Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are also produced by active fractions from Ganoderma lucidum on human leukemia cells (Calvino et al., 2010). Hericium erinaceus (Yamabushitake) mushroom-induced apoptosis of U937 human monocytic leukemia cell has been reported to be via an effect on cell proliferation that involves activation of mitochondria-mediated caspase-3 and caspase-9 but not caspase-8 (Sung Phil et al., 2011). Extracts from Agaricus bisporus and Phellinus linteus have also been shown to induce proapoptotic effects in the human leukemia cell line K562 (Shnyreva et al., 2010).

Extracts from Agaricus bisporus (Jagadish et al., 2009), Agaricus blazei (Gao et al., 2007), Hypsizigus marmoreus (Mizumoto et al., 2008) and other mushrooms have been shown toinhibit cell proliferation of HL-60 leukemia cells and other leukemia human cell lines via the induction of apoptosis. Mechanisms by which apoptosis is induced include down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene (Gao et al., 2007), regulation of Bcl-2 and caspase-3 (Jin et al., 2007), cleavage of poly (ADP-ribose) polymerase and pro-caspase 3 (Bae et al., 2009), mitochondrial membrane potential loss and caspase activation (Mizumoto et al., 2008), release of mitochondrial cytochrome c and subsequent activation of caspase-9 and caspase-3 (Hsu et al., 2008b) and via the signal transduction kinases Akt and Erk (Calvino et al., 2010). These extracts appear to exert tumor-selective cytotoxicity, with studies reporting no significant cytotoxic effects on normal cell lines (Lau et al., 2004).

A nonlectin glycoprotein (PCP-3A) isolated from the fruiting body of the edible golden oyster mushroom Pleurotus citrinopileatus has been shown to stimulate human mononuclear cells to secrete cytokines TNF-alpha, IL-2, and IFN-gamma, which subsequently inhibited the growth of U937 human myeloid leukemic cells (Chen et al., 2010a).

Kinase inhibitors have been used for the treatment of chronic myeloid leukemia (CML) in humans. Despite high rates of clinical response, CML patients can develop resistance to these kinase inhibitors mainly due to point mutations within the Abl kinase domain of the fusion protein. A crude extract of the mushroom Daedalea gibbosa has been reported to inhibit kinase activity of Bcr-Abl kinase, and the active component has been identified as oleic acid (Khamaisie et al., 2011).

It is interesting to note that aqueous and aqueous/ethanolic extracts of Hericium erinaceus (Yamabushitake) mushroom were able to induce apoptosis in U937 humanmonocytic leukemia cells, however, acidic and alkaline extracts with similar proximate compositions were both inactive (Kim et al., 2011a).

Liver Cancer & Mushrooms

Animal model studies

In vivo rodent studies have reported hepato-protective effects on both chemically-induced liver toxicity and hepato-carcinogenesis by extracts from Agaricus blazei (Barbisan et al., 2002, Pinheiro etal., 2003) and Pleurotus pulmonarius (Wasonga et al., 2008).

In vitro studies (human cell lines)

Lucidenic acids (triterpenoids) isolated from Ganoderma lucidum (Weng et al., 2007), hyperbranched β-glucan, extracted from Pleurotus tuberregium (Tao et al., 2006) and extracts from Cordyceps sinensis (Wu et al., 2007b) and Chaga (Inonotus obliquus) mushrooms (Youn et al., 2008) have been shown to inhibit the proliferation of HepG2 human hepatocellular carcinomas. As reported above for human leukemia cell lines, such extracts appear to be have tumor-selective cytotoxicity, without significant effects on normal human liver cell lines (Lin et al., 2003).

Hispolon, an active phenolic compound of Phellinus igniarius, induces apoptosis and cell cycle arrest of human hepatocellular carcinoma Hep3B cells by modulating ERK phosphorylation. Hispolon inhibited cellular growth of Hep3B cells in a time-dependent and dose-dependent manner, through the induction of cell cycle arrest at S phase measured using flow cytometric analysis and apoptotic cell death. Hispolon-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A and E and cyclin-dependent kinase (CDK) 2, with concomitant induction of p21waf1/Cip1 and p27Kip1. Exposure of Hep3B cells to hispolon resulted in apoptosis as shown by caspase activation, PARP cleavage, and DNA fragmentation. Hispolon treatment also activated JNK, p38 MAPK, and ERK expression. Inhibitors of ERK (PB98095), but not those of JNK (SP600125) and p38 MAPK (SB203580), suppressed hispolon-induced S-phase arrest and apoptosis in Hep3B cells. These findings establish a mechanistic link between the MAPK pathway and hispolon-induced cell cycle arrest and apoptosis in Hep3B cells (Guan-Jhong et al., 2011, Huang et al., 2011). Hispolon has also been shown to suppress SK-Hep1 human hepatoma cell metastasis by inhibiting matrix metalloproteinase-2/9 and urokinase-plasminogen activator through the PI3K/Akt and ERK signaling pathways (Huang et al., 2010b).

A lectin isolated from Agrocybe aegerita has been shown to bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro, and to exert an anti-hepatoma effect in vivo via inhibition of tumor growth and extending the survival time of tumor bearing mice (Jiang et al., 2012). Mechanistic studies of apoptosis in human hepatocellular carcinoma cells have shown that Agaricus blazei Murill is able to act as an enhancer to sensitize doxorubicin (Dox)-mediated apoptotic signaling, and this sensitization can be achieved by enhancing intracellular Dox accumulation via the inhibition of NFkappaB activity. These findings suggest that Agaricus blazei Murill, when combined with low doses of Dox, may have the potential to provide more efficient therapeutic effects against drug-resistant human hepatocellular carcinoma (Lee and Hong, 2011).

It has recently been demonstrated that polysaccharides from Phellinus linteus (PL) inhibit proliferation and colony formation of HepG2 and that the growth inhibition of HepG2 cells was mediated by S-phase cell cycle arrest. Phellinus linteus also markedly inhibited cancer cell adhesion and invasion of the extracellular matrix and PL-induced apoptosis was associated with a reduction in B-cell lymphoma 2 levels and an increase in the release of cytochrome c. The results suggest that PL exerts a direct antitumor effect by initiating apoptosis and cell cycle blockade in HepG2 cells (Wang et al., 2012a).

Lung Cancer & Mushrooms

Animal model studies

An in vivo study in mice with Lewis lung carcinoma treated with an aqueous extract of Hypsizigus marmoreus showed a significant increase in life span when given it by intraperitoneal administration, but not as much by oral administration. The extract inhibited spontaneous tumor metastasis in mice bearing the carcinoma and significantly decreased the number of metastasized nodules (Saitoh et al., 1997).

In vitro studies (human cell lines)

In vitro studies have shown that three triterpene aldehydes, lucialdehydes A – C, from thefruiting bodies of Ganoderma lucidum, possess cytotoxicity against murine and human tumor cells (Lewis lung carcinoma (LLC), T-47D, Sarcoma 180, and Meth-A tumor cell lines) (Gao et al., 2002), while Phellinus linteus has been shown to mediate cell-cycle arrest at a low concentration and apoptosis in response to a high dose in mouse and human lung cancer cells (Guo et al., 2007). Blazein, a steroid isolated from Agaricus blazei Murrill (Himematsutake), has also been reported to induce cell death and morphological change indicative of apoptotic chromatin condensation in human lung cancer LU99 and stomach cancer KATO III cells (Itoh et al., 2008), and an extract from Pleurotus ferulae has been reported to have cytotoxic effects on human lung cancer and cervical cancer cell lines (A549, SiHa and HeLa cells) (Choi et al., 2004a).

Recombinant Ling Zhi-8 (rLZ-8), an immunomodulatory protein cloned from Ganoderma lucidum (Reishi or Ling Zhi) inhibits the proliferation of A549 human lung cancer cells.The antitumor effect was via a modulation of p53 via ribosomal stress. rLZ-8 inhibited lung cancer cell growth in vitro and also in vivo, in mice transplanted with Lewis lung carcinoma cells (Wu et al., 2011a).

A recombinant fungal immunomodulatory protein (termed GMI), has been cloned from Ganoderma microsporum and purified. GMI exhibited an inhibitory effect on epidermalgrowth factor-induced migration and invasion in A549 lung cancer cells. GMI inhibited EGF-induced phosphorylation and activation of EGFR and AKT pathway kinases in a dose-dependent manner, and EGF-induced activation of Cdc42 GTPase was inhibited by GMI, while GMI had little effect on the EGF-induced activation of Rac1 GTPase. GMI also inhibited the EGF-induced microfilament depolymerisation (Lin et al., 2010a).

Oral Cancer & Mushrooms

In vitro studies (human cell lines)

Ethanol extracts (0.9 mg/ml) and hot water extracts (0.7 mg/ml) of Agaricus brasiliensis Mural (ABM) caused morphological changes and significantly reduced viability of human oral cancer CAL 27 Cells after 48 h of treatment. Both extracts were able to induce apoptotic cell death in CAL 27 cells via the release of cytochrome c from mitochondria into the cytoplasm and activation of caspase-3 in vitro (Fan et al., 2011).

The responses of human oral squamous cell carcinoma (OSCC) cells to Lentinan, beta-(1 -> 3)-D-glucan, an extract from Lentinus edodes (0.1 mg/kg/day, 2 times/week), alone and in combination with S-1, an oral antineoplastic agent that can induce apoptosis in various types of cancer cells (6.9 mg/kg/day, 7 times/week)has been studied using a nude mouse xenograft model. Combined therapy of Lentinan and S-1 markedly exerted antitumor effects on human OSCC xenografts and significantly induced apoptotic cells in tumors treated with Lentinan plus S-1, with no loss of body weight being observed in mice treated with the combined therapy (Harada et al., 2010).

Ovarian Cancer & Mushrooms

In vitro studies (human cell lines)

Ganoderma lucidum has been shown to significantly decrease ovarian cancer cellnumbers in vitro in a dose-dependent manner. Ganoderma lucidum also inhibited colony formation, cell migration and spheroid formation. Ganoderma lucidum also induced cell cycle arrest at the G2/M phase, induced apoptosis by activating caspase 3, increased p53 but inhibited Akt expression (Zhao et al., 2011).

Prostate Cancer & Mushrooms

Animal model (mouse) studies

Polysaccharide-K (PSK), an extract of the mushroom Trametes versicolor, has been shown to enhancedocetaxel-induced prostate cancer tumor suppression, apoptosis and antitumor responses in transgenic adenocarcinomas of mouse prostate (TRAMP)-C2-bearing mice. Combining PSK with docetaxel significantly induced higher tumor suppression than either treatment alone, including a reduction in tumor proliferation and enhanced apoptosis. Combined PSK and docetaxel treatment led toa lower decrease in number of white blood cells than docetaxel alone, an effect accompanied by increased numbers of tumor-infiltrating CD4+ and CD8+ T cells. PSK with or without docetaxel significantly enhanced mRNA expression of IFN-gamma compared to control, but did not significantly alter T-regulatory FoxP3 mRNA expression in tumors (Wenner et al., 2012).

A Phellinus linteus extract has recently been reported to sensitize advanced prostate cancer cells to apoptosis in athymic nude mice (Tsuji et al., 2010). In this study, a xenograft assay, together with in vitro assays, were undertaken to evaluate the effect of Phellinus linteus on the genesis and progression of the tumours formed from theinoculation of prostate cancer PC3 or DU145 cells. Although Phellinus linteus treatment did not prevent the formation of the inoculated tumours, the growth rate of the tumors after Phellinus linteus treatment was significantly attenuated. Apoptosis occurred with the activation of caspase 3 in the tumours formed by inoculating prostate cancer DU145 or PC3 cells. The data were in agreement with data from cultured cells. The in vivo study suggested that Phellinus linteus attenuated tumour growth and caused tumour regression by inducing apoptosis.

Animal model (rat) studies

The inhibitory effects of methanol extracts of 19 edible and medicinal mushrooms on 5-alpha-reductase activity have been reported, with an extract of Ganoderma lucidum Fr. Krast (Ganodermataceae) showing the strongest 5-alpha-reductase inhibitory activity.

The treatment of the fruit body of Ganoderma lucidum, or the extract prepared from it, significantly inhibited the testosterone-induced growth of the ventral prostate in castrated rats. The results showed that Ganoderma lucidum might be a useful ingredient for the treatment of benign prostatic hyperplasia (Fujita et al., 2005).

Prostate weight increased significantly (37%) following treatment with a Phellinus linteus extract, and in particular, the stromal component of the prostate increased significantly by 80%. A suppression of transforming growth factor-beta1 expression by 56% was observed with the mushroom extract treatment. It was found that this mushroom extract enlarged the prostate and therefore administration of Phellinus linteus extract should be considered carefully by those with an enlarged prostate (Shibata et al., 2005).

Agaricus bisporus mushroom extract and one of its major components, conjugatedlinoleic acid (CLA) have been shown to decrease DU145 and PC3 prostate tumour size and tumour cell proliferation in nude mice treated with the mushroom extract, whereas tumour cell apoptosis was increased compared to pair-fed controls. Gene network analysis identified alterations in networks involved in cell death, growth and proliferation, lipid metabolism, the TCA cycle and immune response (Adams et al., 2008).

In vitro studies (human cell lines)

Altered androgen receptor (AR) activity caused by point mutations or signalling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage of prostate cancer. It has been demonstrated that a hexane extract prepared from Coprinus comatus ( C. comatus) strain 734 was able to reduce AR levels and prostate-specific antigen gene expression in the LNCaP-treated cell line (Dotan et al., 2011a). A further study from the same group isolated 2 fractions from the same mushroom strain that inhibited AR-mediated reporter activity and reduced the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. One of these fractions (F-32) also inhibited the proliferation and clonigenicity of LNCaP cells and inhibited the binding of AR to the PSA enhancer region and inhibited Akt-mediated AR phosphorylation at Ser 213 (Dotan et al., 2011b). The pharmaceutical value of the Basidiomycota fungi Coprinus comatus has also been reviewed by the same group (Dotan et al., 2010).

PSP, an active component extracted from Coriolus versicolor has been reported to be effective in targeting prostate cancer stem/progenitor cells (CSCs). Treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. PSP treatment also inhibited PC-3 cell tumorigenicity in vivo, indicating that PSP can suppress prostate CSC properties. Transgenic mice (TgMAP) that spontaneously develop prostate tumors, that were orally fed with PSP for 20 weeks did not develop tumours, while 100% of the mice that were fed with water only developed prostate tumors at the end of the 20 weeks, suggesting that PSP treatment can inhibit prostate tumor formation in these mice (Luk et al., 2011).

The effects of Agaricus blazei Murill on the growth of human prostate cancer have been examined in vitro and in vivo. A. blazei, particularly in a broth fraction, inhibited cell proliferation in both androgen-dependent and androgen-independent prostate cancer cell lines. The broth of A. blazei induced lactate dehydrogenase leakage in three cancer cell lines, whereas the activities of caspase 3 and the DNA fragmentation were enhanced the most in androgen-independent PC3 cells. Oral supplementation with the broth of A. blazei (with the higher ratio of beta-glucan) significantly suppressed tumour growth without inducing adverse effects in severe combined immunodeficient mice with PC3 tumor xenograft. The data suggested that the broth of A. blazei may directly inhibit the growth of prostate cancer cell via an apoptotic pathway and suppress prostate tumor growth via antiproliferative and antiangiogenic mechanisms (Yu et al., 2009a).

Beta-glucan, a polysaccharide, of the Grifola frondosa (Maitake) mushroom, has a cytotoxic effect, presumably through oxidative stress, on human androgen-independent prostatic cancer PC-3 cells in vitro, leading to apoptosis (Fullerton et al., 2000). Another study in the same cell line showed similar cytotoxic effects from a water-soluble extract from Pleurotus ostreatus (Oyster), although the active components were not identified (Gu and Leonard, 2006).

Ganoderma lucidum has also been shown to inhibit proliferation in a dose- and time-dependent manner and induce apoptosis in human prostate cancer cells PC-3 (Jiang et al., 2004b). However, the molecular mechanisms responsible for the inhibitory effects of G. lucidum on these prostate cancer cells are still unclear. It has been found that G. lucidum inhibits the early event in angiogenesis, capillary morphogenesis of the humanaortic endothelial cells. These effects are caused by the inhibition of constitutively active AP-1 in prostate cancer cells, resulting in the down-regulation of secretion of Vascular Endothelial Growth Factor and Transforming Growth Factor beta (TGF-beta1) from PC-3 cells. The data suggest that G. lucidum inhibits prostate cancer-dependent angiogenesis by modulating MAPK (mitogen activated protein kinase) and Akt signaling and could have potential therapeutic use for the treatment of prostate cancer (Stanley et al., 2005). Interferon (IFN)-alpha2b has also been shown to have synergistic effects with mushroom extracts in inducing significant reductions reduction in PC-3 cell growth. This appears to be due to a synergistic potentiation leading to a G1 cell cycle arrest (Pyo et al., 2008). Polysaccharides and ethanol extracts of the fruiting bodies of Tremella aurantia mushrooms have also been shown to possess growth-inhibitory activity in LNCaP and PC-3 human prostate cancer cells (Kiho et al., 2010).

Phellinus linteus has been reported to sensitise apoptosis induced by doxorubicin (ananti-cancer drug) in prostate cancer LNCaP cells suggesting that Phellinus linteus may have therapeutic potential to augment the magnitude of apoptosis induced by anti-prostate cancer drugs (Collins et al., 2006). Putrescine-1,4-dicinnamide from the gilled mushroom Pholiota spumosa (Basidiomycetes) has also been reported to inhibit cell growth of an androgen-independent human prostate cancer (PCA) cell line by inducing apoptosis, mediated, at least in part, by the activation of caspase cascades (Russo et al., 2007).

Ergosterol peroxide derived from edible mushrooms has been shown to exert anti-tumor activity in several cancer cells. For example, ergosterol peroxide has been shown to attenuate the growth of prostate cells, at least in part, triggering an apoptotic process in androgen-insensitive (DU-145) human prostate cancer cells (Russo et al., 2010). In a recent study in human multiple myeloma U266 cells, ergosterol peroxide exerted antitumor activity in multiple myeloma U266 cells partly with anti-angiogenic activity targeting the JAK2/STAT3 signalling pathway (Rhee et al., 2012).

Skin Cancer & Mushrooms

Animal model (mouse) studies

Proflamin, isolated from the culture mycelium of Flammulina velutipes (Curt. ex Fr.) Sing. is a weakly acidic glycoproteincontaining more than 90% protein and less than 10% carbohydrate, and has a molecular weight of ~13,000 Da. Proflamin has been shown to be markedly effective against the syngeneic tumours, B-16 melanoma (B-16) and adenocarcinoma 755 (Ca-755) in the mouse. The increases in median survival time of treated mice with B-16 and Ca-755 were 86% and 84%, respectively. Proflamin exhibited no cytocidal effect against the cultured cell lines in vitro. Oral administration of proflaminproduced no lethal or any other apparent adverse effect in mice (Ikekawa et al., 1985).

An acidic polysaccharide from Phellinus linteus has been shown to markedly inhibit melanoma cell metastasis in mice, and directly inhibit cancer cell adhesion to, and invasion through, the extracellular matrix, but it had no direct effect on cancer cell growth. In addition, the authors reported that PL increased macrophage NO production. These results suggest that Phellinus linteus has two anti-metastatic functions – it acts as an immuno-potentiator and as a direct inhibitor of cancer cell adhesion (Han et al., 2006).

An extract of Ganoderma lucidum has also been reported to reduce chemically-induced mammary adenocarcinomas in Sprague Dawley rats and skin tumours Balb/c mice (Lakshmi et al., 2009).

Mushroom oligosaccharides that down-regulate production of immuno-suppressive cytokines by ultraviolet radiation injured keratinocytes appear to be promising agents for the prevention of environmental (sun induced) skin cancer (Pelley and Strickland, 2000).

In vitro studies (animal cell lines)

Lentinula edodes has been shown to reduce cell proliferation and induce apoptosis in CH72mouse skin carcinoma cells via an induction of a transient G1 arrest with no effect in non-tumorigenic (C50) cells (Gu and Belury, 2005). Similarly, reduction of cell proliferation of B-16 melanoma cells by arrest in the G0/G1 phase of the cell cycle, followed by both apoptotic and secondary necrotic cell death has been demonstrated for a methanol extract of Coriolus versicolor (Harhaji et al., 2008). In contrast, proflamin, isolated from Flammulina velutipes, exhibited no cytotoxic effects against B-16 melanoma (B-16) and adenocarcinoma 755 (Ca-cultured cell lines in vitro, but increased the median survival time of mice treated with B-16 and Ca-755 by 86% and 84%, respectively, with no apparent adverse effects (Ikekawa et al., 1985).

An acidic polysaccharide from Phellinus linteus has been shown to markedly inhibit melanoma cell metastasis in mice, and directly inhibit cancer cell adhesion to, and invasion through, the extracellular matrix, with an increase in macrophage NO production but to have no direct effect on cancer cell growth. These results suggest that Phellinus linteus has two anti-metastatic functions—it acts as an immunopotentiator and as a direct inhibitor of cancer cell adhesion (Han et al., 2006).

DNA Damage

Animal model (mouse) studies

It has been suggested that synthetic agaritine (i.e. not extracted from mushrooms) is quickly metabolized in mice and disappears in the plasma, whereas DNA damage after a single administration of synthetic agaritine lasts for a longer time (Kondo et al., 2008). The inference for DNA damage in this study was from a result of a separate in vitro test. While data with a particular marker of oxidative stress showed this effect, a similar experiment with a different marker of oxidative stress did not, thus the authors made these comments based on their results with one particular marker only.

In contrast, recent research (Endo et al., 2010b) has ascribed anti-tumor activity of agaritine (from mushrooms) against leukemic cells, and a recent review (Roupas et al., 2010a) also concluded that agaritine from consumption of cultivated Agaricus bisporus mushrooms poses no known toxicological risk to healthy humans. Another in vivo study demonstrated that crude extracts of Agaricus blazei Murrill significantly reduced DNA damage in liver induced by diethylnitrosamine in adult male Wistar rats (Barbisan et al., 2003), while DNA strand breaking by the carbon-centered radical generated from 4-(hydroxymethyl) benzenediazonium salt from Agaricus bisporus has been reported in the mouse (Hiramoto et al., 1995).

A polysaccharide protein complex (PPC-Pr) isolated from the mushroom Phellinus rimosus has been shown to have a protective effect (at doses of 5 and 10 mg/kg bodyweight intraperitoneally for 5 days consecutively) against oxidative stress induced by gamma radiation (4 Gy) in Swiss albino mice. PPC-Pr treatment enhanced the declined levels of antioxidants and demonstrated a DNA protective effect (as determined by a Comet assay) as well as significantly increasing the survival rate of animals (Joseph et al., 2012). An earlier study from the same group at the same doses of PPC-Pr had reported its effect on alleviating gamma radiation-induced toxicity in the Swiss albino mouse model (Joseph et al., 2011).

In vitro studies

In vitro studies have shown that a heat-labile protein from Agaricus bisporus protectsRaji cells (a human lymphoma cell line) against H2O2-induced oxidative damage to cellular DNA (Shi et al., 2002). Similar protective effects against H2O2-induced oxidative damage to cellular DNA have been demonstrated with cold (20ºC) and hot (100ºC) water extracts of Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. No protective effects were observed with mushroom derived preparations from Flammulina velutipes, Auricularia auricula, Hypsizygus marmoreus, Lentinula edodes, Pleurotus sajor-caju, or Volvariella volvacea (Rocha et al., 2002). Similar reductions in DNAfragmentation (Comet assay), compared with H2O2 as a positive control, have been reported from Chaga mushroom (Inonotus obliquus)(Park et al., 2004), while an aqueous extract from Agrocybe cylindracea strain B has also been shown to protect against DNA damage in HepG2 cells (Wang et al., 2004). Some edible mushrooms therefore represent a valuable source of biologically active compounds with potential for protecting cellular DNA from oxidative damage, while other mushroom varieties do not.

Beta-glucan from Agaricus brasiliensis has been reported to be devoid of mutagenic activity and to provide a significant dose-dependent protective effect against DNA damage in the dose range 20-80 mg/ml (Angeli et al., 2006). Furthermore, a possible chemoprotective effect of β-glucan extracted from Agaricus blazei against DNA damage induced by benzo[a]pyrene, using the comet assay (genotoxicity) and micronucleus assay with cytokinesis block (mutagenicity) in a human hepatoma cell line (HepG2) has suggested that b-glucan did not exert a genotoxic or mutagenic effect, but that it did protect against DNA damage via binding to benzo[a]pyrene or by the capture of free radicals produced during its activation (Angeli et al., 2009).

Strong DNA protective effects from oxidative damage have been reported for protein extracts from selenium-enriched Ganoderma lucidum (Se-GLPr), and this effect increased with increasing Se content (Zhao et al., 2004). Polysaccharide extracts from Se-enriched G. lucidum have also been shown to protect DNA from hydroxyl radical oxidative damage in a dose dependent manner (Zhao et al., 2008). A water-soluble polysaccharide from Ganoderma lucidum was protective against hydroxyl radical-induced DNA strand breaks (Kim and Kim, 1999), and radioprotective properties of an aqueous extract of Ganoderma lucidum against radiation-induced plasmid pBR322 DNA strand breaks have been demonstrated that may be due to inhibition of lipid peroxidation (Pillai et al., 2006).

Oral administration of Ganoderma lucidum extract (GLE) to tumor-bearing Swiss albino mice along with exposure to gamma radiation has been shown to result in tumour regression. Single-cell gel electrophoresis (comet assay) on cells of normal and tumour tissues from tumour-bearing animals treated with GLE and radiation, revealed that there was significant reduction in radiation-induced damage to cellular DNA in normal tissues compared to the tumour, indicating preferential protection to normal tissues and possible use as an adjuvant in radiotherapy, for tumour regression and prevention of radiation-induced cellular damage in normal tissues (Gopakumar et al., 2010).

Supplementation with Agaricus blazei, carried out under pre-treatment, simultaneous treatment, post-treatment and pre-treatment +continuous conditions, has shown that A. blazei did not have genotoxic activity but showed antigenotoxic activity (Comet assay).Supplementation caused an increase in the number of monocytes and in phagocytic activity, suggesting that supplementation increases a proliferation of monocytes, consequently increasing phagocytic capacity especially in the groups pre-treatment, simultaneous and pre-treatment+continuous. The data suggest that A. blazei could promote immunomodulation which can account for the destruction of cells with DNA alterations that correlate with the development of cancer, since this mushroom was demonstrated to have a preventive effect against pre-neoplastic colorectal lesions evaluated by the aberrant crypt foci assay (Ishii et al., 2011).

Polysaccharides isolated from Ganoderma lucidum have been reported to enhance the repair of radiation induced DNA strand breaks in human cells after 120min of exposure (Pillai et al., 2010).