The Cancer Preventive Effects of Edible Mushrooms

Abstract

An increasing body of scientific literature suggests that dietary components may exert cancer preventive effects. Tea, soy, cruciferous vegetables and other foods have been investigated for their cancer preventive potential. Some non-edible mushrooms like Reishi (Ganoderma lucidum) have a history use, both alone and in conjunction with standard therapies, for the treatment of various diseases including cancer in some cultures. They have shown efficacy in a number of scientific studies. By comparison, the potential cancer preventive effects of edible mushrooms have been less well-studied. With similar content of putative effective anticancer compounds such as polysaccharides, proteoglycans, steroids, etc., one might predict that edible mushrooms would also demonstrate anticancer and cancer preventive activity. In this review, available data for five commonly-consumed edible mushrooms: button mushrooms (Agaricus bisporus), A. blazei, oyster mushrooms (Pleurotus ostreatus), shiitake mushrooms (Lentinus edodes), and maitake (Grifola frondosa) mushrooms is discussed. The results of animal model and human intervention studies, as well as supporting in vitro mechanistic studies are critically evaluated. Weaknesses in the current data and topics for future work are highlighted.

INTRODUCTION

Mushrooms have been consumed as part of the diet in some countries for thousands of years. In the United States, mushroom consumption is increasing. The National Agricultural Statistics Service (NASS) estimated that the 2010–2011 US mushroom crop was 862 million pounds and had a value of USD 1 billion, an increase of 8% from the previous year [1]. Agaricus bisporus mushrooms including white and brown button and portabella mushrooms accounted for approximately 96% of the total mushroom sales value. In the US, sales of brown button mushrooms and some specialty mushrooms such as oyster mushrooms (Pleurotus ostreatus) increased significantly in 2009–2010 season. Mushrooms are considered a healthy food due to their high (>20%) protein and low (<3%) fat content (72–85% polyunsaturated fatty acids) as well as their content of essential amino acids (lysine and leucine) and vitamins such as thiamine, riboflavin, niacin, biotin and ascorbic acid [2].

In some cultures, mushrooms have been used as traditional medicines to treat diseases including diabetes and cancer, and to stimulate the immune system. Epidemiological data to support this concept, however, is mixed. Three studies conducted in Asia (one in China; two in Korea) showed an inverse relationship between mushroom consumption and breast cancer incidence in women [3–5]. By contrast, two prospective studies in Europe found no inverse association between vegetable consumption (including mushrooms) and ovarian and urothelial cancer risk [6, 7]. The anticancer activity of edible and specialty/medicinal mushrooms has been studied in clinical trials, animal studies, and in vitro cell line studies, with some of the earliest scientific research performed on Boletus edulis in the 1950’s [8].

In this review, we intend to discuss the results of research on the anticancer effects of edible mushrooms over the past two decades, emphasizing clinical trials and animal studies as well as supporting mechanistic studies. Our goal is to critique the literature, point out pressing questions and stimulate further work in the field.

CANCER PREVENTIVE EFFECTS OF EDIBLE MUSHROOMS

Agaricus bisporus

A. bisporus includes three commonly consumed mushrooms: white button, brown button, and portobello mushrooms. These mushrooms have a convex cap with free hymenium and the stipe has a ring. The gill tissue of these mushrooms are pink to brown. The spore print is brown. White and brown button mushrooms can be distinguished by color, and Portobello In the US, A. bisporus is the dominant edible mushroom and accounts for over 90% of the market. Currently, white button mushroom is the most widely consumed, but the popularity of portobello and brown button is growing. Compared to other edible mushrooms, A. bisporus has been less well investigated for its anticancer activity. No studies have been published on the anticancer activity of portabella, and only one publication reported the anticancer activity of a hot water extract of brown button mushrooms against MCF-7 breast cancer cells [9]. Some in vitro and in vivo studies have reported the anticancer activity of white button mushrooms such as its inhibition on breast cancer cell growth as an aromatase inhibitor [10].

White button mushrooms first attracted scientific attention in cancer research in the 1980’s. Agaritine, an aromatic hydrazine, was proposed as a potential carcinogen by Toth and colleagues in 1981 (Fig. 1) [11, 12]. No carcinogenic effect, however, was detected in Swiss mice following subcutaneous injection with this compound [13]. By contrast, oral administration of white button mushroom powder (3 d/week, 4.8 g/d for female and 4.2 g/d for males) to the same model resulted in increases in tumors of the fore stomach, glandular stomach, duodenum, and ovaries compared to control mice [14, 15]. The doses used in these two studies were comparable: 2 – 3 mg agaritine/mouse for subcutaneous injection and 1.7 – 3.4 mg/mouse for the oral administration [14, 16, 17]. The differing results from subcutaneous and oral studies might due to the metabolism of agaritine. In vitro, the mutagenicity of agaritine was increased in the presence of tyrosinase and rat hepatic cytosol [18]. In rodents, agaritine loses a glutamyl moiety by γ-glutamyl transpeptidase, and then the free hydrazine may be oxidized into benzene diazonium ion, which is a mutagen in the Ames test [19].

Figure 1.

Structures of some of the putative bioactive components derived from edible mushrooms.

A recent in vitro study first reported the anticancer activity of agaritine (IC₅₀=2.7 – 16 μg/ml) isolated from Agaricus blazei Murill in U937, K562, MOLT4, and HL60 leukemic cells [20]. However, the rationale of agaritine’s metabolism in human beings has not been fully investigated [18, 19]. Considering the low content and instability of agaritine and incomplete knowledge of its mechanism of carcinogenesis, no conclusive statement on the carcinogenetic potential of white button mushrooms can be made.

Studies on the anticancer effects of white button mushrooms against colon cancer, breast cancer, and prostate cancer models have been reported. White button mushrooms have shown anticancer activity against both human breast and prostate cancer xenografts in nude mice. Treatment for 6 wk with 4X mushroom extract significantly lowered the tumor weight in MCF-7aro-bearing nu/nu mice compared to water-treated controls. Histopathological analysis showed that mushroom extract inhibited tumor cell proliferation as measured by down-regulation of Ki-67, but did not induce apoptosis [21]. The same group reported that oral gavage with 6X mushroom extract inhibited the growth of DU145 or PC3 human prostate cancer xenografts in BALB/c nu/nu athymic mice [22]. Tumor growth inhibition was related to induction of apoptosis as measured by increased cleaved caspase-3. Microarray analysis of tumor tissue showed that the mushroom extract altered the expressions of genes involved in cell death, cell growth, lipid metabolism, and immune response [22]. These two studies are difficult to evaluate because the authors did not clearly quantify the dose of mushroom extract (i.e. mg/kg b.w.). Conjugated linoleic acid (CLA) was suggested as the putative anti-cancer compound in the mushroom extract, however, no direct evidence was provided to show that CLA is in the extract is responsible for the anti-cancer activity.

The mammary cancer preventive effects of the selenium-enriched white button mushrooms was assessed in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced Sprague–Dawley rats [23]. Treatment with 2% selenium-enriched white button mushrooms in the diet enhanced glutathione-S-transferase (GST) activity and reduced DMBA-induced DNA adducts in mammary tissues compared to control animals, indicating that selenium-enriched white button mushrooms worked by preventing initiation of cancer, however, longer-term carcinogenesis studies are needed.

Besides direct antitumor activity, immune modulatory effects of white button mushrooms have been proposed as potential anti-cancer mechanism in animal models [24]. C57BL/6 mice fed with 2 and 10% (w/w) white button mushroom powder in the diet for 10 wk had enhanced natural killer (NK) cell activity (enhanced production of interferon (IFN)γ and tumor necrosis factor (TNF)α) compared to control mice fed with standard diet. These results suggest that white button mushroom promotes innate immunity. Further studies are needed to confirm this result and establish the connection between immune modulation and the cancer inhibition of white button mushrooms.

The cancer-related mechanisms of action of white button mushrooms have also been explored in vitro. From the above animal studies, white button mushrooms have been shown to inhibit cellular proliferation or induce apoptosis [21]. However, various anticancer mechanisms of white button mushrooms have been demonstrated in in vitro studies. White button mushroom-derived lectin (40μg/mL), a granular protein produced by plants or mushrooms under stress, has been shown to accumulate around nucleus through internalization and resulted in blockade of nuclear protein import, resulting in inhibition of HT-29 human colon cancer cell proliferation [25, 26]. A heat-stable extract from white button mushrooms was also shown to inhibit the proliferation of aromatase-transfected MCF-7aro human breast cancer cells and this was related to inhibition of aromatase activity. By contrast, the extract had no effect on normal breast cell line MCF-10A [10]. In human prostate cancer LNCaP cells, the same white button mushroom extract also initiated apoptosis [22].

Although these studies suggest the value of A. bisporus as a cancer preventive or therapeutic agent, more research, especially mechanism-based animal studies and human clinical trials are needed. Similarly more detailed information on the anti-cancer mechanisms of action and the identity of the active chemical constitutes is needed.

Epidemiological data on the relationship between consumption of white button mushrooms and cancer risk is very limited. In a case-control study of pre- and post-menopausal women performed in southeast China, an inverse association between breast cancer incidence and white button mushroom and green tea consumption was established. The odds ratio (OR) was 0.36 (95% confidence interval [CI] =0.25–0.51) for daily intake ≥10g fresh white button mushrooms and 0.53 (95%CI = 0.38–0.73) for daily intake ≥ 4g dried mushrooms [3].

Although no clinical trials have been reported on the anticancer activity of white button mushrooms, there is an ongoing phase I study at the City of Hope Medical Center (Duarte, CA, USA) in patients with recurrent prostate cancer after local therapy to study the safety and feasibility of white button mushrooms as a treatment. Similar as animal study, there is no epidemiological data on the relationship between consumption of portabella or brown button mushrooms and cancer risk.

Agaricus blazei

A. blazei Murrill (ABM) has been consumed as an edible mushroom in Brazil, Japan, and some other Asian countries. It has also been traditionally used in Brazil for the treatment of a variety of diseases including diabetes, atherosclerosis, cancer, and heart diseases and to modulate immunity [27].

The anticancer activity of ABM has been demonstrated in a number of animal and cell culture studies. A significant amount of mechanistic data has been generated in these models. Tumor inhibitory activity has been observed against fibrosarcoma [28], colon cancer [29], myeloid leukemia [30], prostate cancer [31], and lung cancer [32]. A number of bioactive compounds in ABM have been studied. These include polysaccharides as well as compounds including agaritine[33] blazein[34], blazeispirol A[35], and sodium pyroglutamate [32] (Fig. 1).

A summary of the anti-cancer studies on these compounds from ABM is listed in Table 1.

Table 1.

Effective Anticancer Compounds in A. blazei Murrill

Compound	Experimental Model	Effects	References
(1–6)-8-D-Glucan-Protein Complex	Meth A fibrocarcinoma-bearing mice	• Immune modulation • T cell-mediated response to eliminate tumor cells	[36]
Low-molecular-weight polysaccharide	Sarcoma180-bearing mice	• Antiangiogenesis • Decrease expression of VEGF	[37]
Sodium pyroglutamate	Lewis lung carcinoma-bearing mice	• Apoptosis • Immune modulation	[32]
Blazein	LU99 human lung and KATO III stomach cancer cell lines	• Apoptosis • Initiate DNA fragmentation	[34]
Blazeispirol A	Hep 3B human hepatoma cell line	• Apoptosis • Sub-G1phase cell cycle arrest	[35]
Agaritine	U937 leukemic cell line	• Apoptosis	[33]

Oral administration of ABM was shown to inhibit sarcoma180 [28] and Meth A fibrosarcoma xenografts in mice [36]. Daily oral administration of 200mg/kg ABM for 14 d inhibited the growth of sacroma180-tumors by 33% in BALB/c mice. Tumor inhibition was related to antiangiogenic effects measured as decreased expression of mRNA and protein levels of vascular endothelial growth factor (VEGF) [37]. Antiangiogenic effects were also observed in human prostate cancer xenograft-bearing mice treated with 0.1% and 0.2% of ABM extract in the drinking water. A decrease in CD31-positive micro vessels was observed in tumor tissues of treated mice by immunohistochemical analysis [31].

ABM has also been shown to induce apoptosis on a number of tumor models. SCID mice bearing DU145/PC3 human prostate cancer xenografts given 0.1 and 0.2% ABM extract in drinking water had increased tumor cell cytosolic cytochrome c, procaspase 3 and 7 expression, and PARP cleavage [31]. Apoptosis induced by ABM has also been confirmed in PC3 human prostate cancer [31], LU99 human lung cancer and KATO III stomach cancer cells [34], Hep 3B human hepatoma cells [35], and U937 human leukemia cells in vitro [33]. In Hep 3B cells, treatment with blazeispirol A induced apoptosis through increased expression of Bax, increased caspase 9 and 3 cleavage, and increased PARP cleavage, and increased expression of cytosolic AIF and HrtA2/Omi (caspase-independent pathway) [35] has also been reported. Although a number of researchers have examined the anticancer effects of ABM, a limited number of mechanistic biomarkers have been reported. No clear signaling pathways have been proposed and further mechanistic studies are needed to determine how the anti-angiogenic and pro-apoptotic effects are induced.

Immune modulation has also been proposed as an anticancer mechanism of ABM. In 1, 2-dimethylhydrazine (DMH)-induced mice, dietary supplementation with 10% ABM powder suppressed aberrant crypt foci (ACF) incidence and multiplicity by greater than 30 %. A decrease in the number of initiated cells was also observed. Treated mice had increased monocyte proliferation and improved phagocytic activity by cells in the spleen. This improved immune function was believed to result in enhanced of DNA damaged cells [29]. By contrast, no suppression of ACF development was observed in DMH-initiated Wistar rats treated with 5% ABM for 4 or 20 wk [38, 39]. The negative results might due to the lower dose of ABM or an unknown species-specific response to ABM treatment. This immune modulatory effect of ABM needs to be further confirmed and investigated in other tumor models and animal species.

ABM has also been used as an alternative supplement to conventional cancer therapy. Although no clinical trials have been conducted in the US, ABM has been studied in clinical trials in Asia. ABM and its derivative products have been studied alone or as an adjunct therapy to radiation and chemotherapy. The results of these published clinical studies are mixed with regard to the effectiveness as a cancer therapy.

A 6-month open-label study of 32 prostate cancer patients following radical prostatectomy found no significant anticancer activity from ABM treatment [40]. A clinical study of 39 cancer patients using ABM extract in combination with chemotherapy (carboplatin+VP16/taxol) for 9 wk, reported that NK cell activity in ABM-treated patients was significantly higher (P<0.002) than non-treated patients [41]. Less chemotherapy-associated side effects such as lack of appetite, alopecia areata, general weakness, etc. were reported in ABM-treated patients [41]. A survey of Japanese cancer patients in 2007 found that patients reported emotional and physical-well being benefits from ABM beverage than the relief from specific cancer symptoms [42].

Several studies have reported adverse effects of ABM in cancer patients. Three cases of severe hepatic damage in advanced cancer patients (two patients with breast cancer and one with ovary cancer) were reported in 2006 [43]. One case has also been reported of increased serum 5-S-cysteinyldopa (5-S-CD) level, a marker for malignant melanoma cancer, in a malignant melanoma cancer patient who consumed ABM following β-INF therapy [44]. A phase I clinical trial on the safety of ABM in 78 cancer survivors reported adverse effects in 9 patients [45]. These included nausea, diarrhea, and abnormal levels of laboratory parameters such as hemoglobin (P=.03), hematocrit (P=.005), total cholesterol (P=.02), etc. The authors claimed ABM is a safe supplement in most cancer patients, however, further studies with larger sample sizes are needed.

Pleurotus ostreatus

Pleurotus ostreatus, also known as oyster mushroom, is a common edible mushroom consumed worldwide. P.ostreatus has an offset cap with decurrent hymenium. The hymenium has gills and the stipe lacks a ring. The cap color ranges from tan to white, and the spore print is white. The anticancer activity of oyster mushrooms has been examined in several animal models. In ICR mice treated with N-butyl-N’-butanolnitrosoamine (BBN), oral administration of oyster mushrooms reduced urinary bladder carcinoma incidence by 45% compared to control mice [46]. The authors found that mushroom-treated mice had enhanced ex vivo lymphocyte-mediated cytotoxicity against P-815 mastocytoma cells compared to BBN only-treated mice [46]. Whether the anticancer activity of oyster mushrooms is due to immune modulation still needs to be further studied.

Treatment of sarcoma180-bearing Swiss albino mice with 5mg/kg water soluble proteoglycans from oyster mushrooms for 6 d caused arrest of tumor cells in G₀/G₁ phase. Proteoglycans were found to activate NK cells and macrophages in mushroom-treated mice compared to the control group [47].

In DMH-induced Wistar rats, dietary supplementation with 10% pleuran (a β-glucan from oyster mushrooms) suppressed the incidence ACF by 60%. Pleuran intake also significantly reduced the level of conjugated dienes, a marker of oxidative stress, by 40% in the colon [48].

Oyster mushroom extracts have been found to inhibit the growth of several human cancer cell lines including PC-3 human prostate cancer cells [49], HT-29, SW480 [50], and HCT-116 human colon cancer cells [51], and MCF-7 and MDA-MB-231 human breast cancer cells [52]. Treatment with 200μg/mL oyster mushroom extract for 6 h resulted in DNA fragmentation in PC-3 cells [49] and 0.25% oyster mushroom extract enhanced the expression of Bax and the release of cytochrome c into the cytosol of HT-29 cells [51]. Treatment of SW480 cells with oyster mushroom extract resulted in increased reactive oxygen species (ROS) production, depletion of reduced glutathione (GSH), and mitochondrial dysfunction, suggesting a potential role for pro-oxidative effects in the anticancer activity of oyster mushrooms [50]. Cell cycle arrest at the G₁/S transition was observed in HT-29 and MCF-7 cells following treatment with 1mg/mL methanol extract of oyster mushrooms. Increased p21 expression and decreased phosphorylation of Rb were also observed [52].

No clinical trials on the anticancer activity of oyster mushrooms have been published. Two epidemiological studies, however, have reported an inverse association between consumption and breast cancer risk in Korean women [3, 4]. In one case-control study, the OR was 0.35 (95% CI = 0.13–0.91) for the highest quartile of mushroom intake in pre-menopausal women compared to the lowest quartile. This inverse association was specific for women who were estrogen receptor (ER) and progesterone receptor (PR) positive [5]. We believe this result indicates the possible hormone receptor-mediated anticancer mechanism of oyster mushrooms, although the issue was not discussed by the authors. It is unclear, however, if the protective effect is strictly due to oyster mushrooms, because the authors also included other mushrooms including Lentinus edodes (Shiitake), A. bisporus, and Flammulina velutipes (winter fungus) in the analysis. A second case-control study found a similar inverse association between mushroom consumption and breast cancer occurrence in post-menopausal women [4], but as with the first study, the results are confounded by inclusion of other mushrooms.

In summary, oyster mushrooms have shown anticancer activity in several animal model studies and in cancer cell lines. Further studies are needed to investigate the anticancer mechanisms in vivo, and human intervention studies of oyster mushrooms alone or in combination with conventional chemotherapy are needed to establish efficacy in humans.

Lentinus edodes

Based on animal studies, Lentinus edodes or shiitake mushrooms are believed to exert anticancer effects via stimulation of the immune system. Shiitake mushrooms have a convex cap and free hymenium with gills. The top of the cap is brown to purple brown and the gills are white and the stipe is bare. The spore print of these mushroom range in color from white to buff. The most intensively investigated anticancer compound in L. edodes is lentinan. This polysaccharide has a backbone of (1→3)-β-D-glucan with side chains of β-D-(1→3)- and β-D-(1→6)-linked D-glucose residues [53]. The molecular weight of lentinan is in the range of 400 – 1,000 kD and its oral bioavailability is low. Thus, it has often been administered intravenously. The molecular weight [54] and the triple helix structure [55] of lentinan are suggested as determinants of its anticancer activity.

A polysaccharide fraction from L. edodes has been found to inhibit tumor growth in sarcoma180-bearing Swiss albino mice by 90% following subcutaneous injection with 5mg/kg/d for 10 d [56]. Lentinan from L. edodes has been to have similar antitumor effects in the same animal model when given at 1mg/kg/d by intraperitoneal (i.p.) injection for 10 d [57]. When mice were co-treated with anti-lymphocyte serum, the tumor inhibition of lentinan was reduced by 44%. These results suggest a critical role for lymphocytes in the antitumor effect of shiitake [57]. The role of immune response in lentinan’s anticancer activity was also demonstrated in a nude mouse model [58]. In that study, lymphocytes were collected from AKR mice fed lentinan for 7 d and then injected in nude mice before inoculation with human colon cancer cells. In nude mice injected with lymphocytes from shiitake-treated AKR mice, smaller tumor volume was observed compared to the mice with no lymphocyte injection [58]. In another study, B16 murine melanoma cells were inoculated in the footpads of both C57BL/6 (immune competent) and nude (immune compromised) mice [59]. Dietary supplementation of 1% shiitake mushroom extract inhibited tumor growth by 60% in C57BL/6 mice compared to the controls, whereas no inhibitory effect was observed in nude mice. The authors propose that shiitake mushrooms work via a T cell-dependent anticancer mechanism [59].

Given this observed activity in mouse model studies, it is unclear what the impact of poor bioavailability is on anticancer potential. Additionally, other compounds such as the protein lentin (molecular weight: 27.5kD) isolated from L. edodes has been shown to inhibit L1210 murine leukemia cell proliferation in vitro [60], and should be investigated further.

In addition to effects on immune surveillance, in vitro studies have demonstrated that shiitake can also directly induce apoptosis [61]. An ethyl acetate extract prepared from shiitake (100μg/mL) induced apoptosis by increasing expression of pro-apoptotic Bax in MDA-MB-453 and MCF-7 human breast cancer cells. This extract also enhanced expression of p21 (100μg/mL) and reduced expression of cyclin D1 and cyclin-dependent kinase (Cdk4) (400μg/mL), resulting in G₀/G₁ phase cell cycle arrest [61].

Shiitake has been used as a complementary treatment to conventional cancer therapies in Asia, and especially in Japan. The safety of shiitake supplement has also been tested in a Phase I study following US Food and Drug Administration (FDA) guidelines. Active hexose correlated compound (AHCC), containing α-glucan as the main constituent, was given to 26 healthy subjects at a dose of 9g/day (a dose higher than that used in routine clinical application) for 14 d. Mild and transient adverse effects such as headache, cramps in the toes, mild diarrhea and bloating were reported by 4 subjects [62]. In many clinical trials, lentinan has been given as the treatment. The improved prognosis has been observed in lentinan-treated breast cancer patients without any severe adverse effects [63].

Shiitake mushrooms or lentinan have demonstrated anticancer activity when used as an adjunct to conventional anticancer therapies, such as radiation or chemotherapy. Gastrointestinal cancer and breast cancer patients who received intravenous administration of lentinan at 1mg/person twice a week or 2mg/person once a week combined with chemotherapies such as mitomycine C plus 5-fluorouracil (MF) or 5 fluorouracil plus tegafur (FT) had significantly longer life span (50–100% longer in lentinan-treated cancer patients than non-treated patients) than the patients who received MF or FT alone (p<0.05) [64]. Life span was also prolonged in patients with advanced or recurrent gastrointestinal cancer who received lentinan combined with chemotherapeutical agents (MF or FT) in a Phase III study [65].

By contrast, a study of prostate cancer patients found that treatment with shiitake extract (1g/kg, t.i.d.) for 6 mos had no effect on serum levels of prostate specific antigen [66]. The effectiveness of shiitake in conjunction with conventional cancer therapy is largely positive. The negative results in the prostate cancer study could be due to the lack of additional conventional cancer therapy. It could be also due to the route of administration. As we discussed above, the oral bioavailability of lentinan is low. In most other studies that had positive outcomes, patients were intravenously injected with lentinan [64, 65, 67, 68].

Shiitake is hypothesized to stimulate immune system and that this underlies its anticancer activity. The immune modulatory effects have been demonstrated in some cancer patients. In a small scale study of 10 breast cancer patients, oral supplementation with 9g/d shiitake modulated chemotherapy-induced loss of NK cells [69]. In another clinical trial involving 81 lung cancer patients in China, NK cell activity was enhanced in chemotherapy-treated patients treated intravenously twice weekly with lentinan (1mg/person) for 8 wk compared to patients received chemotherapy only [67]. The immune modulatory effects of shiitake have also been demonstrated in a Phase III/IV placebo-controlled trial involving 98 HIV-positive patients in US. A trend of increased CD4 cell number and neutrophil activity was observed in some patients intravenously injected weekly with lentinan of 2 – 10 mg/person for 8 wk or 1 – 5 mg/person weekly for 12 wk [68]. Although the immune modulatory effects of shiitake have been demonstrated in several human studies, the effects on long-term anticancer outcomes still need to be investigated in humans.

Grifola frondosa

Maitake mushrooms (G. frondosa) is morphologically characterized by an offset cap and decurrent hymenium without gills. The cap is dark brown in color and the mushroom lacks a stipe. The maitake spore print is white. The anticancer activity of maitake was first demonstrated by Yadomae and colleagues [70]. The authors found that the polysaccharide fraction from maitake fruiting bodies and β-1–3-glucan from cultured maitake mycelium [71] both inhibited tumor growth in Sarcoma180-bearing ICR mice when given by i.p., intravenous, or intratumoral (i.t.) injection at doses of 0.5 – 5mg/mouse/d for 10 d. However, the anticancer effect was not observed in mice treated by oral administration [71]. As with shiitake, the putative anticancer mechanism is believed to be immune modulation. D-fraction was the earliest and the mostly-investigated effective fraction isolated from maitake. This fraction contains beta-glucan polysaccharides and proteins with molecular weight around 1,000 kDa and was first isolated by Nanba and colleagues in 1984 [72]. Subsequently, a more purified fraction called MD-fraction with better bioactivity was developed by the same group. Since then, most clinical trials and animal studies have used either D-fraction or MD-fraction as materials to investigate the anticancer activity of maitake mushrooms.

In MM46 carcinoma-bearing C3H/HeJ mice, daily i.p. injection with 4mg/kg D-fraction for 3 d was found to inhibit tumor growth by 50% and increase NK cell activity by 30% in both spleen and blood cells. Enhanced blood and splenic macrophage activity (2-fold) as well as enhanced interleukin (IL)-12 production (2-fold) in spleen cells have also been observed [73]. With the same animal model, another study showed that i.p. injection of maitake β-1,3-glucan at 500μg/mouse/d for 5 d induced macrophage activity and initiated cytostasis of MM46 carcinoma [74]. However, these studies only examined the acute immune stimulation and antitumor effects by polysaccharide fraction from maitake.

In Sarcoma180-bearing mice given 5 daily i.p. injections of 4 mg/d maitake polysaccharide fraction, tumor volume was reduced 95% by day 40 compared to controls. Inhibition of sarcoma180 growth was also observed when these mice were reinoculated with fresh sarcoma180 cells [75]. Increased spleen cell number and enhanced antigen specific response by peritoneal exudate was observed in these mice, indicating the antitumor effect of maitake is via host-mediated immune mechanism [75]. Oral administration of maitake-derived polysaccharide at 50–150mg/kg/d for 9 d inhibited S180 carcinoma by 30–40% in tumor-bearing mice [76]. In a granulocytopenia mouse model, daily injections of MD-fraction (8mg/kg, i.p.) for 7 d enhanced granulocytes production in spleen by 3-fold compared to controls [77].

Immune modulation through restoration of T lymphocyte subtype balance (Th-1/Th-2 cells) has also been proposed as an anticancer mechanism of D-fraction. Imbalance of Th-1/Th-2 ratio is associated with increased carcinogenesis [78]. In one Th-2 cell dominant mouse model, injection with D-fraction (5 mg/kg, i.p.) for 19 d inhibited tumor growth by 80%, restored Th-1 cell balance, and enhanced IFNγ production by 1.4-fold in CD4 cells [79]. Another Th-2 cell dominant mouse model (colon 26 carcinoma-bearing mice) was injected with 7.8mg/kg, i.p. D-fraction for 19 d and the dendritic cells (DCs) were collected from the whole spleen and lymph nodes on day 20. D-fraction enhanced the production of Thu-1 cytokines such as IFNγ (>2-fold) in both spleen and lymph node DCs. When injected into colon 26 carcinoma-bearing mice once a week for 5 wk, DCs from D-fraction treated mice inhibited tumor growth by greater than 95% compared to mice treated with DCs from control animals [80]. Daily injection with D-fraction (4 mg/kg, i.p.) for 17 d also enhanced cytokine production by Th-1 and Th-2 cells in non-tumor bearing C3H/HeJ mouse [76, 81]. A low molecular weight protein fraction from maitake (7.5 ng/mouse/d i.p.) for 7 d has been also found to enhance IFNγ production in colon 26 tumor-bearing mice by 2-fold compared to the saline-injected controls [82].

The safety of maitake polysaccharides has been examined in a phase I/II clinical trial involving 32 postmenopausal breast cancer women treated with one of five doses (0.2, 1, 3, 6 or 10 mg/kg/d) for 34 mos. No dose-dependent toxicity was observed in these patients. One patient at 1 mg/kg/d withdrew from the study due to grade I nausea and joint swelling, and another patient at 10mg/kg withdrew because of grade I allergic reaction [83]. In 1998, the FDA approved a Phase II pilot study of D-fraction as an Investigational New Drug (IND) application with an exemption of the requirement for a Phase I toxicology study in patients with advanced prostate and breast cancer [84].

Case studies have demonstrated some positive results in the anticancer effects of maitake [85]. For example, a lung cancer patient diagnosed with stage IV lung cancer had improvement to stage III-A after consuming 100 mg MD-fraction and 6 g maitake mushroom tablets daily for 60 mos. A breast cancer patient was diagnosed with metastasis to the lung after surgery and chemotherapy. After 20 mos of treatment with 125 mg MD-fraction and 4 g whole maitake daily, the lung tumor was eliminated [85]. Finally, in four liver cancer patients, increased IL-2 production, CD4+ cell count and decreased bilirubin level were observed following daily consumption of 40 – 150 mg MD-fraction and 4 – 6 g maitake tablet [85].

Larger scale trials have supported the immune modulatory effect of maitake mushrooms. A small scale clinical trial in 8 stage II-IV cancer patients reported enhanced NK cell activity after oral administration of 100 mg/d D-fraction for 34 mos [86]. Similar results were observed in a second clinical trial of 10 stage II-IV cancer patients in 2003 (lung, breast, gastric, and liver cancer). The patients have been given different doses of D-fraction twice a day (40/80 mg, 50/100mg, and 75/150mg) for 63 mos [87]. A phase I/II clinical trial of 32 postmenopausal breast cancer women for 34 mos found that intake of 5–10mg/kg maitake extract increased IL-2, IL-10, TNFα, and IFNγ production by T cells [83].

Maitake mushrooms have also shown synergistic anticancer effect when used in conjunction with other therapies. In a clinical trial of patients with polycystic ovary syndrome, a risk factor for endometrial cancer [88], maitake mushroom SX-fraction (a extract containing water-soluble glycoprotein with molecular weight of 20 kD) alone was able to induce ovulation in some patients, but when combined with clomiphene citrate, the induction of ovulation was observed in the patients who failed with either clomiphene citrate or SX-fraction treatment alone [82, 89].

Maitake mushroom and its polysaccharide fraction have been shown to be effective in conjunction with conventional cancer therapy. One discrepancy between clinical trials and animal studies is the route of administration. Most animal studies have used i.p. injection: studies with oral administration have not been reported inhibition. By contrast, in clinical trials, maitake mushrooms have been given by oral consumption and activity has been observed. These contradictory results make interpretation of mechanistic studies difficult. Further studies on the bioavailability of D-fraction may clarify these issues. There are other potentially effective small molecules in the fractions which should also be studied for their anticancer activity. For example, one paper reported a new low molecular weight protein fraction isolated from maitake increased cytokine production in normal mice; this fraction also inhibited tumor growth in colon 26 carcinoma-bearing mice [82]. This inhibition of tumor cell growth was also associated with enhanced plasma levels of IL-12 and IFNγ, suggesting a role for immune stimulation [82].

CONCLUSIONS

Polysaccharides (mainly β-glucan), protein (lectin), and small molecules (agaritine, blazein, blazeispiol A, ect.) are the three major categories of putative anticancer compounds in these five edible mushrooms that have been examined in most studies to date. From the limited in vitro and in vivo mechanistic studies, some edible mushrooms appear to inhibit cancer cell growth via induction of apoptosis, inhibition of cell cycle progression, modulation of immune function, and inhibition of angiogenesis. Pre-clinical studies in animal models and a relatively limited number of clinical trials have shown mixed concerning the anticancer effects of commonly consumed edible mushrooms. For some such as maitake and shiitake, their effectiveness alone or as adjuncts to conventional cancer therapy has been demonstrated in some studies. The activity of others, such as A. blazei, is not supported by the available animal model and human data. For still others, such as A. bisporus, there is insufficient evidence to draw conclusions about putative anticancer activity. We recommend the following as important and promising research directions regarding the anticancer activity of edible mushrooms:

1. Additional effort is needed to establish the identity of the putative anticancer components in edible mushrooms. A number of different classes of compounds, some of which are represented in multiple species such as β-glucan and lectin, have been proposed. Which compounds are necessary and sufficient for activity of a given species, however, has not been established. Similarly, it is unclear whether the same compounds are responsible for activity across cancer cell type or if different compounds are responsible for activity against different cell types.

2. Careful animal models studies with mechanism-based endpoints and using a dose-response design are needed to clearly establish the efficacy and mechanisms of action of various edible mushrooms against different cancer types. Further, if dietary mushrooms are proposed to have a role in the prevention of human cancer, the animal models studies should reflect this hypothesis by use of the oral route of administration. The bioavailability of the putative anticancer compounds must be determined in vivo. Although parentral administration is useful to establish efficacy, it is difficult or impossible to relate the results of these studies to the effect of diet on human disease.

3. A limited amount of epidemiological data is available regarding the anticancer or cancer preventive effects of edible mushrooms. Large-scale population-based studies and case-control studies with careful assessment of exposure would be useful to determine if these foodstuffs play a role in the prevention or in affecting the outcome of human cancer.

4. Finally, pending further promising pre-clinical efficacy and safety data, human intervention studies in populations with elevated risk of cancer (e.g. breast cancer survivors) or in patients with early stage disease are needed to clearly establish the usefulness of edible mushrooms or their constituents in the prevention or treatment of cancer.

Abbreviations:

ABM

Agaricus blazei Murrill

ACF

aberrant crypt foci

BBN

N-butyl-N’-butanolnitrosoamine

CLA

conjugated linoleic acid

DCs

dendritic cells

DMBA

7,12-dimethylbenz(a)anthracene

DMH

1,2-dimethylhydrazine

ER

estrogen receptor

FT

5 fluorouracil plus tegafur

GSH

glutathione

GST

glutathione-S-transferase

IFN

interferon

IL

interleukin

i.p.

intraperitoneal

i.t.

intratumoral

MF

mitomycine C plus 5-fluorouracil

NK cells

natural killer cells

PR

progesterone receptor

ROS

reactive oxygen species

5-S-CD

5-S-cysteinyldopa

TNF

tumor necrosis factor

VEGF

vascular endothelial growth factor