Research: Mushrooms & Breast Cancer –
What is the Connection?

Anti-tumour effects, primarily in human cell lines, have been reported from polysaccharides extracted from a variety of mushrooms. The polysaccharides belong to the betaglucan family of compounds and appear to exert their antitumourigenic effects via enhancement of cellular immunity e.g. via activation of T helper cells and induction of the production of interferon (IFN)-gamma and certain interleukins. Anti-tumour 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 synthase gene expression, which is then followed by nitric oxide production in macrophages via activation of the transcription factor, NF-kappaB. Activation of NK cells is likely via interferon-gamma and interleukin mediated pathways.

Yunzhi (Coriolus versicolor) has been reported to modulate various immunological functions in vitro, in vivo, and in human clinical trials, while Danshen (Salvia miltiorrhiza) has been shown to benefit the circulatory system by its vasodilating and anti-dementia activity. A clinical trial has been carried out to evaluate the immunomodulatory effects of Yunzhi-Danshen capsules in post-treatment breast cancer patients. Eighty-two patients with breast cancer were recruited to take Yunzhi (50mg/kg body weight, 100% polysaccharopeptide (PSP)) and Danshen (20mg/kg body weight) capsules every day for 6 months. Blood samples were collected every 2 months for the investigation of immunological functions. Flow cytometry was used to assess the percentages and absolute counts of human lymphocyte subsets in whole blood. The plasma level of soluble interleukin-2 receptor (sIL-2R) was measured by enzyme-linked immunosorbent assay (ELISA). The results showed that the absolute counts of T-helper lymphocytes (CD4+), the ratio of T-helper (CD4+)/T suppressor and cytotoxic lymphocytes (CD8+), and the percentage and the absolute counts of B-lymphocytes were significantly elevated in patients with breast cancer after taking Yunzhi-Danshen capsules, while plasma sIL-2R concentration was significantly decreased, and therefore regular oral consumption of Yunzhi-Danshen capsules may be of benefit for the promotion of immunological function in post-treatment of breast cancer patients (Wong et al., 2005).

A recent case-controlled epidemiological study of 362 Korean women, between the ages of 30 and 65 years who were histologically confirmed to have breast cancer, has reported that the consumption of dietary mushrooms may decrease breast cancer risk in post-menopausal women, but not in premenopausal women (Hong et al., 2008). In contrast, a recent study has suggested a decreased risk of breast cancer from mushroom consumption by premenopausal women (Shin et al., 2010). This study evaluated the association between mushroom intake and the risk of breast cancer according to hormone receptor status among 358 breast cancer patients and 360 cancer-free controls. Intake of mushrooms was assessed using a quantitative food frequency questionnaire. Greater mushroom intake was related to lower risk of breast cancers among premenopausal women for the highest vs. the lowest quartile intake. 

The association was stronger for premenopausal women with estrogen receptor (ER)+/progesterone receptor (PR) + tumours than those with ER-/PR- tumours, suggesting that this effect may be more robust among women with hormone receptor positive tumours.However, a recent case-controlled study in China with 1009 female patients aged 20–87 years with histologically confirmed breast cancer concluded that higher dietary intake of mushrooms decreased breast cancer risk in both pre- and postmenopausal Chinese women and an additional decreased risk of breast cancer was observed from a synergistic effect of mushrooms and green tea (Zhang et al., 2009). In response to this study of Zhang et al., it has been suggested that vitamin D2 could be one of the protective phytonutrients against breast cancer as mushrooms are rich in 4 ergosterol, generating vitamin D2 when exposed to ultraviolet B (UVB) light and ergocalciferol being bioavailable and increasing serum 25(OH) vitamin D2 levels in humans (Furlanetto, 2009).

A polysaccharide extract from Grifola frondosa (Maitake extract) has shown immunomodulatory effects in preclinical studies. In a phase I/II dose escalation trial, 34 postmenopausal breast cancer patients, free of disease after initial treatment, were enrolled sequentially in five cohorts. The primary endpoints were safety and tolerability. No dose-limiting toxicity was encountered. There was a statistically significant association between Maitake and immunologic function. Increasing doses of Maitake increased some immunologic parameters and depressed others; the doseresponse curves for many endpoints were non-monotonic with intermediate doses having either immune enhancing or immune suppressing effects compared with both high and low doses. Oral administration of the polysaccharide extract from Maitake mushroom was therefore associated with both immunologically stimulatory and inhibitory measurable effects in peripheral blood. Cancer patients should be aware that botanical agents may depress as well as enhance immune function (Deng et al., 2009).

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 postmenopausal women by reducing the in situ production of estrogen   (Grube et al., 2001).

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), have anti-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 dosedependent, 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). 5 Ganoderma lucidum suppresses the invasive behaviour of breast cancer cells by inhibiting the transcription factor NF-kappaB. It has been shown that Ganoderma lucidum inhibits proliferation of breast cancer MDA-MB-231 cells by downregulating Akt/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 the result 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 stressinduced 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 6 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 has also 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 human breast 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 40µg/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 proapoptotic 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% antiproliferative 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 of breast 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. 7 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). 

Extracts from Agaricus bisporus 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/aromatasepositive 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 testosteronedependent 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 tumour cell proliferation and tumour weight with no effect on the rate of apoptosis (Chen et al., 2006).

A study of the effects of Ganoderma lucidum (Basidiomycetes) polysaccharide (GL-PS) extract on tumour 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).