Edible and Medicinal Mushrooms as Promising Agents in Cancer

have very weak effects when administered orally.


Introduction
Conquering cancer is one of the major challenges facing mankind in the 21st century. The advancement of diagnostic techniques has made discovering miniscule tumors feasible, and early treatment of many types of cancers has consequently become a reality. However, while the development of anticancer drugs progresses, the number of people diagnosed with cancer continues to rise. The drug, tamoxifen, has been approved in the US to prevent breast cancer relapse. In addition, cancer prevention has become an important part of conquering cancer, with both primary and secondary prevention strategies. The former entails the prevention of cancer itself, while the latter involves the prevention of death once an individual has already developed cancer.
Edible mushrooms such as Lentinula edodes (shiitake) and Grifola frondosa (maitake) have been known from ancient folklore to possess properties that enhance biological defense responses (immune functions), and have been used in people with decreased immune function such as those with cancer, allergies and other disorders, and in elderly people. Many of these mushrooms contain compounds called β-glucans, which are high molecular weight polysaccharides of glucose linked together by glycosidic bonds. β-glucans are contained in mushrooms, yeast, fungi, and higher plants. In Japan, several mushroom-derived pharmaceutical products have been developed, and include schizophyllan from Schizophyllum commune, krestin from Trametes versicolor, and lentinan from L. edodes an anticancer polysaccharide from shiitake. In South Korea, meshima, a mycelia culture of Phellinus linteus, was developed as an anticancer drug. Antitumor activities of polysaccharides and peptide polysaccharides in these mushrooms have been reported. In addition to polysaccharides, unique substances such as sterols and triterpenes are reportedly present in mushrooms. Some of these compounds are promising anticancer agents. Please refer to a review published elsewhere for a description on herbal medicine extracts that have been anticipated for their cancer prevention effects [1]. In this chapter, we will introduce the anticancer activities of polysaccharides as well as the cancer prevention activities of sterols and triterpenes.
with oral administration, there was a period of time in which it was used alone after its release in 1977. However, it is now evident that it has no effect by itself, and is now used in conjunction with other drugs. Krestin is thought to exhibit its antitumor actions by acting on the immune response mechanism that has decreased due to a cancer-bearing state. Krestin has a mean molecular weight of 9.4 × 10 4 , and its sugar chain moiety consists of glucose (74.6%), galactose (2.7%), mannose (15.5%), xylose (4.8%), and fucose (2.4%), but mostly glucose in the form of β-glucans. The glucans have main chain β1 → 4 bond, and side chain β1 → 3 and 1 → 6 bond structures, and it has been suggested that branching occurs per number of sugar residues. Proteins and sugar chain moeities in Krestin are linked with each other by either O-or Nglycosidic bond [15]. In addition, coriolan, another antitumor polysaccharide derived from Trametes versicolor, was reported in 1971 [16].
P. linteus belongs to Hymenochaetaceae family, and is called souou in traditional Japanese medicine, and has been highly valued since ancient times. It has been referred to as the "mythical" mushroom since it grows extremely slowly in nature and artificial cultivation is also difficult. Research in South Korea succeeded in the mass cultivation of P. linteus Yoo (HKSY-PL2) strain, which has been shown to be more effective than most other strains. P. linteus has properties to enhance the natural healing capability of the body, and was developed as a pharmaceutical product called meshima. Mycelia culture of P. linteus activated dendritic cells and macrophages through increased secretions of interleukin 12 (IL-12), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) by T-cells, and enhanced the antitumor effects of NK cells [17]. A proteoglycan generated by P. linteus acted as an immunostimulant and disrupted the Reg IV/EGFR/Akt signaling pathway, thereby exhibiting tumor-inhibitory effects [18]. In addition, polysaccharides from P. linteus activated the P27kip1-cyclinD1/E-CDK2 pathway and induced S-phase cell cycle arrest in HT-29 cells, resulting in cellular damage [19].
Through their immunostimulatory properties, mushroom-derived polysaccharides and glycoproteins augment anticancer drugs, alleviate side effects, and contribute greatly to quality of life (QOL) improvement.

Chemical carcinogenesis and two-stage carcinogenesis theory
It has been acknowledged that many types of cancers are caused by environmental carcinogenic agents. In 1915, Yamagiwa and Ichikawa succeeded in inducing cancer by rubbing coal tar on rabbit ears [20]. The significance from this study was the skin cancer had metastasized to the rabbit lung. In 1941, Berenblum et al. applied carcinogenic agent benz[a]pyrene (B[a]A) and croton oil (seed oil of Croton tiglium) on mouse skin, and proposed a two-stage carcinogenesis theory that tumorigenesis occurs similarly to when B[a]A is applied continually [21,22]. Specifically, changes due to a carcinogenic agent were termed initiation, and changes due to croton oil were termed promotion. Later, Hecker reported the cancer-promoting ingredient of croton oil as 12-O-tetradecanoylphorbol-13-acetate (TPA). Many of these experiments are conducted using initiators 7,12-dimethylbenz[a]anthracene (DMBA) and TPA [23,24]. Fujiki et al. later reported on many mouse skin tumor promoters such as teleocidin [25]. Cancer begins when cells transform into latent cancer cells after undergoing initiation by receiving initiators or radiation. Subsequently, these cells become cancer cells after a long period of promotion process by promoters. Finally, after modifications through a process termed progression, the cells acquire the ability to divide infinitely, thereby clinically morphing to cancer. These steps occur in a continuous manner, and cannot be strictly distinguished from each other. When considering primary prevention, it is realistic to suppress the promotion process, which requires a long period of time and is known to be reversible to some degree. In addition, it has also become evident that cancer develops via similar mechanisms in many organs. Furthermore, TPA is known to activate Epstein-Barr virus (EBV). Although the prevalence of EBV is extremely high in Africa, the incidence of Burkitt's lymphoma greatly differs depending on the village [26]. It has been revealed that villages with greater incidence regularly utilized Euphorbia tirucalli and phorbol-esters, which are constituents of Euphorbia tirucalli and closely related to TPA. It is suggested these phorbol-esters are involved in the onset of Burkitt's lymphoma [27,28].

Screening for cancer preventative substances
We are conducting a screening for an antitumor substance using a method in which the suppressive effect against tumor promoter-induced inflammation is examined as a positive outcome index [29]. This method was utilized by Hecker et al. when they isolated and identified TPA and this method has been confirmed to be advantageous with high correlation as it employs a carcinogenesis experiment and skin from inbred (syngeneic) mice. Specifically, when TPA is applied on the auricle of female ICR mice, maximum swelling was observed 6-10 hours later. The mushroom extracts suppressed the TPA effects, as seen by swelling inhibition, and were confirmed by two-stage carcinogenesis experiments on mouse skin. We induced inflammation with TPA in mice and investigated methanol extracts of 27 edible mushrooms, 8 mushroom supplements, and 3 medicinal mushrooms, discovering the presence of promising mushrooms as shown in Table 1. Specifically, inhibitory effects were observed in: Russula delica, Lactarius deliciosus, Hypsizigus marmoreus (H. marmoreus), Mycoleptodonoides aitchisonii (M. aitchisonii), Naematoloma sublateritium for edible mushroom; Inonotus obliquus (chaga), meshima, Ganoderma lucidum (reishi), deer horn shape Ganoderma amboinense (rokkaku reishi), Pleurotus cornucopiae (golden oyster mushroom) for mushroom supplements; and Poria cocos (poria) and polyporus as medicinal mushrooms [30]. Of these mushrooms, the application of methanol extracts of H. marmoreus [31], M. aitchisonii [30], poria [32], chaga [33], and meshima [34] suppressed the promotion process. These results indicated that edible and medicinal mushrooms are effective cancer preventing foods. In addition, there is a method in which the suppressive effect against the EBV activation that is involved in the onset of Burkitt's lymphoma is examined as a positive outcome index [35]. Substances that were confirmed to have inhibitory effects through this method are thought to contribute to cancer prevention in those infected with EBV.   Figure 3-B shows the mean number of tumors at 20 weeks, where M. aitchisonii group presented 1.6 tumors in contrast to the vehicle control group that exhibited 11.2 tumors, confirming a 63% inhibitory effect [30]. Methanol extracts of H. marmoreus similarly suppressed the tumor promotion process [31].

Cancer preventative effects of edible mushroom
A screening for suppressive ingredients was, therefore, conducted; using inhibitory effects against TPA-induced inflammation as an index, active ingredients were isolated and their chemical structures were elucidated. The active ingredients were ergosterol (1) and ergosterol peroxide (2) (Figure 4), which are normal ingredients of mushrooms, and these were stronger than non-steroidal anti-inflammatory drug indomethacin as shown by their 50% inhibitory effects (ID 50 : 756 and 467 nM/ear, respectively vs. 908 nM/ear). These two sterols have been demonstrated to suppress the promotion process in mouse skin two-stage carcinogenesis experiments [31,36]. Other sterols (6-10) have been reported to inhibit the TPA-induced EBV activation (Table 2.) [37].

Cancer preventative effects of mushroom supplements
Mushroom supplements, such as meshima, chaga, and almond mushroom, are all believed to be beneficial for cancer, and utilized based on the wishes of cancer patients and their families. As shown in Table 1, supplements including reishi, rokkaku reishi, meshima, and chaga strongly suppressed TPA-induced inflammation [30]. Methanol extracts of Meshima and chaga strongly suppressed the promotion process in experiments involving DMBA and TPA carcinogens [33,34]. Furthermore, chaga and meshima suppressed the promotion process through oral administration [38,39].

Compound IC 50
Lucidenic acid F (20)   Piptoporus betulinus is a fungus in the Polyporaceae family and the surface of its fruiting body had been used as a strop for razor blades. It is known that the Iceman, as evidenced by a mummy from 5,000 years ago found in the Tyrol region glacier, carried around this mushroom to prevent wound suppuration [52,53]. Lanostane-type triterpenes (Figure 7) isolated from this mushroom suppressed TPA-induced inflammation [54].

Cancer preventative effects and active ingredients of medicinal mushrooms
Of the medicinal mushrooms, polyporus (Polyporus umbellatus; Polyporaceae family) is an herbal medicine that possesses diuretic effects, but is also known to suppress TPA-induced inflammation. Screening for the active ingredients of this mushroom resulted in the isolation of insect metamorphosis hormone sterols, and the structures of eight compounds including new compounds polyporoid A (58), polyporoid B (59), and polyporoid C (60) were elucidated ( Figure 8.) As shown in Table 6, the effects of these compounds in inhibiting TPA-induced inflammation (ID 50 ) were 117-682 nM/ear, which were greater than that of indomethacin [55].
The sclerotia of Poria cocos (Polyporaceae family) are referred to as poria, and due to their diuretic properties, and they are formulated in traditional Japanese medicine prescriptions. Additionally, they are also commonly formulated in traditional Japanese medicine prescriptions that are used as adjuvants. The oral administration of Juzentaiho-to and Rikkunshi-to, Japanese Kampo medicines, suppressed cancer promotion in mouse skin two-stage carcinogenesis experiments [56,57]. It has been shown that, for an effect to appear, the immune response that is decreased during carcinogenic process be activated [57]. Of the formulated ingredients in these prescriptions, hoelen showed the strongest effect in suppressing TPA-induced inflammation [58]. A screening for the active ingredients of hoelen was therefore conducted, and multiple lanostane-type triterpene acids were isolated and identified ( Figure 9) [32]. Of the poria-derived triterpenes, pachymic acid (71), 3-O-acetyl-16α-hydroxytrametenolic acid (70), dehydropachymic acid (79), 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid (75), dehydroebuliconic acid (81), and poricoic acids A (97) and B (94) had inhibitory effects against TPA-induced inflammation (ID 50 : 31-83 nM/ear), that were greater than that of indomethacin but similar to that of hydrocortisone (ID 50   hydroxytrametenolic acid (70), and poricoic acid B (94), all of which showed strong inhibitory effects, a mouse skin two-stage carcinogenesis experiment using DMBA and TPA demonstrated that they exhibited suppressive effects that were similar to that of the aforementioned ergosterol (1), ergosterol peroxide (2) and other triterpenes, even when 10% of the dosage of the latter compounds were administered [59]. These compounds have a carboxyl group (COOH) at the carbon 21 position (on side chain), and their suppressive effects decreased 90% when the COOH-group was methylated. It was discovered that COOH at the carbon 21 position plays an important role for activation [32]. Akihisa et al. isolated many new lanostanetype triterpene acids from poria, and reported their suppressive effects in TPA-induced EBV activation (Table 8) [60][61][62]. Moreover, they confirmed that 16-deoxyporicoic acid B (93), poricoic acid C (95), and 25-methoxyporicoic acid A (102) suppress the promotion process [60,61]. Of these compounds, poricotriol A was revealed to induce apoptosis and possess antitumor effects [63]. Pachymic acid and dehydrotumulosic acid strongly suppress PL-A 2, which is related to inflammation [64].

Conclusion
Mushroom polysaccharides and glycoproteins have antitumor mechanisms such as activating various immunocompetent cells and reinforcing the tumor aggressiveness of the host. Many mushroom-derived polysaccharides have very weak effects when administered orally.
However, with the advancement in food technology, the development of these polysaccharides as food products is progressing and their development as oral pharmaceutical products is also anticipated.
Poria and reishi are listed in the first treatise of Shennong Ben Cao Jing, and viewed as herbal medicines that help maintain health. Although some mushroom triterpenoids show strong suppressive effects similar to that of hydrocortisone, most result in a moderate antitumour promotor effect. It is expected that these triterpenoids, such as pachymic acid, may inhibit phospholipase A 2 . Nonetheless, since these mushrooms are edible and are used as supplements and herbal medicines, they are considered to have extremely low or no toxicity. Therefore, these triterpenoids from poria and reishi are a promising group of compounds. In particular, pachymic acid, ganoderic acid T, and lucidenic acid B, are leads in the search for cancer prevention drugs; the development of cancer prevention drugs with properties akin to tamoxifen is desired. When developing a preventative drug, the safety of the substance must first and foremost be considered.
There are many other challenges, such as further elucidating the mechanism, ascertaining the appropriate intake level, and supplying large amounts of the compound. The cooperation and collaboration of researchers from various fields will be necessary to address these issues.