Inhibitory effect of edible and medicinal mushrooms on TPA-induced inflammation in mice.
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 . In this chapter, we will introduce the anticancer activities of polysaccharides as well as the cancer prevention activities of sterols and triterpenes.
2. Mushroom-derived anticancer polysaccharides
Research on mushroom-derived β-glucans began when Chihara, Hamuro, and others at the National Cancer Center Research Institute in Japan isolated and purified lentinan, a β-1,3-glucan with branched chains formed by β-1,6-glycosidic bonds, from L. edodes in 1968 [2, 3]. Subsequently, many efficacy studies on lentinan, primarily concerning its antitumor activities, have been reported [4-6]. Ikekawa et al. intraperitoneally administered aqueous extracts of six types of edible mushrooms, and demonstrated their antitumor effects on cancer cell line sarcoma S-180 .
Upon such discoveries, polysaccharides lentinan and schizophyllan, glycoprotein krestin, and P. linteus mycelia extract meshima have been utilized as anticancer drugs.
Lentinan (Figure 1) demonstrated an effect to prolong the survival of patients with inoperable and relapsed stomach cancer in combination with a chemotherapeutic agent in a human double-blind controlled clinical trial. It has been revealed that its oral consumption, however, does not exhibit efficacy. In 1985, this compound was approved as an anti-malignant tumor agent (injectable solution), and has been prescribed to cancer patients as a pharmaceutical product. Subsequently, the antitumor effects of various mushroom extracts that contain β-glucan were reported in animal experiments . However, most of these studies administered mushroom extracts that contain β-glucan to animals via injection, and there are very few reports that showed its effect via oral consumption. There is, however, one such rare report; an epidemiological study that suggests mushroom intake via oral consumption may be effective . Intraperitoneal administration of lentinan suppressed 3-methylcolanthrene-induced tumor expression . In Lentinula edodes, α-(1,4)-glucan binds with TLR-4, thereby inducing monocyte differentiation and exhibiting cytotoxic effects in A549 human lung carcinoma cells .
Schizophyllan derived from Schizophyllum commune (Figure 2) is typically structured with β1 → 3 linkage and on rare occasions with β1 → 6 linkage between D-glucose monomers . Due to such structure, a rigid triple-helical structure is formed. In addition, this compound is used in anticancer drugs since it possesses antitumor activities . However, it is administered via intramuscular injection, and its effects via oral route in the manner of food consumption have not been elucidated. Although the mechanism of the antitumor activities of β-glucans including schizophyllan is not completely understood, it is thought that they activate macrophages and natural killer (NK) cells through respective β-glucan receptors, induce a helper T1 cell-dominant immune response state, and consequently exhibit antitumor activities [13, 14].
Krestin, an anti-malignant tumor agent, is a protein-bound polysaccharide derived from the mycelia of Trametes versicolor CM-101 strain. Since this drug does not cause serious side effects 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 × 104, 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 N-glycosidic bond . In addition, coriolan, another antitumor polysaccharide derived from Trametes versicolor, was reported in 1971 .
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 . A proteoglycan generated by P. linteus acted as an immunostimulant and disrupted the Reg IV/EGFR/Akt signaling pathway, thereby exhibiting tumor-inhibitory effects . 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 .
Through their immunostimulatory properties, mushroom-derived polysaccharides and glycoproteins augment anticancer drugs, alleviate side effects, and contribute greatly to quality of life (QOL) improvement.
3. 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 . 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 . 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 . 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].
4. 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 . 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 . Of these mushrooms, the application of methanol extracts of H. marmoreus , M. aitchisonii , poria , chaga , and meshima  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 . Substances that were confirmed to have inhibitory effects through this method are thought to contribute to cancer prevention in those infected with EBV.
|Scientific name||IR (%)|
|Pleurotus cornucopiae var. citrinopileatus||52**|
5. Cancer preventative effects of edible mushroom
Figure 3 illustrates the inhibitory effects of M. aitchisonii in mouse skin, two-stage carcinogenesis experiments. Specifically, Figure 3-A indicates the tumor incidence, where the vehicle control group showed the first tumor appearance in week 5 and tumor development in 93% of the mice in week 20. In contrast, mice that were given M. aitchisonii (M. aitchisonii group) showed the first tumor appearance in week 5 and tumor development in 53% of the mice in week 20. 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 . Methanol extracts of H. marmoreus similarly suppressed the tumor promotion process .
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 (ID50: 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.) .
|Ergosterol peroxide (2)||525|
6. 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 . 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].
Lanostane-type triterpenes depicted in Figure 5 were isolated and identified from chaga, and these triterpenes are known to show inhibitory effects in TPA-induced EBV activation (Table 3) [40, 41]. Eight types of lanostane-type triterpenes were isolated as active ingredients, and using the inhibitory effects against TPA-induced inflammation as an index, their 50% inhibitory effects (ID50: 125-458 nM/ear) indicated that they are stronger than non-steroidal anti-inflammatory drug indomethacin (908 nM/ear) (Tasble 4) . Of these triterpenes, inotodiol (13) and 3β-Hydroxylanosta-8,24-dien-24-al (15) suppressed the tumor promotion process [40, 41].
|Inotolacton B (19)||265|
Reishi belongs to the Ganodermataceae family, and is cut into appropriate sizes to be brewed in hot water and consumed as an extract since the fruiting body is woody and not suitable for direct consumption, or is consumed as medicinal alcohol. It has been described in Shennong Ben Cao Jing (or The Classic of Herbal Medicine) compiled in the Eastern Han Dynasty (25-220), as a life-prolonging miracle drug that nourishes life, and since then, it has been used for various medicinal purposes in China. Akihisa et al. isolated multiple lanostane-type triterpene acids from its fruiting body, and reported that they suppress EBV activation as shown in Table 5 [42-44]. Of these compounds 20-Hydroxylucidenic acid N (21) suppressed the promotion process in mouse skin two-stage carcinogenesis . With regards to triterpenes from reishi, ganoderic acid T (49) exhibited anticancer activities by inducing apoptosis in metastatic lung cancer cells mediated through mitochondria dysfunction and p53 expression . In addition, ganoderic acid T (49) suppressed the nuclear translocation of NF-κB and expression of MMP-9 and iNOS, thereby inhibiting invasion by cancer cells . Ganoderic acid DM (46) displayed anticancer activities by inducing G1-phase cell cycle arrest and apoptosis in MCF-7 cancer cells . Ganoderic acid A (44) and ganoderic acid H (42) suppressed breast cancer cell invasion by inhibiting AP-1 and NF-κB and consequently down-regulating Cdk4 expression . Ganoderic acid Me (48) inhibited tumor invasion by suppressing MMP2/9 expressions . Lucidenic acid B (26) exhibited anti-invasive activity through suppressing TPA-induced NF-κB and AP-1 DNA-binding activities thereby downregulating MMP-9 expression in HepG(2) cells . Lucidenic acid B (26) induced apoptosis through mitochondrial cytochrome release and the activations of caspase-9 and caspase-3 .
|Lucidenic acid F (20)||352|
|Methyl lucidenate F (21)||285|
|Lucidenic acid D2 (22)||287|
|Methyl l ucidenic acid D2 (23)||290|
|Lucidenic acid A (24)||280|
|Methyl l ucidenate A (25)||287|
|Lucidenic acid B (26)||354|
|Methyl lucidenate Q (27)||283|
|Methyl lucidenate L (28)||275|
|Lucidenic acid E2 (29)||280|
|Methyl l ucidenate E2 (30)||288|
|Lucidenic acid N (31)||332|
|Methyl l ucidenate C (32)||331|
|Lucidenic acid P (33)||286|
|Methyl l ucidenate P (34)||293|
|20-Hydroxy lucidenic acid F (35)||339|
|20-Hydroxy lucidenic acid D2 (36)||350|
|20-Hydroxy lucidenic acid E2 (37)||290|
|20-Hydroxy lucidenic acid N (38)||288|
|20-Hydroxy lucidenic acid P (39)||288|
|20(21)-Dehydrolucidenic acid A (40)||350|
|Methyl 20(21)-dehydrolucidenate A (41)||357|
|Ganoderic acid F (42)||293|
|Ganoderic acid C1 (43)||336|
|Ganoderic acid A (44)||291|
|Ganoderic acid C2 (45)||290|
|Ganoderic acid DM (46)||352|
|Ganoderic acid T-Q (47)||281|
|Ganoderic acid E (52)||281|
|Methyl ganoderate F (53)||289|
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 .
7. 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 (ID50) were 117-682 nM/ear, which were greater than that of indomethacin .
|Polyporoid A (58)||531|
|Polyporoid B (59)||682|
|Polyporoid C (60)||184|
|Polyporusterone A (61)||141|
|Polyporusterone C (62)||289|
|Polyporusterone B (63)||117|
|Polyporusterone G (64)||207|
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 . Of the formulated ingredients in these prescriptions, hoelen showed the strongest effect in suppressing TPA-induced inflammation . A screening for the active ingredients of hoelen was therefore conducted, and multiple lanostane-type triterpene acids were isolated and identified (Figure 9) . 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 (ID50: 31-83 nM/ear), that were greater than that of indomethacin but similar to that of hydrocortisone (ID50: 69 nM/ear). With regards to pachymic acid (71), 3-O-acetyl-16α-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 . 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 . Akihisa et al. isolated many new lanostane-type triterpene acids from poria, and reported their suppressive effects in TPA-induced EBV activation (Table 8) [60-62]. 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 . Pachymic acid and dehydrotumulosic acid strongly suppress PL-A2, which is related to inflammation .
|Tumulosic acid (69)||440|
|3-O-Acetyl-16α-hydroxytrametenoic acid (70)||31.1|
|Pachymic acid (71)||83.2|
|3β-Hydroxylanosta-7,9(11),24-trien-21-oic acid (75)||59.4|
|Dehydropachymic acid (79)||38.0|
|Dehydroeburiconic acid (81)||57.9|
|Polyporenic acid C (82)||201|
|3-Epidehydrotumulosic acid (84)||188|
|Poricoic acid B (94)||35.1|
|Poricoic acid A (97)||56.1|
|Poricoic acid AM (98)||148|
|Poricoic acid D (100)||243|
|Eburicoic acid (68)||465|
|Pachymic acid (71)||286|
|16α-Hydroxyeburiconic acid (72)||348|
|16α,25-Dihydroxyeburiconic acid (73)||299|
|25-Hydroxy-3-epitumulosic acid (74)||238|
|3-Epidehydrotrametnolic acid (75)||464|
|Dehydroebricoic acid (76)||460|
|15α-Hydroxydehydrotumulosic acid (78)||268|
|Dehydropachymic acid (79)||284|
|Dehydrotrametenonic acid (80)||310|
|Dehydroebriconic acid (81)||405|
|16α,25-Dihydroxydehydroeburiconic acid (83)||340|
|16α,27-Dihydroxydehydrotrametenoic acid (85)||269|
|5α,8α-Peroxydehydrotumulosic acid (87)||202|
|Poricoic acid HM (91)||219|
|25-Hydroxyporicoic acid H (92)||202|
|16-Deoxyporicoic acid B (93)||262|
|Poricoic acid C (95)||273|
|Poricoic acid CM (96)||332|
|Poricoic acid AM (98)||195|
|25-Hydroxyporicoic acid C (99)||201|
|Poricoic acid D (100)||198|
|Poricoic acid DM (101)||207|
|25-Methoxyporicoic acid A (102)||268|
|26-Hydroxyporicoic acid DM (103)||187|
|6,7-Dehydroporicoic acid H (104)||193|
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 A2. 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.
Dr. Michio Takido, my mentor and professor emeritus at School of Pharmacy, Nihon University, passed away on August 25, 2014. My research and attitude towards this research were greatly influenced by his guidance. This article is dedicated to Dr Takido with profound gratitude.