cancer review

International Immunopharmacology 7 (2007) 701–724www.elsevier.com/locate/intimp

Review

Immunomodulating and anticancer agents in the realm ofmacromycetes fungi (macrofungi)

Mohammad-Fata Moradali a, Hossein Mostafavi b,⁎,Shirin Ghods a, Ghorban-Ali Hedjaroude a

a Plant Protection Department, Faculty of Agriculture, Tehran University, Karaj, Iranb Organic Chemistry and Biochemistry Department, Faculty of Chemistry, Tabriz University, Tabriz, Iran

Received 26 November 2006; received in revised form 7 January 2007; accepted 8 January 2007

Abstract

Nowadays macrofungi are distinguished as important natural resources of immunomodulating and anticancer agents and withregard to the increase in diseases involving immune dysfunction, cancer, autoimmune conditions in recent years, applying suchimmunomodulator agents especially with the natural original is vital. These compounds belong mainly to polysaccharidesespecially β -D-glucan derivates, glycopeptide/protein complexes (polysaccharide-peptide/protein complexes), proteoglycans,proteins and triterpenoids. Among polysaccharides, β (1→3)-D-glucans and their peptide/protein derivates and among proteins,fungal immunomodulatory proteins (Fips) have more important role in immunomodulating and antitumor activities.Immunomodulating and antitumor activity of these metabolites related to their effects to act of immune effecter cells such ashematpoietic stem cells, lymphocytes, macrophages, T cells, dendritic cells (DCs), and natural killer (NK) cells involved in theinnate and adaptive immunity, resulting in the production of biologic response modifiers. In this review we have introduced themedicinal mushrooms' metabolites with immunomoduling and antitumor activities according to immunological evidences and thendemonstrated their effects on innate and adaptive immunity and also the mechanisms of activation of immune responses andsignaling cascade. In addition, their molecular structure and their relation to these activities have been shown. The importantinstances of these metabolites along with their immunomodulating and/or antitumor activities isolated from putative medicinalmushrooms are also introduced.© 2007 Elsevier B.V. All rights reserved.

Keywords: Macrofungi; Immunomodulating; Antitumor; Glycopeptide/protein complexes; Fips

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7021.1. Macrofungi characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7021.2. Macrofungi in medicine as immunomodulating and antitumor agents . . . . . . . . . . . . . . . . . . . . . 702

2. Immunomodulating and antitumor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7033. Effects of macrofungi metabolites on hematopoietic stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705

⁎ Corresponding author. Tel.: +98 4113393120; fax: +98 4113340191.E-mail address: [email protected] (H. Mostafavi).

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702 M.-F. Moradali et al. / International Immunopharmacology 7 (2007) 701–724

4. Effects of macrofungi metabolites on the innate (non-specific) immunity . . . . . . . . . . . . . . . . . . . . . . 7054.1. Effect on macrophages and APC (Antigen-presenting cell) activation . . . . . . . . . . . . . . . . . . . . . . 705

4.1.1. Effect on dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7054.1.2. Effect on NK cells (cytotoxicity and interferon production) . . . . . . . . . . . . . . . . . . . . . 7064.1.3. Complement system and macrofungi metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . 706

5. Effects of macrofungi metabolites on the adaptive (specific) immunity . . . . . . . . . . . . . . . . . . . . . . . 7065.1. Macrofungi metabolites as antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7065.2. Biologic response modifiers activation (cytokine production) and macrofungi metabolites . . . . . . . . . . 707

6. Major immunomodulating and antitumor agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7086.1. 1-Polysaccharides and glyco-conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7086.2. 1-1-Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708

6.2.1. D- and MD-fraction from G. frondosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7096.2.2. 1-2-Glycoproteins (polysaccharide-protein complexes) . . . . . . . . . . . . . . . . . . . . . . 7116.2.3. 1-3-Glycopeptides (polysaccharide-peptide complexes) . . . . . . . . . . . . . . . . . . . . . . 712

6.3. 1-4-Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7126.3.1. GLIS from G. lucidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712

7. The structure-activity relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7128. Chemical and structural modification of polysaccharides and their derivates . . . . . . . . . . . . . . . . . . . . . 7139. β-D-Glucan receptor(s) and signaling cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714

10. 2-Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71511. 3-Fungal immunomodulatory proteins (Fips) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716

11.1. Fips structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71611.2. Fips immonumodulating activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717

12. Clinical and experimental evidences for anticancer properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71713. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720

1. Introduction

1.1. Macrofungi characteristics

Fungi are classified in the independent kingdom Fungiamong thorough organisms. They have the cells with truenucleus (Eucaryotes), and reproduce by spores. They areC-heterotrophic and obtain the essential nutrients throughbreaking down organic substances and absorbing them.The fungal body (thallus) is either a single cell or athreadlike structure (hypha). A large number of branchinghypha together constitutes the mycelium which givesraise to usually multiform spore-producing cells. In mosttypes of fungi, spore-producing cells (basidia, asci,conidiophores) form a part of special structure made uphyphal tissue and called the fruit body (sporocarp).Although the fruit bodies are given to extraordinaryvariations in shape, size and coloring, these do remainfairly constant in individual fungus groups. The “mush-room” is a popular term that applied for the fungus hasobservable fruit body with the naked eye. Nowadays,fungi kingdom includes main five phyla includingChytridiomycota, Zygomycota, Glomeromycota, Asco-mycota and Basidiomycota. They differ with each otherprincipally in ontogenesis, phylogeny, sexual and asexualreproduction, how producing spores, elements constitut-

ing cell wall etc. The mushrooms are also called“Macromycetes” (a Latin name) in opposite the term“Micromycetes” (a Latin name for the fungi that areinvisible with the naked eye). Among the Fungi kingdomtheMacromycetes arranged in the phylumBasidiomycotaand a few numbers in the Ascomycota and evolutionaryBasidiomycetes are known as the higher fungi. In humanlife fungi are well known and they have important roles indifferent fields close to human life as friend or foe.

1.2. Macrofungi in medicine as immunomodulating andantitumor agents

Applying fungi as medicines dates back to 3000 BCwhen macrofungi are used to remedy diseases bymankind especially in the traditional oriental therapiesand after discovering of Penicillin (1929), fungi wereregarded as rich sources of natural antibiotics and otherbioactive compounds. Macrofungi such as Ganodermalucidum, Lentinus edodes, Fomes fomentarius, Fomi-topsis officinalis and many others have been used toremedy different diseases for hundreds of years inChina, Japan, Korea and the Slav regions [1]. Now-adays, constituent molecules of macrofungi organellesand secondary metabolites are known as bioactive com-pounds that belong to polysaccharides, glycoproteins,

Table 1The different groups of bioactive compounds isolated from macrofungi

Main compoundgroup

Example Medicalpotentiality

Reference

Polysaccharides Grifolan Immunomodulator [61]Lentinan Antitumor [111]Schizophyllan Antiviral [112]

Antimicrobial [113]Polysaccharide-

peptidePSP Antitumor [35]

PSK Antiviral [114]Antimicrobial [115]Cytotoxic [116]

Proteins Fips Immunomodulator [87]Ganoderic acids [117]Ganoderiol Anti-HIV activity [118]Ganoderenic acids antitumor [119]Lucidenic acids Cytotoxic [119]

Terpenoids Ganolucidic acids Histamine releaseinhibition

[120]

Lucidumols Antihypertension(ACEinhibitor)

[121]

Ganoderols Anti-inflammatoryApplanoxidic acids

Steroids Polyoxygenatedderivates ofergosterol

Cytotoxic [122]

Antitumor [123]antibacterial [124]

Fatty acids linoleic acid Antimutagenic [125]palmitic acid antibacterial [126]11-octadecanoicacid

Organicgermanium

Bis-β-carboxyethyl-germaniumsesquioxide

Antitumorimmunomodulating

[127]

Nucleotides Adenosine Platelet aggregateinhibition

[128]

Polyacetyleniccompounds

Biformyne,agrocybin Nemotinicacid, marasmin,quadrifidins

As antibiotic [129]

703M.-F. Moradali et al. / International Immunopharmacology 7 (2007) 701–724

proteoglycans, terpenoids, fatty acids, proteins, lectins,etc that possess certain medicinal properties (Table 1).Particularly, they can be added to the diet and usedorally that they are considered as a safe and usefulapproach for disease treatment. These compounds arefound in fruiting bodies, mycelia and spores and culturebroth of macrofungi. Several major substances withimmunomodulating and/or antitumor activity have beenisolated from them. These include mainly polysacchar-ides, glycopeptide/protein complexes, proteoglycans,proteins, triterpenoids.

Immunomodulating activity of compounds isolatedfrom medicinal macrofungi, related their effects to act onimmune effecter cells such as hematpoietic stem cells,lymphocytes, macrophages, T cells, DCs, and NK cells

involved in the innate and adaptive immunity, resulting inthe production of cytokines. The therapeutic effects ofthese compounds such as antitumor and antiinfectiveactivity and suppression of autoimmune diseases havebeen associated in many cases with their immunomodu-lating effects. Immunomodulators (biological responsemodifiers) can be effective agents in treating and preven-ting diseases and illnesses andwith regard to the increase indiseases involving immune dysfunction and cancerand along with them infectious diseases in recent years,medical researchers and clinicians are interested inImmunotherapy as well as the discovery of novelimmune-potentiators and compounds with powerfulremedy potential without side effects, pathogenic resis-tance or affecting normal cell division especially foranticancer and antiviral agents. In this review article wehave introduced the macrofungi metabolites with immu-nomoduling and antitumor activities and then havedemonstrated their effects on innate and adaptive immunityand also mechanisms of activation of immune responsesand signaling cascade. In addition, theirmolecular structureand their relation to these activities have been shown. Theimportant instances of these metabolites along with theirimmunomodulating and/or antitumor activities isolatedfrom putative medicinal macrofungi are also introduced.Hence we have mentioned the scientific names of someputative medicinal macrofungi that are more studied forfinding immunomodulatory and antitumor properties.

2. Immunomodulating and antitumor activity

Compounds that are capable of interacting with theimmune system to upregulate or downregulate specificaspects of the host response can be classified as immu-nomodulators or biologic response modifiers. Whethercertain compounds enhance or suppress immune res-ponses can depend on a number of factors, including dose,route of administration, and timing of administration ofthe compound in question. The type of activity can alsodepend on their mechanism of action or the site of activity[2]. In this way these compounds' affect on the differentcells types involved hematpoietic stem cells, innate (non-specific) and adaptive (specific) immune systems and alsocytokine networks and signaling pathways. Many com-pounds isolated from macrofungi that are known asimmonumodulators that almost related to polysaccharidesand their peptide or protein derivates and Fips. Also insome cases triterpenoids have closed to immunomodulat-ing activity. These compounds are also worthwhile an-tiinfective and antitumor agents.

A wide range of antitumor activity is proven for thesefungi. Numerous reports have documented the ability of


these compounds especially β-D-glucan derivates to non-specifically activate cellular and humoral components ofthe host immune system so that they increase functionalactivity of macrophages, mononuclear cells, and neutro-phils [3–5]. Although the body's defense against microbialattack and against spontaneously arising malignant tumorcells comprises a dynamic orchestrated interplay of innateand acquired immune responses. Innate immunity (havingmacrophages, neutrophils, NK and DCs as gatekeepers), isregulated by chemical-messengers or cytokines and byactivation of inflammatory and acute phase responses [6].The mononuclear phagocyte system (e.g., macrophagesand monocytes), DCs and certain lymphocytes (e.g., NKcells) play a number of important roles including the re-cognition and destruction of abnormal cells. Stimulatedmacrophages and NK cells produce cytokines such asinterferons, interleukins and others that are targeted to-wards destroying cancer cells. Specific immunity to ab-normal cells or tissues includes humoral (e.g. generatesantibodies) and cell-mediated immunity (also promotesinflammatory responses and ultimately kills infected or

Fig. 1. Mechanisms of antitumor activity of lentinan as a β-D-glucan (accordiNK, natural killer cells; IL-1, -2 and -13, interleukin-1, -2, -13; CSF, colonycytolytic T lymphocyte; CTL, cytolytic (cytotoxic) T lymphocyte; BL, B ly

abnormal cells). So a fully functional immune response iscritical to the recognition and elimination of tumor cells.With regard to effects of macrofungi metabolites and ext-racts on antitumor effector cells, in the recent years, me-dical researchers have attempted to identify macrofungithat have compounds that are capable to stimulate com-ponents of immune system toward discovering more ef-fective antitumor agents especially with natural originationand with more safety. In this way the basic strategy under-lying immunomodulation is to identify aspects of the hostresponse that can be enhanced or suppressed in such a wayas to augment or complement a desired immune response.For example lentinan is understood as a pure β-D-glucanderivate isolated from L. edodes that affects and stimulatesthe key immune mechanisms mediate destruction of tumorcells (Fig. 1). Such mechanisms are also looked upon forpeptide or protein derivates of β-D-glucan [6]. Of course, itshould not be forgotten that there are other pathways andmechanisms that macrofungi can prevent and suppresstumor cell growth. In this way, it has proved that differentcompounds of G. lucidum act via different pathways

ng to Chihara [6]). Mac, macrophages; TL(H), T-lymphocyte (helper);-stimulating factor; MAF, macrophage-activating factor; PC-TL, pre-mphocyte.


recognized as enhancing detoxification of carcinogens,increased expression and activity of Phase II enzymes,inhibition of organ exposure of carcinogens due to reducedabsorption or increased excretion, decreased expressionand activity of Phase I enzymes, decreased formation oftoxic metabolites and adduct formation with macromole-cules, antioxidative and radical-scavenging effect, antipro-motion effect, antiproliferation, induction of differentiation,direct cytotoxicity, induction of cell-cycle arrest, antipro-liferation and modulation of signaling transduction mole-cules, antiprogression and tumor growth inhibition,antimetastasis, and anti-angiogenesis [7].

Chihara was one of the first ones that reported theantitumor properties of the macrofungi. He stated thatlentinan “was found to almost completely regress the solidtype tumors of Sarcoma 180 and several kinds of tumorsincluding methylchloranthrene-induced in synergic host-tumor system A” [8]. The antitumor effect of lentinan wasoriginally confirmed by using Sarcoma 180 transplantedin CD-1/ICD mice. Later, it showed prominent antitumoractivity not only against allogenic tumors such as Sarcoma180, but also against various synergic and autochthonoustumors, and it prevented chemical and viral oncogenesis.

3. Effects of macrofungi metabolites on hematopoieticstem cells

Many cells involved in the immune response arederived from undifferentiated hematopoietic stem cellsin the bone marrow and fetal liver. These differentiateinto various cell lineages under the influence of mi-croenviromental factors such as cell-to-cell interactionsand the presence of soluble or membrane-bound cyto-kines. In other words hematopoiesis is regulated byseveral different cytokine growth factors produced bybone marrow stromal cells, T cells, and other cell types.Various metabolites, especially polysaccharides isolatedfrom medicinal mushrooms that showed to affect bone-marrow cells (BMCs) and to induce hematopoiesis. It isproved that these metabolites are able to differentiate theBMCs to Colony-forming units granulocytes-macro-phages (CFU-GM) and erythroid burst-forming units(BFU-E) that will be explained in the follow sections [9].

4. Effects of macrofungi metabolites on the innate(non-specific) immunity

4.1. Effect onmacrophages and APC (Antigen-presentingcell) activation

Macrophages are involved at all stages of the immuneresponse. They function in phagocytosis, antigen proces-

sing and presentation, secretion of cytokines, andantibody-dependent cell-mediated cytotoxicity.

The effects of macrofungi extracts and metaboliteson macrophages have been extensively studied in vitroand in vivo. It has been proven that some macrofungiextracts can activate macrophages to produce variousmediators. Polysaccharides isolated from these fungihave a role as APCs and stimulate them to producecertain cytokines and subsequence of these events is theactivation of other immune cells. Also after contact ofthese antigens with macrophages, they mediate process-es as APCs that involved in the cell-mediate immunitythat will be explain in later sections.

Macrophages have an important role in the initialresponse to infection before T- and B-cell enhancedimmunity can act. They act as a rapid protective that canrespond before T-cell-mediated amplification has takenplace. Then they take part in the initiation of T-cellactivation as APCs. APCs leads to T cells respond toantigen and they release cytokines which activatemacrophages. Following macrophage activation, anti-microbial, antitumor and production of other cytokinestake place. Macrofungi extracts can mediate theseevents. For instance, a glycoprotein, PSPC, isolatedfrom Tricholoma giganteum could restore and increasephagocytic function of macrophages of the tumor-bearing mice. It could also exhibit indirect cytotoxicityagainst P815 and L929 by activating macrophages torelease the mediators, such as nitric oxide (NO) andtumor necrosis factor α (TNF-α) [10].

4.1.1. Effect on dendritic cellsDCs are bone marrow-derived immune accessory

cells that are found in epithelial and lymphoid tissuesthat are morphologically characterized by thin membra-nous projections. They also function as APCs for nativeT lymphocytes. Different macrofungi metabolites espe-cially β-D-glucans and their derivates are capable ofinducing the maturation of bone marrow-derived DCsand also increase the membrane molecules in them.Similar to effect of these polysaccharides on macro-phages as an antigen, DCs also affected by them andstimulation of cell-mediate immunity occurs. Subse-quently processes activated in this immunity leads toactivation of antimicrobial and antitumor effector cellsand thus these compounds can be effective to inductionof tumor immunity. Besides activating native T cells,they can directly activate native and memory B-cells.DCs at different stages of differentiation can regulateeffectors of innate immunity such as NK cells and NK Tcells. The induction of tumor immunity can be initia-ted by the effectors of innate immunity and further


developed by cells of adaptive immunity, with DCsplaying a central regulatory role [9].

4.1.2. Effect on NK cells (cytotoxicity and interferonproduction)

NK cells are a subset bone marrow-derived lympho-cyte that are capable of lysing or killing infected ortumor cells. Cytotoxicity and cytokines production suchas Interferon-γ (IFN-γ), TNF-α, and granulocytemacrophage-colony stimulating factor (GM-CSF) thatcan modulate natural and specific immune responses aretwo relevant functions of NK cells. They osmoticallylyse target cells and induce apoptotic cell death. Theyalso have an important role in the inflammatoryresponse so that they can enter sites of inflammationwhere they can be stimulated by a cytokine Interleukin-(IL-) 12 that is produced by activated macrophages.Some macrofungi metabolites exhibit stimulatingeffects on NK cells. They are capable to stimulatesecretion of IL-2 that as known as stimulating ofproliferation of NK cells. Because of NK cells serve as arapidly acting first-line defense mechanism that isinvolved in the early destructive events followingtumor implantation, stimulation of these cells by thesemetabolites is very important in tumor immunity. Notonly IL-2 production induced by these metabolite butalso induction of cytokines and immune factors occurs[1,2,6]. For instance, oral administration of SSG (apolysaccharide) isolated from Sclerotinia sclerotiorumto mice had enhanced the activities of NK cells in spleen[11].

4.1.3. Complement system and macrofungi metabolitesComplement is a system of serum and cell surface

proteins that interact with one another and with othermolecules of the immune system to generate importanteffectors of innate and adaptive immune responses.Components of the complement system (i.e. activatedcomponents C3a, C3b through to C9) mediate andamplify immune reactions. Following the release ofchemotactic factors and histamine C3a this inducesconsiderable inflammation and tissue damage at thesites of reactions with antibodies. Residual C3bcomponent bound to the antigen–antibody complexesattaches to C3b receptors present on macrophages andthus acts as an opsonin, promoting enhanced phagocy-tosis. Although there are a few reports concerning theeffects of macrofungi metabolites on complementsystem but it is reported that some β-D-glucans (e.g.lentinan) and glycoproteins and glycopeptides arecapable of activating a complement system and inhibittumor growth via alternative pathway [9]. It is eluci-

dated that inhibitory activity of GU-P (a polysaccharideisolated from Grifola umbellata) and PSK on sarcoma180 solid tumor growth is due to the C3 activation [12].Also, an alkali extract isolated from cultured myceliumof G. lucidum activated classical and alternative path-ways of a complement system [7].

5. Effects of macrofungi metabolites on the adaptive(specific) immunity

5.1. Macrofungi metabolites as antigens

Affecting of macrofungi metabolites on the cells,cytokines and other factors involved in the adaptiveimmunity are well known especially for β-glucans andtheir derivates and proteoglycans. Concerning theseaffecting, it is understood that mentioned metabolites,act as the antigens, play role in the first step of activationsignaling pathway in the APCs. Subsequent of thisactivation and signaling is humoral- and cell-mediateimmunity induction. As mentioned above, in virtue ofappearing of certain different receptors on the APCs anddisplaying of Th cells to recognize and interact withantigen-major histocompatibility complex (MHC) IImolecule complex, various cytokines (IL-12 and IL-4)are produced and they affect on activation of B cells, Tccells, macrophages and other T cells that their results areas inflammation, antimicrobial and antitumor activities(Fig. 2). For example, a partially purified polysaccharideisolated from Antrodia camphorata via promoting aTh1-dominant state and killer activities and increasingIL-12 secretion act as an antitumor fungal polysaccha-ride [13]. Also it is showed that β-glucans have variouseffects on the Th1- or Th2-dependent antibody sub-classes and, in particular, that SSG form S. sclerotioruminduces the development of Th1 cells via the IL-12pathway. Stimulating Th1 to produce INF-γ is anotherimmunomodulating activity that this group of macro-fungi can cause [14]. Activation Th1 immune responsedue to the increased level of IL-12 and -18 bypolysaccharides isolated from Agaricus brasiliensis isalso reported [15].

Thymus-derived regulatory T-cell populations, in-cluding naturally occurring CD4+CD25+T cells andinducible IL-10 or TGF-β-producing TR/TH3, developin the periphery from TH cells depending on thetolerance-inducing micro-environment in which theseT cells reside [9]. The downstream immune responseis chosen depending on which subtype of T cell isactivated, which means that the proportion of theactivated sub-types influences phylaxis immunity andantitumor immunity. This control system is also affected

Fig. 2. Schematic representation of the affecting of immunomodulatory macrofungi metabolites on the adaptive immune system leading to activationof antimicrobial and antitumor pathways. Ag, antigen; PPCs, polysaccharide-peptide/protein complexes; FIPs, fungal immunomodulatory proteins;APC, antigen-presenting cell; TLR, toll-like receptor; CR1, complement receptor 1; AC, activated complement; NF-kB, nuclear factor kappa B; CD,cluster designation; MHC II, major histocompatibility complex; TCR, T-cell receptor; NO, nitric oxide; IL, interleukin; IFN-γ, interferon γ; LT,lymphotoxin; TH, helper T cell; NK cell, natural killer cell; CTL, cytotoxic T lymphocyte.


by the production of IL-1β, IL-12, and IL-18 by APC[9,16].

5.2. Biologic response modifiers activation (cytokineproduction) and macrofungi metabolites

Cytokines are known as biologic response modifiersthat modulates inflammation, immunity and hematopoi-esis. They are produced during the activation and ef-fector phases of innate and adaptive immunity andserves to mediate and regulate immune and inflamma-tory responses. Many macrofungi metabolites especiallypolysaccharides and their derivates and Fips affect on

cytokine production (known as multi-cytokine indu-cers). Hence these compounds are also known as bio-logical response modifiers as known as cytokines. Thesecompounds with affecting cytokine production are cap-able to induce various changes in immune system. It isproved that these metabolites can increase messengerribonucleic acids (mRNAs) encoding cytokines. Forexample, the antitumor activity of GL-B (a polysaccha-ride from Ganoderma) was derived from promotingmRNA expression of TNF-α and IFN-γ, resulting inTNF-α and IFN-γ release [17]. Synthesis of cytokineshappened following recognition of these metabolitesby cell surface receptors. This event leads to the

Table 2Some cytokine-production stimulating or suppressing compounds belong to different groups isolated from macrofungi

Agent Group IL-1 IL-2 IL-3 IL-4 IL-6 IL-8 IL-12 IL-18 LT TNF-α IFN-γ GM-CSF MIF

D-fraction PS SU ST ST ST STGrifolan PS ST ST STPG101 PS ST SU STlentinan PS ST ST ST ST ST ST ST STPL PS SU SU STSSG PS STPSK GP ST ST ST ST ST STPSP GP ST STLZ-8 Fip ST STFip-vvo Fip ST ST ST

ST, stimulating; SU, suppressing; PS, polysaccharide; PPC, GP, glycopeptide; Fip, fungal immunomodulating protien.


transcription of genes responsible to code cytokines.Cytokines often influence the action of other cytokinesand act upon many different cell types. For instance, it isshowed that many β-D-glucans induce to produce IL-12.IL-12 is known as a macrophage-derived activator ofNK cell cytolytic function, although it is appreciated tobe a potent inducer of IFN-γ production by Tcell as wellas NK cells. Consequently, macrofungi metabolites areable to induce to stimulate or suppress cytokinesproduction (Table 2) that mediates innate immunityand includes antiviral cytokines (e.g. IL-12 and IL-15),pro-inflammatory cytokines (e.g. TNF-α, IL-1, IL-6 andchemokines) and regulatory cytokines (e.g. IL-10).

To evaluate immunomodulating activity of macro-fungi extracts contain polysaccharides, it is seen thatL. edodes and Grifola frondosa increased TNF-αproduction approximately from 500–700 pg/ml and300–500 pg/ml and NO production 4–7 μM and 5–7 μM respectively. They finally decreased TNF-α levelto 500 and 260 pg/ml and NO level to 5 and 4 μMrespectively through the growth periods in murine peri-toneal macrophages [18]. GLP known as a polysaccha-ride from G. lucidum could induce a marked increase inthe gene expression levels of interleukin IL-lα (2-fold),IL-lβ (3-fold), TNF-α (2-fold), IL-12 p35 (up to 6-fold),and IL-12 p40 in the splenocytes. In the macro-phages, GLP promoted a remarkable increase in thegene expression levels of IL-lβ (2.5- to 3-fold), TNF-α(up to 6-fold), and GM-CSF (up to 2-fold) [19].

Studies on the lentinan showed that it increase thegene expression level of IL-1β and IL-1α cell lines [20].Also, IL-1 is not produced without this stimulation andthis cytokine is known to have a wide range of biologicalactivities on many different target tissues, including Bcells, T cells and monocytes. Increasing of production ofIL-1 causes inducing of production of various othercytokines such as TNF-α and this cytokine induces lysisof malignant cells and regression of some animal tumor

[20]. It is indicated that the β-glucan-related polysac-charides of the macrofungi activate macrophages torelease NO which is an important chemical messengerfor the induction of many biological responses [7].

6. Major immunomodulating and antitumor agents

6.1. 1-Polysaccharides and glyco-conjugates

Numerous bioactive polysaccharides, glycoproteins,glycopeptides, and proteoglycans from macrofungi areconsidered as immunomodulators affecting on prolifer-ation and differentiation of immune cells and cytokines,interleukins and receptors production due to recognitionthese compounds by the certain receptors located on theleukocytes and other immune cells that lead to enhancethe innate and cell-mediate immune responses. In virtueof these activities also induction of different types ofantitumor effector cells such as cytotoxic T cells, NKcells and macrophages occur. In addition to theseactivities some of them possess antiviral, antibacterial,antifungal and antiprotosoal activities [21]. Macrofungiare rich in such components in their cell wall structures.These compounds structurally are divided as following:

6.2. 1-1-Polysaccharides

Polysaccharides are carbohydrate polymers that can befound abundantly in higher fungi cell wall. They involvedifferent chemical compositions including β-glucans,hetero-β-glucans, heteroglycans, α-manno-β-glucan com-plexes that are found in macrofungi and all haveshowed immunomodulating and antitumor properties(Table 3). Among them β-glucans are most importantpolysaccharides with immunomodulating and antitumoractivity. β-glucans are glucose polymers that can exist as anon-branched (1→3)-β-linked backbone or as a (1→3)-β-linked backbone with (1→6)-β-branches (Fig. 3) and they

Table 3Some immunomodulating and antitumor polysaccharides isolatedfrom macrofungi

Scientific name Bioactive compound Reference

Lentinusedodes

Galactoglucomannan [130]

Inonotusobliquus

Xylogalactoglucan [109]

Polyporusconfluens

Xyloglucan [109]

Flammulinavelutipes

Galactomannoglucan [131]

Ganodermatsugae

Glucogalactan, Arabinoglucan [132]

Ganodermalucidum

β-(1→3)-glucuronoglucan,Mannogalactoglucan

[133]

Hericiumerinaceum

Galactoxyloglucan, Mannoglucoxylan,Glucoxylan, Xylan

[109]

Pleurotuspulmonarius

Xyloglucan, Mannogalactoglucan,Mannogalactan, Glucoxylan

[134]

Grifolafrondosa

Mannoxyloglucan, Xyloglucan,β-(1→6); β-(1→3)-glucan,Mannogalactofucan

[132]

Agaricusblazei

Mannogalactoglucan, β-(1→6);α-(1→3)-glucan, α-(1→4); β-(1→6)-glucan, α-(1→6); α-(1→4)-glucan,

[109]

Riboglucan, Glucomannan, β-(1→2);β-(1→3)-glucomannan, β-(1→6);β-(1→3)-glucan

[133]


occur as a primary component in the cell walls of higherfungi in the great deals. Heteroglucans side chains containglucuronic acid, xylose, galactose, mannose, arabinose

Fig. 3. Schematic representation of the molecular structure of β-(1→3)-D-glglucan (c), polysaccharide peptide/protein complex (d).

and ribose that may combined with other components.Glycans are other polysaccharides that have been found inmacrofungi. These polysaccharides, in general, containunits other than glucose in their backbone. They areclassified as galactans, fucans, xylans, and mannans bythe individual sugar components in the backbone.

More than 100 types of polysaccharides withbiological activities have been isolated from the fruitbody and mycelia of G. lucidum and most of them havea molecular weight range from 4×105 to 1×106 in theprimary structure [22]. Lentinan, schizophyllan (alsocalled SPG, sonifilan, sizofiran), grifolan or GRN, SSG,GLPS, D- and MD-fraction, PL, PG101, CA1, SCG aremacrofungi polysaccharides are more studied forimmunomodulating and/or antitumor activities.

6.2.1. D- and MD-fraction from G. frondosaMD-fraction enhances BMCs growth and differenti-

ation into the CFU-GM. This compound that obtained byfurther purification of D-fraction has enhanced directlyCFU-GM response of BMCs progenitors and has en-hanced recovery of the CFU-GM response after doxo-rubicin induced hematopoietic suppression [23]. Thus,these results can suggest that this compound can reducehematopoietic suppression induced by chemotherapy.

In different studies, it is elucidated that antitumoractivity of D-fraction and MD-fraction relate to theireffects on immune cells and factors that have importantrole in the antitumor activity. These compounds are

ucan (a), α-(1→3)-D-glucan (b), 1,6-Monoglucosyl-branched 1,3-β-D-


capable of releasing IL-12 from macrophages and DCsand acts on T lymphocytes and NK cells to stimulateINF-γ production and cytolytic activity. Monitoring oflevels of NK cell cytotoxic activity in cancer patientsreceiving D-fraction has showed that elevated levels ofcytotoxic activity were maintained for one year. To in-vestigate and elucidate the mechanisms of this activity,examination in the tumor-bearing mice showed that D-fraction can markedly suppress tumor growth withincrease TNF-α and INF-γ production from spleen cellsand a significant increase in TNF-α expression in NKcells [24,25]. Thus clinical study on patient with lungand breast cancer has showed that D-fraction is able toenhance and maintain peripheral blood NK cell activity[24]. Also it is showed their control of the balancebetween T lymphocyte subsets Th1 and Th2. Thiscompound decreases the activation of B cells andpromotes the activation of Th cells, resulting inenhanced cellular immunity. It also induces theproduction of INF-γ, IL-12p70, and IL-18 by wholespleen cells and lymph node cells, but suppressed that ofIL-4. These results suggest that antitumor activity of D-fraction arose affecting on IL-12 production frommacrophage and lead to increase NK cells and establishTH1 dominance which induces cellular immunity [26].

6.2.1.1. Grifolan from G. frondosa. Grifolan is amacrophage activator which augments cytokine pro-duction without dependence on endotoxins [27] and itenhances mRNA level of IL-6, IL-1 and TNF-α ofmacrophages [28].

6.2.1.2. PL from Phellinus linteus. PL is an acidicpolysaccharide that is introduced as an immunomodula-tor, anti-inflammatory and antitumor agent. PL can mo-dulate circulating cytokine responses in lippolysaccharide(LPS)-treated mice and administration of this compoundin vivo decreased IL-2 and TNF-α production insplenocytes and enhance spontaneous cell apoptosis inmacrophages and lymphocytes stimulated with LPS invitro [29]. Also this compound can induce maturation ofbone-marrow-derived DCs and readies them for T cell-mediated immune responses and increase membranemolecules includingMHC class I, II, CD80+, and CD86+,and IL-12p70 in DCs [30]. The inhibitory effect of PL onthe growth of MCA-120 tumor cells was associated withits immunoregulating properties including the inductionof IL-12 and IFN-γ production leading to a TH1 domi-nant state [30]. Also the administration of PL could in-duce immunomodulating and antitumor activities viamaturation of CD11c+CD8+DCs in tumor-bearing mice.Also PL selectively activates murine B cells [31].

6.2.1.3. PG101 from Lentinus lepideus. This com-pound can enhance CFU-GM and BFU-E. These resultsthat proved in the study on radiation-treatedmice suggestthat this compound can recover the radiation-damagedbone marrow system very efficiently along with itincreases the levels of IL-1β, IL-6, and GM-CSF and alsoreduce the level of TNF-α [32]. Such damages includingdysregulation of cytokine expression and increase ofthe level TNF-α in the tissue injury are caused by ra-diation. Thus PG101 can be as a supplement or a majortherapeutic for bone marrow system is damaged.

6.2.1.4. Lentinan from L. edodes. Many researcheshave been done on immunomodulating and antitumoractivity of lentinan. This compound is known as a T cell-oriented adjuvant [33]. The skewing of TH1/TH1balance to TH1 by lentinan is directed through thedistinctive production of IL-12 versus IL-6, IL10, andPGE2 by peritoneal macrophages depending on intra-cellular glutathione redox status [34]. Lentinan inducesnon-specific cytotoxicity in macrophages and stimulatescytokine production and is able to activate the alternatecomplement pathway in vitro and it opsonised with C3bis considered to bind to complement receptor type 1 or 3(CR1 or CR3) on monocytes. It does not bind tolymphocytes. Lentinan augments the secretion of IL-1,colony-stimulating factors (CSF), migration inhibitionfactor (MIF), IL-3, IL-6, IFN-γ and generation ofcytotoxic T cells (CTLs) and NK cell activity in thepresence of IL-2 [35].

Study on preoperative intratumor administration oflentinan for gastric cancer cases showed that lentinanadministered directly into tumors decreased the ratioof suppressor–inducer T cells and suppressor T cellstended to increase the ratio of CTLs and IL-2 productionof lymph node lymphocyte [36]. It has been shown toaugment the activities of natural killer cells, lympho-kine-activated killer cells and CTLs. It can also activatemacrophage differentiation and increases response indelayed-type hypersensitivity against tumor antigen[37].

Results obtained of patient orally administeredL. edodes fruit body suggest that tumor growthinhibition by this macrofungi is at least partly due tothe enhanced production of the superoxide anion bymacrophages or prevention of deterioration of immuno-phagocytosis on the tumor-bearing state. The cytotoxi-city is potentiated by oral administration of L. edodesfruit bodies is that of T-cells, so that it accelerate theinduction of cytotoxic T cells and are very similar to themechanisms of lentinan injected intraperitoneally intomice. Its action is host mediated [38] (Fig. 1).

Table 4Some structural characteristics of β-D-glucans and glycopeptides andglycoproteins isolated from macrofungi

Compound Original Molecularstructure

Molecularweight (KDa)

Schizophyllan Schizophyllumcommune

1,6-Monoglucosyl-branched 1,3-β-D-glucan

350

Grifolan Grifolafrondosa


500

D-fraction Grifolafrondosa


1000

Lentinan Lentinusedodes


500

SSG Sclerotiniasclerotiorum


≥2000

PSK Coriolusversicolor

1,3 and 1,6-monoglucosyl-branched 1,4-β-D-glucan with bindingto aspartic, glutamicand other acidicamino acids

100

PSP Coriolusversicolor

Resemble to PSKstructure but is richedin glutamic andaspartic acids

100

PSPC Tricholomalobayense

Consisted ofgalactose, glucose,mannose, fucose,arabinose andrhamnose withbinding to aspartic,glutamic and otheracidic amino acids

150


6.2.1.5. SSG from S. sclerotiorum. — It is reportedthat SSG can induce the development of TH1 cells viathe IL-12 pathway [39].

6.2.1.6. GLPS from G. lucidum. GLPS has activatedBALB/c mouse B cells and macrophages, and also couldinduce IL-1beta production by peritoneal macrophages[40]. It could promote the maturation of cultured murinebone marrow-derived DCs and the immune responseinitiation by DCs [41].

6.2.1.7. β-glucan fraction CA1 from Sparassis crispa.Oral administration of the CA1 has enhanced the hema-topoietic response in cyclophosphamide (CY)-induced leu-kopenic mice assessed by white blood cell count. The rateof leukocyte recovery in CY-administered mice was dif-ferent in each population, such as granulocyte, monocyte,NK cell, Â cell, Ò cell, and so on. Administration of CA1modulated the recovery rate of each population. In in vitro,CY-treated spleen cell culture, IL-6 and INF-γ productionwas enhanced by CA1 treatment. These facts stronglysuggested that the enhanced hematopoietic response byCA1 is due to enhanced cytokine production [42].

6.2.1.8. SCG from S. crispa. It is proved that SCGenhances the hematopoietic response. In cyclopho-sphamid-(CY)-induced leukopenic mice, SCG recov-ered monocytes and granulocytes in the peritonealcavity, liver, spleen, and bone marrow. The ratio of NKcells and γδT cells in the liver, spleen, and peritonealcavity was also increased [43].

6.2.1.9. Galactomannan from Morchella esculenta.This compound enhances macrophage activation. At3.0 μg/mL the galactomannan increased nuclear factorkappa B (NF-kB)-directed luciferase expression in THP-1 human monocytic cells to levels of 50% of thoseachieved by maximal activating concentration (10.0 μg/mL) of LPS [44].

6.2.1.10. Fucogalactan from Sarcodon aspratus. Thiscarbohydrate at 50 μg/mL could increase the release ofTNF-α and NO in macrophages of mice in vitroapproximately 4.3 times than lentinan at 500 μg/mL[45]. It is suggested that fucogalactan can contribute toantitumor activity in tumor-bearing host as well asimmunomodulating effects.

6.2.2. 1-2-Glycoproteins (polysaccharide-proteincomplexes)

Polysaccharide polymers can reach maximum com-plexity when they are covalently attached to other

molecules such as polypeptide and proteins etc.Glycoproteins also consist of a protein core thatsurrounded with numerous glucan chains (Fig. 3) asthese chains bind to protein moiety through O- or N-glycosidic bones (Table 4). β-glucan-protein, α-glucan-protein, heteroglycan-protein complexes are antitumorglycoproteins that found in macrofungi.

Among glycoproteins isolated from macrofungiPSPC and ATOM are well known for immunomodulat-ing and/or antitumor activities.

6.2.2.1. ATOM from Agaricus blazei. It is suggestedthat tumor growth inhibitory effect of ATOM is ap-parently due to immunological host-mediated mechan-isms so that it is caused by increasing the number ofperitoneal macrophages, the phagocytosis of polysty-rene latex beads and the proportion of the third


component of complement (C3)-positive fluorescentcells in the tumor-bearing mice [46].

6.2.3. 1-3-Glycopeptides (polysaccharide-peptidecomplexes)

This group is structurally similar to glycoproteins buthas a smaller chain of amino acids (Fig. 3). PSP and PSK(Table 4) are well known for immunomodulating andantitumor activities and they are commercially consideredto develop immunomodulating and anticancer agents.

6.2.3.1. PSK and SPS from Coriolus versicolor. PSKis classified as biological response modifier that stim-ulates T-cell activation and induce IFN-γ and IL-2 pro-duction. The biological activity is characterized bytheir ability to increase white blood cell counts, IL-2production and delayed-type hypersensitivity reactions.

PSK stimulates production of differentiation-inducingfactor (DIF) frommacrophages, and activates the functionof polymorphonuclear leukocyte (PMN). PMN plays aprominent role in the overall course of infectious diseasesand it has a role in the defense against intra- and extra-cellular pathogens such as bacteria, viruses and someprotozoa. Also, it is clarified PSK induces gene expressionof some cytokines such as TNF-α, IL-1, IL-8 and IL-6 invivo and in vitro [47]. It is reported that PSK promotes thephenotype and functional maturation of DCs derived fromhuman CD14+ mononuclear cells and can resolve theimmunosuppressive state of a cancer-bearing host andmay be associated with DCs maturation directly [48].

PSK injected directly into human stomach tumorsprior to surgery was taken up specifically by DCslocated in and around the tumors [49]. Aside, thiscompound activates killer cells in vivo. Instillation ofPSK into a human gastric tumor mass prior to respectivesurgery causes T cells around the site to become tumor-infiltrating and develops significantly enhanced cyto-toxic "killer" activity directed at the tumor. Similarfindings have been obtained with a 14-day course ofPSK in bladder cancer patients [50]. PSK also activateshuman NK cells in culture at concentrations reached inthe blood by normal oral dosing of 3 g per day [50,51].

Similar to PSK, PSP is classified as biological responsemodifier that stimulates T-cell activation and induces IFN-γ and IL-2 production. In mice with suppression of IL-2production from cyclophosphamide toxicity, PSP supple-mentation has restored IL-2 production to normal [52].Also it is known to stimulate DCs and macrophage orother immune phagocytic activity in vivo. When micewere given charcoal intravenously, then fed PSP, both thephagocytic activity of cells in the blood and the clearanceof the charcoal from the circulation were significantly

accelerated [53]. PSP stimulates lymphokine-activatedkiller (LAK) cell proliferation by itself at relatively lowconcentrations and in the absence of IL-2 [54].

6.2.3.2. Polysaccharide-polypeptide complexes fromG. lucidum. The fungus G. lucidum (in fruit bodyand spore) contains kinds of glycopeptide complexeswith antitumor and immunostimulating activities.Some of them stimulate the expression of cytokines,especially IL-1, IL-2 and INF-γ [55,56].

6.3. 1-4-Proteoglycans

This group of glyco-conjugates is proteoglycans thatis known as a special class of glycoproteins that areheavily glycosylated. They consist of a core protein withone or more coavalently attached glycosaminoglycanchain(s).

Glycosaminoglycan molecules are long unbranchedpolysaccharides containing a repeating disaccharideunit. The disaccharide units contain either of twomodified sugars-N-acetylgalactosamine (GalNAc) orN-acetylglucosamine (GlcNAc) and an uronic acidsuch as glucuronate or iduronate. Glycosaminoglycanmolecules are highly negatively charged molecules,with extended conformation that imparts high viscosityto the solution. Proteoglycans are remarkable for theirdiversity includes different cores, different numbers ofglycosaminoglycans with various lengths and composi-tions. GLIS is well known as a macrofungi proteoglycanthat has immunomodulating activity. This compoundcontains carbohydrates and protein in a ratio of 11.5:1 sothat the carbohydrate portion is formed by seven differentmonosaccharides, predominantly D-glucose, D-galactose,and D-mannose in the molar ratio of 3.0:1:1 [57].

6.3.1. GLIS from G. lucidumGLIS is proteoglycan that is introduced as a B-cell

stimulating factor. This compound stimulates B lym-phocyte activation, proliferation, differentiation andproduction of immunoglobulin. The activation of Bcells by GLIS may be associated with the expression ofprotein kinase C (PKC) α and PKC γ in B cells. Thiscompound stimulates the proliferation of mouse spleenlymphocytes, resulting in a three-fold to four-foldincrease in the percentage of B cells [57].

7. The structure-activity relationship

PSs have high capacity for carrying biological infor-mation because they have great potential for structuralvariability. Despite the structural and functional


similarities of these glucans, they differ in theireffectiveness against specific tumors and in their abilityto elicit various cellular responses, particularly cytokineexpression and production [58]. The structure of β-D-glucans has a relationship with the binding characteristicswith receptors that the triple helical solution conforma-tion,molecular weight, branching ratio, solubility inwaterand charge of the glucan polymer are importantdeterminations in receptor ligand interaction and anti-tumor activity [59]. For example it is proved that TNF-αrelease by macrophage is induced only by β-D-glucanswith high molecular weights and lower branching ratios[59,60]. Also, it is showed that high molecular weightglucans appear to be more effective in antitumor activitythan those of lowmolecular weight [61]. Also bioactive β-D-glucans present a triple-strand helical structure withright winding [62]. Triple helix conformation of β-glucans(Fig. 4) and presence of hydrophilic groups located on theoutside surface of the helix are important for theirbiological activities whether immunomodulating orantitumor activity. Also strong antitumor activities arefound in various hetero-β-D-glucans having a (1→3)-β-D-glucan chain as the active site such as β-D-glucan,glucurono-β-D-glucan, arabinoxylo-β-D-glucan, xylo-β-D-glucan, manno-β-D-glucan and xylomanno-β-D-glucan, aswell as their polypeptide complexes.

Fig. 4. Schematic representation of the triple helix structure of β-(1→3)-D-glu(1→3)-D-glucan formed as triple helix structure.

8. Chemical and structural modification ofpolysaccharides and their derivates

Furthermore, it is proved that increased watersolubility favors enhanced antitumour activity whilethe location of substitute groups would also beimportant. For example, when the alkali-insoluble,branched (1→3)-β-D-glucan isolated from Auriculariaauricula-judae was modified by controlled, periodateoxidation, borohydride reduction, and mild, acidhydrolysis, resulted in increasing water solubility byhaving covalently linked polyhydroxy groups attachedat O-6 of the (1→3)-linked D-glucosyl residues, andexhibited potent antitumor activity significantly, whilein the native state had no such activity [63].

They cause to improve such polysaccharides to moreeffect and their clinical qualities. The main proceduresused for chemical improvement are: Smith degradation(oxydo–reducto-hydrolysis), formolysis, and carboxy-methylation [21]. For example, the carboxymethylgroup with low degree of substitution (DSb0.28) (DSis a measure of the average number of hydroxyl groupson each anhydroglucose unit (AGU) which arederivatized by substituent groups) is introduced asthe best choice on the improvement of immunostimulat-ing activity of (1→3)-α-D-glucan [64] (Fig. 3) and

cans comparison to other helix structures (a), the higher structure of a β-


modification of D-glucosyl group of side chains of β-D-glucans to polyol groups can enhance antitumor activity[65]. Another finding for carboxymethylation was donefor a polysaccharide with linear structure of (1→3)-α-D-glucan isolated from Amanita muscaria so that degreeof substitution (DS) of carboxymethyl groups was 0.95and the substituents were located at O-2, at O-4, at O-6,at O-2 and O-6, and at O-4 and O-6 on glucose, resultingpotent antitumor activity against sarcoma 180 in mice,although the native polysaccharide had little effect [66].

Enzymatic reactions also can improve polysacchar-ides activities. Amylase, cellulose, and protease areenzymes that used for this purpose. In this way, linearlow molecular α-(1→4)-glucans obtained after enzy-matic reduction of side chains and protein component(active hexose correlated compounds) that showsimmunomodulating and anticancer properties [67].

9. β-D-Glucan receptor(s) and signaling cascade

The innate immune system identifies infectiousagents or compounds by using of pattern-recognitionreceptors. These receptors recognize the macromole-cules of pathogens and they called pathogen-associatedmolecular patterns. Because of polysaccharide's largemolecular mass, these compounds cannot penetrate cells,so the first step in the modulation of cellular activity isbinding to immune cell receptors. It is proved that thereare fungal pattern-recognition molecules for the innateimmune system. The mechanism by which the innateimmune system recognizes and responds to fungal cellwall carbohydrates is a very complex and multifactorialprocess [68]. It is clarified that several β-glucan receptorsmediate these activities such as Complement receptor3(CR3, αM β2-integrin, CD11b/CD18) [69,70], lactosyl-ceramide, glycosphingolipd [71], scavenger receptors[72], dectin-1 [73], and tool-like receptors (TLR)-2 andTLR-4 [74].

CR3 occurs on monocytes, macrophages, neutroph-lis, NK cells and follicular DCs and is an importantreceptor and adhesion molecule. They mediate a varietyof adhesive and cytotoxic functions that are dependentupon its ability to bind a multiplicity of ligands such asiC3b, ICAM-1, clotting factory X, certain bacteria andfibrinogen [69].

It serves as the leukocyte β-glucan receptor [75]through one or more lectin sites located on the portion ofthe α-subunit (CD-11b) known as the CD11b I-domainthat contains the binding sites for iC3b, ICAM, andfibrinogen [69]. Thus β-glucans mediate cytotoxic andphagocytic responses by binding to these sites and areone of the first microbial response modifiers for which

the cellular mechanism of action has been defined at thespecific receptor level. Elsta et al. had found thatopsonized zymosan particles as a derivation of β-glucans stimulate platelet-activating factor synthesis bymonocytes and they had suggested that CD11b/CD18facilities binding of the particle and that a β-glucanreceptor transduces the activation signal [76]. platelet-activating factor has been implicated in the pathogenesisof septic shock, anaphylaxis, asthma, immune-mediatedtissue injury, and other inflammatory conditions.

Lentinan has been found to bind to monocytes viaCR1, CR3 and may also bind via a beta-glucan receptor.Lentinan induces non-specific cytotoxicity in macro-phages and stimulates cytokine production and is able toactivate the alternate complement pathway in vitro andit opsonised with C3b is considered to bind to CR1 orCR3 on monocytes. However, lentinan does not bind tolymphocytes [35].

β-D-glucan can also help override the normalresistance of iC3b-opsonized tumor cells to the cytotoxicactivation of phagocyte and NK cell CR3, allowing thisimportant effecter mechanism of the complement systemto function against tumor cells in the same way that itnormally functions against bacteria [70].

Dectin-1 is a type II transmembrane protein andmammalian cell surface receptor for β-1, 3-D-glucan andhas the typical amino acid sequences of C-type lectins[77] and is important for recognizing fungal invasion[78]. It is broadly expressed with highest surfaceexpression on monocytes, macrophages and neutro-phils. Also it expressed by dendritic cells and a sub-population T cells at lower levels [79]. C-type lectinsplay important roles in the innate immune response byrecognizing microbial saccharides.

In the study on dectin-1 to recognize the schizo-phyllan, it is proved that dectin-1 has a carbohydraterecognition domain consisting of six cysteine residuesthat are highly conserved in C-type lectins. C-typelectins recognize sugar ligands through this carbohy-drate recognition domain with Ca2+ dependence [78].This receptor is specific for the (1→3)-β-D-glucosyllinkage. SPG possess a 1,6-monoglucosyl branch on a(1→3)-β-D-glucosyl main chain, which forms triplehelix conformation in a physiological solution [80]. Thebiologic response transduced by dectin-1 is dependentupon the TLR pathway. It is determined that dectin-1 incombination with a β-glucan-enriched zymosan deriv-ative enhances TLR-2-mediated cell activation [81].Also it is indicated that for the activation of NF-kB andinduction of TNF-α, dectin-1 needs to cooperate withTLR-2 (Fig. 5), possibly as part of a heterodimer withTLR-6 [82]. NF-kB comprises a family of inducible

Fig. 5. Schematic representation of the β-glucans recognition by certain receptors on the cell surface and activation of NF-κB leading to transcriptionof many genes in both innate and adaptive immune responses.


transcription factors that serve as important regulators ofthe host immune and inflammatory response. NF-kBfactors are important in the transcription of many genesin both innate and adaptive immune responses.

It has been reported that dectin-1 ligationwith zymosanresults in tyrosine phosphorylation of the receptor'simmunoreceptor tyrosin-based activating motif (ITAM)-like signaling motif, generating intracellular signals thatmediate phagocytosis and activation of nicotinamideadenine dinucleotide phosphate (NADPH) oxidase [83].In addition to the inflammatory response, dectin-1 binds tolymphocytes and augments their mitogenic response bycross-linking T-cell receptors [84].

Recognition of β-glucans by antigen-presenting cellssuch as DCs or macrophages can augment pathogen-specific T-cell activation. Thus β-glucans may be a linkbetween innate and adaptive immune responses. Despitethe intracellular events that occur what after glucan-receptor binding has not been fully clarified. Howevermore studies are required to determine which receptor(s)

are essential of the various immunobiological effectsascribed to β-glucans.

10. 2-Terpenoids

Terpenes are composed from 5-carbon units namedisoprene that they are not with saponification property.Hitherto, many terpenes are isolated from plants, fungi,and marine organisms etc. In among terpenes, triterpe-noids have been found exclusively from macrofungi thatare famous compounds with biological activities andmedicinal properties and they are produced in higherfungi (Basidiomycetes). Among triterpenoids highlyoxidized lanostane-type triterpenoids are the further typeof triterpenes are isolated from family Polyporaceae andGanodermataceae (Fig. 6). They have different biolog-ical activities especially antiinfectives, cytotoxic andimmunomodulating activities. Ganoderic acids, Gano-derenic acids, Ganodermic acids, Applanoxidic acids,Ganoderals, Ganoderols, Lucidone, Ganodermanontriol,

Fig. 6. Some lanostane-type triterpenoids isolated from Ganoderma lucidum.


Ganodermanondiol, are of basidiomycetous triterpe-noids [85]. Structures of these compounds have alanostane skeleton and they are classified into 10 groupsbased on their carbon numbers and state of oxidation.

The researches done on G. lucidum have showed thatantitumor activity of such triterpenoids is similar to β-D-glucans do in some pathways. These compounds, forinstance, could activate NF-kB pathway and modulateRas/Erk, c-myc, CREB protein and mitogen-activatedprotein kinases [86]. So, these activations can lead toother immune activations against tumor cells.

In spite of fact that many triterpenoids have beenknown of macrofungi, few works have been done todiscovery mode of action of anticancer and immuno-modulating effects of terpenoids.

Fig. 7. Comparison of sequences of

11. 3-Fungal immunomodulatory proteins (Fips)

11.1. Fips structure

Hitherto, four proteins with relatively similar struc-ture isolated from macrofungi that have showed immu-nomodulating activity. These proteins, Fips, are foundmerely in the mushrooms including LZ-8, Fip-gts, Fip-fve and Fip-vvo isolated from G. lucidum, Ganodermatsugae, Flammulina velutipes and Volvariella volvacea,respectively. Fips have a molecular mass of 13 kDaand share high amino acid sequence homology. Alig-ment of these proteins revealed 44 % identity and 42%homology for approximately 110 amino acid residues(Fig. 7). They are rich in β-structure by secondary

Fips isolated from macrofungi.

Fig. 8. Amino acid sequence and secondary structure of Fip-gts (thisstructure was predicted by Lin et al. [87]).


structure prediction, and contain seven β-strands,two α-helixes, and one β-turn (Fig. 8). The amphi-pathic α-helix is a common structural motif, which isfound in a number of functional proteins or peptidesand involved in various functions such as glucagonbinding to its receptor, plasma apolipoproteins solu-bilization of lipids, antimicrobial peptide disintegra-tion of bacterial cells, and signal peptide targeting tomitochondria [87].

11.2. Fips immonumodulating activities

Fips are mitogenic in vitro for human peripheralblood lymphocytes (hPBLs) and mouse splenocytes,and induce a bell-shaped dose-response curve similar tothat for lectin mitogens. Activation of hPBLs with Fipsresults in the increased production of IL-2, IFN-γ andTNF-α molecule associated with ICAM-1 expression.They can also act as immunosuppressive agents. Theseproteins could prevent systemic anaphylactic reactionsand significantly decrease foot-pad edema during theArthus reactions in vivo [87]. Study on LZ-8 showedthat it is similar to the variable region of immunoglob-ulin heavy chain both in its sequences and in itspredicted secondary structure by sequencing studies. Itappears to be related to an ancestral protein of theimmunoglobulin superfamily [88,89]. LZ-8 is a potentT-cell activator, mediating its effects via cytokineregulation of integrin expression. LZ-8 increases pro-duction of IFN-γ, TNF-α and IL-1 β [90]. It is proved invivo, LZ-8 prevents the production of systemicanaphylaxis reaction in mice if it has been administeredrepeatedly, and reduction of antibody production isthe suggested mechanism. The mechanism of action of

LZ-8 on antibody production is unclear but two types ofFc receptors have been reported; one on the cell surfaceof basophiles and mast cells with high affinity for IgE,and another on the cell surface of T or B lymphocyteswith low affinity. The latter low affinity Fc receptor-bearing lymphocytes may be important for the regula-tion of selective IgE production by secreting two IgEbinding factors, one with potentiating and the otherwith suppressing activity toward IgE production [88].Also an immunomodulating protein named Fip-gts hasbeen purified from G. tsugae that it has the same aminoacid sequence as LZ-8. Using deletion analysis, asequence of about 10 amino acids of the N-terminalamphipathic alpha-helix domain of Fip-gts has beenidentified to be responsible for the immunomodu-latory activity. This region may play an important rolein the formation of homodimers for binding to cellsurface receptors to exert its immunomodulatoryactivity [87]. Also it is reported that Fip-vvo inducedmost Th1-specific cytokines (IL-2, INF-γ, and lympho-toxin (LT)) and one Th2-specific cytokine (IL-4)within 4 hours in mouse spleen cells [91]. These resultssuggest that Fip-vvo principally acts on Th1 cells and toa lesser extent on Th2 cells in the early evet of acti-vation. Also it is proved Fip-fve selectively stimulates aTh1 response in hPBMCs and trigger Th1 cytokineproduction and oral administration of Fip-fve duringallergen sensitization could induce a Th1-predominantallergen-specific immune response in mice and protectthe mice from the systemic anaphylaxis-like symptomsafter subsequent oral challenge with same allergen. Itis worth noting that Fip-fve could be administratedorally and retains its activity, while most protein drugscannot [9,92].

12. Clinical and experimental evidences for anticancerproperties

Although, mode of action of many anticancercomponents have not been known yet, clinical andexperimental evidences can help us move on theseworks. Beyond these evidences, there are mechanismsact that one of them is immunostimulating effect thatcaused by anticancer compounds.

Almost worked studies to investigate anticancerpotentiality of macrofungi metabolites have been donefor Sarcoma 180 and Ehrlich carcinoma in white mice.But mammalian cell cultures also have been used forthis purpose. In many experiments, study of antitumoractivities on Sarcoma 180 and Ehrlich carcinoma haveshowed great antitumor activity to regression tumorsgrowth even as much as 100% inhibition (Table 5).

Table 5Tumor growth inhibitory effect of some macrofungi hot water extractin clinical and experimental studies

Species Against Sarcoma180 (%)

Against Ehrlishcarcinoma (%)

Auricularia auricula 70–90 60–80A. delicate 42–70 80A. mesenterica 42–60 60Tremella sp. 60–100 70–100Phlogiotis helvelloides 100 100Tremellodon gelationsum 90 90Cantharellus sp. 60–100 60–90Craterellus sp. 60–90 60–90Clavaria sp. 60–90 60–100Clavulinopsis sp.LentariaRamaria formosa 60–70 60–70R. botrytisR. flavaHydnum sp. 70 90Polyporus occidentalis 80 100P. umbellatus 70Ganoderma applanatum 64.9G. tropicum 70G. tsugae 77.8Armillariella mellea 70 80Clitocybe fragrans 70–80 70–80Flammulina velutipes 81–100 80Lepista nuda 90 100Mycena pura 60 70Tricholoma gambosum 70–90 70–90Buletus edulis 100 90Agaricus bisporus(edible mushroom)

90 100

Coprinus atramentarius 100 100Pholiota nameko 100 100


The purified polysaccharide has been shown inanimal studies to produce strong tumor regression andeven the disappearance of sarcoma tumors in 5 weeks,ascite hepatoma 134, and Ehrlich carcinoma as well as anumber of other experimentally induced cancers inallogenic, syngeneic, and autologous hosts [8].

In clinical studies for anticancer property, lentinan hassucceeded in prolonging the overall survival of cancerpatients especially those with gastric and colorectalcarcinomas [93–95]. The study evaluated the ability oflentinan to modulate Th1 and Th2 responses in patientswith digestive cancers. It apparently can cancel Th2-dominant condition in patients with digestive cancersandmay improve the balance between Th1 and Th2 [96].In another clinical study, It was effective in metastaticprostate cancer when incorporated into hormonochem-otherapy, so that the five-year survival rate of treatedpatients was 43% according to the KaplanMeier method,while that of control patients was 29% ( pb0.05) [97].

Also it could be effective for the patients with advancedor recurrent breast cancer as an agent for supportivetherapy. In that, life span prolongation effect of lentinanhad also been observed with statistical significance [98].

Schizophyllan has also been shown to increase overallsurvival of patients with head and neck cancers [99].Grifolan-D(R) had completed (N95%) cell death of prostatecancer cell in vivo conditions with GD≥480 μg/ml [100].

PSK and PSP had controlled various carcinomas inexperimental animals and humans. PSP is active againstEhrlich ascites carcinoma, P388 leukemia and sarcoma180 [101] and also it enhance the transcription of tumornecrosis factor gene in mouse peritoneal macrophages,IL-6 and IL-2 and interferons [102].

Also, in an experimental study it is showed that PSPcan be associated with slower deterioration in patientwith advanced non-small cell lung cancer. In this ex-periment, after 28-day treatment, there was a significantimprovement in blood leukocyte and neutrophil counts,serum IgG and IgM, and percent of body fat among thePSP, but not the control, administrated patients [103].PSP also activates killer cells in situ in the living cancerpatient. In the Phase II and III double-blind trials, PSPhas significantly raised NK cytotoxic activity, IL-2levels, and significantly improved the CD4 helper/CD8suppressor T-cell ratio. Altogether, these are the primarycomponents of anticancer immunity [51]. In Phase IIand Phase III trials in China, PSP significantly enhancedimmune status in 70 to 97% of patients with cancers ofthe stomach, esophagus, lung, ovary, and cervix. Inthese studies, PSK and PSP increased the number ofimmune cells and facilitated CDs and cytotoxic T-cellinfiltration of tumors [104].

Studies are conducted for antitumor activities of somemacrofungi in the National Cancer Center, Japan. Basedon these studies, extracts prepared from Hypsizygusmarmoreus and F. velutipes and contain polysaccharidesand glycoproteins showed positive effects on the cachexiaof advanced cancer patients. These extracts had bettereffect than methylacetoxyprogestrone in clinical re-sponse, performance status, and quality of life [105].

Ganoderic acids U, V,W, X and Y [85] and lanostane-type triterpenes isolated from spores of G. ucidum, theGanoderic alcohols Lucidumol A ((24S)-24, 25-dihy-droxylanost-8-ene-3, 7-dione) and Lucidumol B (β,(24S)-lanosta-7, 9(11)-diene-3β, 24, 25-triol), Ganoder-manondiol, Ganoderiol F and Ganodermanontriolshowed cytotoxic effect on Meth-A (sarcoma) andLLC tumor cells and lucidumol A exhibited the mostpotent cytotoxicity (ED50 value 2.3 ìg/ml) against LLCtumor cells and Ganodermanondiol (ED50 value 3.4 μg/ml) against Meth-A cells [106].


13. Conclusion

The immune system is the body's ultimate defenseagainst infectious diseases, tumor and cancer growth and itis complex containing many interacting blood cells, pro-tein and chemicals. A healthy immune system containselements that are in balance with one another and in acompromised immune system, the components areunbalanced and unable to protect the body against harmfulagents or processes. The potential immunomodulatoryeffect of antimicrobial agents was postulated as early asthe late 19th century with regard to quinine-inducedphagocyte modulation and the emergence of newpathogen/diseases, microbial resistance to classical anti-microbial agent, and the increased number of immuno-compromised individualsmakes new therapeuticweaponsincreasingly important. Applying immunomodulatorsparticularly is important when immune system does notfunction optimally e.g. in young children, the elderly andpatients especially cancer and AIDS patients (againstopportunistic infections) and surgical procedures. Immu-nomodulators found in the various organisms that inamong them macrofungi (known as medicinal mush-rooms) known as important resources of these agents.Polysaccharides, glycoproteins, glycopeptides, Fips, andtriterpenoids are major immunomodulating agents isolat-ed from macrofungi. Moreover, antitumor and antimicro-bial activities are associated with immunomodulating andimmunostimulating activity of these substances. Withregard to that, nowadays, immunotherapy for cancer ismore attended via stimulation of the immune system,using of such agents and targeting the immune system toeliminate tumor cells are rapidly developing. In this way,macrofungi metabolites are well known as antitumoragents that act with affecting on antitumor effector cellsand factors and immune mechanisms that involved in theantitumor activity. Polysaccharides are a structurallydiverse class of molecules that are found throughoutnature. They have a considerable capacity for carryingbiological information due to their structural variability.Their variability arises from the almost endless combina-tions that the sugar units can join together to form complexsugars. This makes them very flexible which allows themto exert the regulatory mechanisms of various cell-to-cellinteractions in higher organisms. It is cleared thatmacrofungi are much more important resources of β-D-glucans and their derivates and other polysaccharide thatare known as immunomodulator and antitumor agents andusing of them in virtue of their safety is important inbiomedical science.Macrofungi polysaccharides and theirpeptide/protein derivates are among the emerging newagents that could directly support or enhance functional

autologous hematopoietic stem cells recovery [23].Overall, the major immunopotentiation effects of theseagents include mitogenicity, stimulation of hematopoieticstem cells, activation of alternative complement pathway,and activation of immune cells such as lymphocytes,macrophages, DCs, NK cells, Th cells, Tc cells and Bcells. On the other hands, their antitumor activity alsoassociated with such activation and enhancement ofimmune system power. In this way, activation ofmacrophages due to secretion different cytokines such asIL-1, IL-6, IL-8, TNF-α, and NO and affecting on cell-mediate immunity are much important pathways to act[107]. In preventivemedicine, defense against invasion byforeign bodies is dependent on enhancing the naturalimmune system, including activation of macrophages andNK cells. Because of ability of some macrofungiimmunomodulating agents to enhance NK cells activityin cancer patients these are also known as biologicalresponses modifiers. The effects that cause production ofcytokines such as IL-1β IL-6, IL-8, TNF-α and NO lead toinflammatory responses, but in other cases macrofungiextracts inhibit the production of NO, IL-1β and TNF-α,resulting anti-inflammatory effects occur. Thus thesemacrofungi might be relevant for clinical use forinflammatory diseases, including endotoxemia or sepsis[9]. It is suggested that the immunomodulating action ofthese agents is valuable as a mean of prophylaxis, a mildand noninvasive form of treatment, prevention ofmetastatic tumors, and as a co-treatment with chemother-apy [1]. The enhancement or potentiation of host defensemechanisms has been recognized as a possible means ofinhibiting tumor growth without harming the host [9]. Onthe other hand, Fips as fungal immunomodulatoryproteins, so far, are merely discovered in this group offungi. These proteins possess relatively similar structureand also have shown considerable similarity to thevariable region of immunoglobulin heavy chain both intheir sequences and in their secondary structure [89].Although their details of mode of action and affectingmechanisms are still not clearly understood but also it isclarified that these substances act with affecting ondifferent cells of the immune system that are responsiblefor immune responses in the innate (non-specific) andadaptive (specific) immunity.

The first step of action of these metabolites isrecognition of them by certain receptors located ondifferent immune cells and activation of signal trans-duction pathways [108]. Details of recognition of suchsubstances whether polysaccharides and their derivatesand Fips is not yet cleared but also some reports andexperiments have introduced some β-glucan receptors.It is clarified several β-glucan receptors mediate these


activities such as Complement receptor 3 (CR3, αM β2-integrin, CD11b/CD18) [69], lactosylceramide, glyco-sphingolipd [71], scavenger receptors [72], dectin-1[73], and TLR-2 and TLR-4 [74]. Recognition microbesand their mediating molecules by macrophages aremediated by TLRs that are specific for differentcomponents of microbes. NF-kB as inducible transcrip-tion factor is served as important regulators of the hostimmune and inflammatory response after recognition ofmicrobial products by receptors. The biologic responsetransduced by dectin-1 is dependent upon the TLRpathway. It is determined that dectin-1 in combinationwith a β-glucan-enriched zymosan derivative enhancesTLR 2-mediated cell activation [81]. Also it is indicatedthat for the activation of NF-kB and induction of TNF-α,dectin-1 needs to cooperate with TLR-2, possibly as partof a heterodimer with TLR-6 [82].

The cytoplasmic domain of dectin-1 also has consec-utive acidic amino acid residues that are a putativeinternalizing signal sequence for the lysosomal endosomeand it is also has a putative ITAM-like region consisting ofan YXXL amino acid sequence [77]. This ITAM can bephosphorylated by stimulation with particulate β-glucans.It has been reported that this phosphorylation can beinvolved in superoxide production by macrophages [81].There for dectin-1 may contribute not only to phagocy-tosis of fungal cells but also to induction of fungicidaleffector molecules [78].

Overall such findings suggest that using of macro-fungi as immunomodulator is important in the fieldinfectious and immunodeficiency diseases especiallywhen immune system does not function optimally. Alsosuch compounds can mobilize the immune system toward off viral, bacterial, fungal and protozoan infec-tions resistant to current antibiotics. Much attention isnow being paid to the use of immune modulators,colony stimulating factors and cytokines as adjunctivetherapy in immunocompromised and infected patients.However, to discover interaction between antitumoragents and the immune system can cause to select moreeffective drugs and, ideally, to combine antitumorefficacy and “biological response modifiers” activity.Consequently, macrofungi can be important resourcesof immunomodulating and antitumor agents as well asthey known as antiviral, antimicrobial, antimutagenic,antihypertension, antiinflammatory, antiallergic etc.Macrofungi growth wildly in forests and rangelandsand nowadays some of them are cultivated by human.They are more regarded as natural products and dietarysupplements and they are produced in various formula-tions in world. Nowadays, different formulations ofmedicinal macrofungi are produced with the intention

of certain aims. For example PSK under the nameKrestin was produced in Japan with the intention ofprevention and remedy of a number of cancers. In 1993,Krestin comprised 25% of the anticancer drug market inJapan and sales totaled US $350 million [109]. Also ananalogous product under the name PSP was developedin China from this fungus (but from other strain) that ina period of 10 years was soled totaled US $75 million or10 billion yen. The market values of G. lucidum-basednatural healthcare products in 1995 were estimated asUS $215 million in Taiwan, US $350 million in China,US $600 million in Korea and US $350 million in Japan[110]. G. lucidum dietary supplement are valued fortheir immunomodulating, anticancer and antiviralproperties. The market value of macrofungi dietarysupplement products worldwide is estimated at US$6 billion per year. The market value of G. lucidummushroom-based dietary supplement alone in 1995 wasestimate at more than US $1.628 billion [110]. Atpresent, L. edodes is one of the five most cultivatededible mushrooms in the world. Its production (2 mil-lion tones) is second only to button mushroom Agari-cus bisporus [8]. These macrofungi almost are appliedas multipurpose medicines. There is an increasinginterest in finding natural biological response modifiersand immunoenhancers or immunomodulators fromthem. In 2003, the value of world macrofungiproduction and medicinal mushroom products wasestimated to be worth approximately 21 billion USdollars. However, in this discipline, there are thepowerful sources to discover novel pharmaceuticalproducts.

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