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Appl Microbiol Biotechnol (2005) 67: 453468DOI 10.1007/s00253-004-1787-z

MIN I-REV IEW

Ben-Zion Zaidman. Majed Yassin. Jamal Mahajna.

Solomon P. Wasser

Medicinal mushroom modulators of molecular targetsas cancer therapeutics

Received: 13 July 2004 / Revised: 27 September 2004 / Accepted: 2 October 2004 / Published online: 23 February 2005# Springer-Verlag 2005

Abstract Empirical approaches to discover anticancerdrugs and cancer treatments have made limited progressin the past several decades in finding a cure for cancer. The

expanded knowledge of the molecular basis of tumorigen-esis and metastasis, together with the inherently vast struc-tural diversity of natural compounds found in mushrooms,

provided unique opportunities for discovering new drugsthat rationally target the abnormal molecular and biochem-ical signals leading to cancer. This review focuses on mush-room low-molecular-weight secondary metabolites targeting

processes such as apoptosis, angiogenesis, metastasis, cellcycle regulation, and signal transduction cascades. Also dis-cussed in this review are high-molecular-weight polysaccha-rides or polysaccharideprotein complexes from mushroomsthat appear to enhance innate and cell-mediated immune re-sponses, exhibit antitumor activities in animals and humans,

and demonstrate the anticancer properties of selenium com-pounds accumulated in mushrooms.

Introduction

Medicinal mushrooms have an established history of use intraditional oriental therapies. Modern clinical practice inJapan, China, Korea, and other Asian countries continues torely on mushroom-derived preparations. Medicinal effectshave been demonstrated for many traditionally used mush-rooms (Ooi and Liu 2000), including extracts of speciesfrom generaAuricularia,Flammulina,Ganoderma,Grifo-

la, Hericium, Lentinus (Lentinula), Pleurotus, Trametes(Coriolus),Schizophyllum, andTremella(Wasser2002).

Over the past two to three decades, scientific and medical

studies in Japan, China, Korea, andmore recently theUnitedStates have increasingly demonstrated the potent and unique

properties of mushroom-extracted compounds for the pre-vention and treatment of cancer.

It is well established that many mushroom-extracted com-pounds are commonly used as immunomodulators or asbiological response modifiers (BRM). The basic strategyunderlying immunomodulation is to identify aspects of thehost response that can be enhanced or suppressed in such away as to augment or complement a desired immune re-sponse. Whether certain compounds enhance or suppressimmune responses depends on a number of factors, includingdose, route of administration, timing of administration of the

compound, mechanism of action, and siteof activity. Knowl-edge of the specific components of cytokine networks andsignaling pathways andtheir role in theregulation of immuneresponses is important in designing strategies to augmentthese responses.

Immunomodulators isolated frommore than 30 mushroomspecies have shown anticancer action in animals (Wasser andWeis 1999). Only a few have been taken to the next step, thatis, objective clinical assessment for anticancer potential inhumans. Of this relative few, all are chemically - or-glucans in nature or peptide-bound polysaccharides. Numer-ous reports have documented the ability of -glucans tononspecifically activate cellular and humoral components of

the host immune system (Tzianabos 2000), but failed todefine their exact mechanism of action until CR3 was iden-tified as the leukocyte membrane receptor for-glucans (Xiaet al.1999).

New discoveries in molecular oncology along with rapidexpansion of our knowledge concerning the processes thatgovern differentiation, apoptosis, immune surveillance, an-giogenesis, metastasis, cell cycle, and signal transductioncontrol have unveiled an abundance of specific moleculartargets for cancer therapy, including a variety of small-mol-ecule compounds that inhibit or stimulate these moleculartargets.

B.-Z. Zaidman. M. Yassin. S. P. WasserBiodiversity and Biotechnology Center of Cryptogamic Plantsand Fungi, The Institute of Evolution,University of Haifa,Mount Carmel,Haifa, 31905, Israel

B.-Z. Zaidman. M. Yassin. J. Mahajna (*)MigalGalilee Technology Center,South Industrial Zone,P.O. Box 831, Kiryat Shmona, 11061, Israele-mail: jamalm@migal.org.il

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Cancer

A neoplasm is an abnormal mass or colony of cells producedby a relatively autonomous new growth of tissue. Most neo-plasms arise from the clonal expansion of a single cell thathas undergone neoplastic transformation. The transformationof a normal to a neoplastic cell can be caused by a chemical,

physical, or biological agent (or event) that directly and irre-

versibly alters the cell genome. Neoplastic cells are char-acterized by the loss of some specialized functions and theacquisition of new biological properties: self-sufficiency ingrowth signals, insensitivity to growth-inhibitory signals, eva-sion of apoptosis, limitless replicative potential, sustained an-giogenesis, and tissue invasion and metastasis. Neoplasticcells pass on their heritable biological characteristics to prog-eny cells.

The biological behavior or clinical course of a neoplasmis further classified as benign or malignant. A malignant neo-

plasm manifests a greater degree of autonomy, is capable ofinvasion and metastatic spread, may be resistant to treatment,and may cause death. A benign neoplasm has a lesser degree

of autonomy, is usually not invasive, does not metastasize,and generally produces no great harm if treated adequately.

Cancer is a generic term for malignant neoplasms. Ana-plasia is a characteristic property of cancer cells and denotesa lack of normal structural and functional characteristics. Atumor is literally a swelling of any type, such as an inflam-mation or other swelling, but modern usage generally de-notes a neoplasm.

Anticancer activity of mushrooms

It has been known for many years that selected mushrooms

of higher Basidiomycetes origin are effective against can-cer. The antitumor activity of the higher Basidiomyceteswas first demonstrated by Lucas et al. (1957), who em-

ployed extracts of fruiting bodies ofBoletus edulisBull.:Fr.and other Homobasidiomycetes in tests against the Sarco-ma 180 line in mice. In the 1960s, calvacin was the mostcommonly cited natural product isolated from the medicinalmushroom and was broadly used in many laboratories as anantitumor agent. Calvacin was isolated from the giant puffball[Calvatia(=Langermannia)gigantea(Batsclh: Pers.) Lloyd]

by Lucas et al. (1958) and showed activity against manyexperimental tumors, including Sarcoma 180, mammaryadenocarcinoma 755, leukemia L-1210, and HeLa cell lines

(Wasser and Weis 1999). There areapproximately 650speciesof higher Basidiomycetes that have been found to possessantitumor activity (Mizuno1995a,b; Wasser2002). Search-ing for new antitumor and other medicinal substances frommushrooms and studying the medicinal value of thesemushrooms has become a matter of great significance.

High-molecular-weight mushroom compounds

Five mushroom preparations have shown clinically signif-icant efficacy against human cancers and are used as BRMs:

lentinan from Lentinus edodes, D-fraction from Grifolafrondosa, schizophyllan (also called SPG, sonifilan, sizo-firan, sizofilan) from Schizophyllum commune, PSK (alsocalled krestin) from Trametes versicolor, and PSP (poly-saccharide peptide), also from T. versicolor. Extensiveresearch has established that Ganoderma lucidum poly-saccharide (GLPS) fractions have immunomodulating

properties, although only a few clinical trials with GLPS

preparations to treat cancer have been reported in interna-tional peer-reviewed journals (Gao and Zhou 2002). Re-cently, an eight-herb mixture, PC-SPES, that includes aGLPS fraction showed promising results in prostate cancertreatment (Lu et al. 2003), but, unfortunately, the mixturewas found to be contaminated with a synthetic estrogen,diethylstilbestrol, which has been used for many years totreat hormone-dependent prostate cancer (Guns et al. 2002).

Lentinan (1985), schizophyllan (1986), and PSK (1977)are approved in Japan as prescription drugs for the treatmentof cancer (Mizuno1999).

All of these preparations are chemically -D-glucans innature or-D-glucans linked to proteins. The biological

activity of-D-glucans is influenced by their solubility inwater (Ishibashi et al. 2001), molecular weight (Okazakiet al.1995; Cleary et al.1999; Mueller et al.2000), branch-ing rate (Cleary et al. 1999; Kataoka et al. 2002), triplehelical solution conformation (Mueller et al. 2000; Falchet al.2000), and-(16)-bonding system in the-(13)major chain (Cleary et al.1999).

There is evidence indicating that-D-glucans induce bio-logical responses by binding to a membrane receptor. Theidentity of the -glucan receptor has been defined as a-glucan inhibitable receptorfor particulate activators of thealternative complement pathway (Czop and Austen1985).Morerecently, another receptor, Dectin-1, wascharacterized

as a -glucan receptor that mediates this activity (Adachiet al.2004; Brown et al.2003).

The ability of complement receptor type 3 (CR3; knownalso as Mac-1, CD11b/CD18, orM2-integrin, function-ing as an adhesion molecule and a receptor for factorI-cleaved C3b, i.e., iC3b, on immune effector cells, such asmacrophage) to recognize-glucans led to the proposal thatthis receptor is the major-glucan receptor on leukocytesand that it mediates all the immunomodulatory effects ofthese carbohydrates (Ross et al. 1999; Xia et al. 1999).When phagocyte CR3 binds to iC3b on bacteria or yeast,

phagocytosis and cytotoxic degranulation are triggeredbecause of simultaneous recognition of iC3b via a CD11b

I-domain binding site and specific microbial polysaccha-rides via a lectin site located COOH-terminal to theI-domain. In contrast, when phagocyte or natural killer(NK) cell CR3 adheres to iC3b on erythrocytes or tumorcells that lack CR3-binding membrane polysaccharides,lysis and cytotoxicity are not stimulated. Cytotoxicity ofneutrophils, macrophages, and NK cell CR3 receptor forneoplastic tissues can be achieved by priming it with-glucans, overriding the normal resistance of iC3b-op-sonized tumor cells (Vetvicka et al. 1997; Yan et al.1999;Ross et al.1999). Moreover, the cytotoxic activation of-glucan-primed NK cell CR3 by iC3b-opsonized tumors

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was accompanied by a tumor-localized secretion of thecytokine tumor necrosis factor (TNF-), interferon (IFN-), IFN-, and interleukin 6 (IL-6) (Ross et al.1999). Furthermore, it has been shown that ligand bindingto the -glucan receptor stimulated nuclear factor B(NF-B) activation in U-937 human myelogenous leuke-mic cells (Battle et al. 1998).

Indeed, many mushroom and yeast-glucans have been

shown to stimulate the mononuclear phagocyte system (e.g.,macrophages, monocytes) and certain lymphocytes (e.g.,

NK cells) to produce cytokines such as IFNs, ILs, and others.These are regarded as the first line in the host defense system,and may themselves successfully eliminate infected or trans-formed cells prior to the establishment of fully fledged hu-moral and cell-mediated immune responses (Borchers et al.1999).

Treatment of human peripheral blood mononuclear cells(PBMC) with Lentinus edodes water-soluble extract in-creased levels of TNF-, IL-1, IL-10, and IL-12 (Jin etal. 2003). The -glucan lentinan induced a marked in-crease in the mRNA levels of IL-1, IL-1, TNF-,

IFN-, and macrophage colony-stimulating factor in peri-toneal exudate cells and splenocytes (Liu et al. 1999).

The ability of grifolan, a purified -(13)-D-glucanfrom Grifola frondosa, to induce various cytokines frommacrophages was examined in vitro. Grifolan enhancedIL-1, IL-6, and TNF- production in the RAW 264.7macrophage-like cell line (Adachi et al. 1994; Okazakiet al.1995). The solubility and molecular mass of grifolanhad an effect on TNF-production (Ishibashi et al. 2001).The D-fraction, a -glucan extracted from the fruiting bodyofG. frondosa, significantly increased TNF-expressed in

NK cells and macrophage-derived IL-12 (Kodama et al.2002a), IFN- in CD4(+) T cells (Kodama et al. 2002b),

and IFN-, IL-12 p70, and IL-18 in spleen and lymph nodecells (Inoue et al. 2002).

Schizophyllan, a -glucan isolated from the culture fil-trate ofSchizophyllum commune increased production ofIFN-and IL-2 in PBMC cultures (Sakagami et al. 1988).A modified single helical conformer of schizophyllan in-duced TNF-and IL-8 production in the U-937 monocyte-like human cell line and TNF- only in PBMC cultures(Hirata et al.1998).

Water extract of PSP extracted fromTrametes versicolorsignificantly increased IL-1and IL-6, while substantiallylowering IL-8 in human promyelocytic leukemic HL-60cells (Hsieh et al.2002a). PSK extracted fromT. versicolor

stimulated the production of IL-1 by PBMC cultures moreefficiently than the production of TNF. More IL-1 wasaccumulated in the cells than in the medium fraction, whereasIL-1 was distributed evenly into both fractions (Sakagamiet al. 1993).

Mushroom trace elements

Mushrooms are a rich source of rare minerals and aminoacids. Mushrooms are also a good source of trace elements

such as copper, zinc, selenium, iron, and molybdenum,which are involved in many biochemical processes sup-

porting life.Many mushroom species accumulate trace elements to a

considerably higher extent than plants. About 40 traceelements have been reported in the literature to date. Anumber of reviews have been published on trace elementcontent in mushrooms (Michelot et al. 1998; Kalac and

Svoboda 2000). Selenium is a very important factor inmaintaining good health and for the function of a largenumber of physiological processes (Chariot and Bignani2003; Rayman and Rayman2002), including a number ofenzymes (Burk 2002). The ability of mushrooms to ac-cumulate selenium depends on their species, inhabitation,

phases of mushroom growth, and precipitation quantity.Various techniques have been employed to determine theselenium concentration of mushrooms (Dernovics et al.2002; Slejkovec et al.2000), which is found to range over0.01220.0 mg/kg dry weight. The results of analyses in-dicate that selenium content varies considerably amongdifferent mushroom species. The highest selenium con-

tents were found in Boletus edulis.During the past two decades, selenium emerged with the

most consistent anticancer effect among a number of mi-cronutrients tested in animal experiments and clinicaltrials (Clark et al. 1996, 1998; Sinha and El-Bayoumy2004). Several chemical forms of selenium have beenused in laboratory studies. The prototype forms are sodi-um selenite, which causes single- and double-strand-breakDNA damage, and selenomethionine, which is relativelynontoxic and non-DNA-damaging. Although several mech-anisms including DNA cytosine methyltransferase inhi-

bition (Fiala et al.1998), antioxidant protection (De Silvaet al.2004), enhanced immune surveillance (Kiremidjian-

Schumacher et al. 2000; Safir et al. 2003), and inhibitionof angiogenesis (Lu and Jiang 2001; Jiang et al. 2000)have been proposed to account for the anticancer effectof selenium (Raich et al. 2001), selective induction ofapoptosis of tumor cells may be of special significance(Ghosh2004).

Suggested molecular mechanisms include inhibition ofthe anti-apoptotic proteins IB kinase (IKK) and NF-B(Gopee et al.2004; Gasparian et al.2002), up-regulation of

pro-apoptotic genes, such asp21CIP1/WAF1,p27KIP1,APO-1,andCaspase-3, while down-regulating cell growth-regula-tory genes, such as c-myc, cyclin D1, cyclin D2, and

proliferating cell nuclear antigen (PCNA) (El-Bayoumy

et al. 2003; Jiang et al. 2002; Venkateswaran et al. 2002;Dong et al.2002; Zhong and Oberley2001).

The recognition that selenium has anti-carcinogenicproperties, with the ability of mushrooms to accumulatethis element, created a market niche for selenium-enrichedmushrooms (e.g.,Agaricus bisporus,Flammulinavelutipes,

Pleurotus ostreatus) that could be used as a new seleniumsource in dietary supplements or as a value-added ingredientfor the formulation of functional foods or nutraceuticals(Anonymous2003; Werner and Beelman2002; Stajic et al.2002; Spolar et al.1999).

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Low-molecular-weight mushroom organic substances

Another source for substances of therapeutic interest is thepool of secondary metabolites produced by a variety ofmushrooms. These substances have their origins as deriva-tives from many intermediates in primary metabolism, butmost of them can be classified according to five main meta-

bolic sources.These are:(1) amino acid-derived pathways,(2)

the shikimic acid pathway for the biosynthesis of aromaticamino acids, (3) the acetatemalonate pathway from acetylcoenzyme A, (4) the mevalonic acid pathway from acetylcoenzyme A which functions in primary metabolism for thesynthesis of sterols, and (5) polysaccharides and peptido-

polysaccharides (discussed in the previous section). Thepolyketide and the mevalonic acid pathways are most ofteninvolved; and they produce a greater variety of compoundsthan the other pathways.

NF-B inhibitors

Rel/NF-B (NF-B) transcription factors are a family ofstructurally related eukaryotic transcription factors that areinvolved in the control of a large number of normal cellu-lar and whole-organism processes such as immune andinflammatory responses, developmental processes, cellulargrowth, and apoptosis. In addition, these factors are activein a number of disease states including cancer, arthritis,chronic inflammation, asthma, neurodegenerative diseases,and heart disease (Kumar et al. 2004).

The activity of NF-B is tightly regulated by interactionwith inhibitory IB proteins. As with the Rel/NF-B pro-teins, there are several IB proteins that have different af-finities for individual Rel/NF-B complexes, are regulated

slightly differently, and are expressed in a tissue-specificmanner.

In most cells, NF-B is present as a latent, inactive, IB-bound complex in the cytoplasm. When a cell receivesany of a multitude of extracellular signals, NF-B rapidlyenters the nucleus and activates gene expression. There-fore, a key step for controlling NF-B activity is the reg-ulation of the IB/NF-B interaction. Almost all signalsthat lead to activation of NF-B converge on the activa-tion of a high-molecular-weight complex that contains aserine-specific IKK. Activation of the IKK complex leadsto the phosphorylation by IKK of two specific serinesnear the N terminus of IB , which targets IB for

ubiquitination and degradation by the proteasome. Theunmasked Rel/NF-B complex can then enter the nucleusto activate target gene expression. One of the target genesactivated by NF-B is that which encodes IB . Newlysynthesized IB can enter the nucleus, remove NF-Bfrom DNA, and export the complex back to the cytoplasmto restore the original latent state (Lipniacki et al. 2004;Yamamoto and Gaynor2004).

Supernormal activation of NF-B has been shown to beassociated with cancer. This is thought to be associated withenhanced transactivation of the RelA subunit of NF-Bvia activation by the hypoxic environment of many tu-

mors, or through the action of inflammatory cytokinesreleased either by the tumor cells themselves or by in-filtrating non-tumor cell types. Active NF-B promotestumor growth by increasing transcription of genes that in-duce cell proliferation, are anti-apoptotic, pro-angiogenic,

pro-metastatic, and are responsible for other cellular mech-anisms required for tumor growth. An important componentof tumor prevention, therefore, involves the inhibition of

aberrant NF-B activity (Ravi and Bedi2004).Two epoxy compounds are involved in modulating the

activity of NF-B: panepoxydone (isolated from Panusconchatus,P. rudis, and later fromLentinus crinitus(Erkelet al. 1996) and cycloepoxydon (isolated fromXylaria strain45-93; Gehrt et al. 1998); together with the lipid-solublethiol compound gliotoxin (an epipolythiodioxopiperazineclass of fungal toxin), originally isolated from the micro-scopic fungus Gliocladium fimbriatum, but also found invarious species of Aspergillus (Watanabe et al. 2004),

Penicillium (Waring et al. 1987), and Candida (Shah andLarsen1991).

Panepoxydone inhibited TNF--induced activation of

NF-B in a promoterreporter assay using COS-7 cellsand was suggested as inhibiting TNF-- or 12-O-tetra-decanoylphorbol-13-acetate (TPA)-induced phosphoryla-tion and degradation of IB (Erkel et al.1996).

Recently, a 5-dehydroxymethyl derivative of epoxyqui-nomicin C (a weak antibiotic isolated from Amycolatopsishaving a 4-hydroxy-5,6-epoxycyclohexenone structure like

panepoxydone from species of the generaPanusand Len-tinus) designated as DHMEQ (formerly DHM2EQ) wastested as a NF-B inhibitor (Matsumoto et al. 2000).DHMEQ inhibited TNF-- and TPA-induced transcrip-tional activity of NF-B in human T cell leukemia Jurkatcells. It also inhibited the TNF--induced DNA-binding of

nuclear NF-B, but not the phosphorylation and degrada-tion of IB. Moreover, DHMEQ inhibited the TNF--in-duced nuclear accumulation of p65, a component of NF-B.It also inhibited TNF--induced nuclear transport of greenfluorescent protein-tagged p65. In contrast, DHMEQ didnot inhibit the nuclear transport of Smad2 and large Tantigen. Also, it did not inhibit TNF--induced activationof c-Jun N-terminal kinase, but synergistically inducedapoptosis with TNF- in Jurkat cells. These data indicatethat DHMEQ is a unique inhibitor of NF-B, acting at thelevel of nuclear translocation (Ariga et al. 2002).

The activity of NF-B was inhibited by DHMEQ inDU145, JCA-1, and PC-3 hormone-refractory prostate can-

cer cell lines. Statistically, significant growth inhibition wasachieved by 20g/ml DHMEQ; and marked levels of ap-optosis were induced 48 h after DHMEQ administration invitro (Kikuchi et al.2003).

The effectiveness of DHMEQ against the advanced KU-19-19 human bladder cancer cell line, in which NF-B isconstitutively activated, was investigated. The DNA-bind-ing activity of NF-B was completely inhibited following26 h exposure to 10 g/ml DHMEQ. Marked levels ofapoptosis were observed 48 h after DHMEQ administration.These results confirmed that DHMEQ inhibited constitu-tively activated NF-B and, consequently, apoptosis was

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induced. However, it was still possible that DHMEQ causedapoptotic cell death through some other mechanism, whichhas not yet been fully investigated (Horiguchi et al. 2003).

Cycloepoxydon inhibited TPA-induced NF-B- andactivator protein-1 (AP-1)-mediated secreted alkaline phos-

phatase (SEAP) expression with 50% inhibition concentra-tion (IC50)valuesof12g/ml and 35g/ml, respectively,in COS-7 and HeLa S3 cells. Cycloepoxydon strongly re-

duced TPA- and TNF--mediated binding of NF-B to ahigh-affinity consensus sequence, which was due to theinhibition of phosphorylation of the protein IB (Gehrt et al.1998).

Pahl et al. (1996) reported that nanomolar concentrationsof gliotoxin inhibit the activation of NF-B in response toa variety of stimuli in T and B cells. The effect of gliotoxinwas specific because, at the same concentrations, gliotoxindid not affect activation of the transcription factor NF-ATor interferon-responsive signal transducers and activatorsof transcription. Likewise, gliotoxin did not alter the activ-ity of the constitutively DNA-binding transcription factorsOct-1 and cyclic AMP response element-binding protein

(CREB) and the activation of the protein tyrosine kinasesp56lck and p59fyn. Very high concentrations of gliotoxinprevented NF-B DNA-binding in vitro. However, in in-tact cells, inhibition of NF-B did not occur at the level ofDNA binding. Rather, gliotoxin appeared to prevent deg-radation of IB, NF-Bs inhibitory subunit.

Recently, Vigushin et al. (2004) demonstrated that glio-toxin inhibited proliferation of six breast cancer cell linesin culture with a mean IC50 value of 328289 M. In-tracellular farnesylation of lamin B and geranylgeranyla-tion of Rap1A were inhibited in a dose-dependent manner.In randomized controlled studies using the N-methyl-N-nitrosourea rat mammary carcinoma model, gliotoxin had

pronounced antitumor activity in vitro and little systemictoxicity when administered to ten animals at 10 mg/kg bysubcutaneous injection weekly for 4 weeks, comparedwith ten controls. Single doses up to 25 mg/kg were welltolerated.

Spores or dried fruiting bodies ofGanoderma luciduminhibit constitutively active transcription factors AP-1 and

NF-B in breast MDA-MB-231 and prostate PC-3 cancercells. Furthermore,G. luciduminhibits both the expressionof uPA and uPA receptor and the secretion of uPA, whichresults in the suppression of the migration of MDA-MB-231and PC-3 cells. These data suggest that spores and un-

purified fruiting bodies of G. lucidum inhibit invasion of

breast and prostate cancer cells by a common mechanismand could have potential therapeutic use for cancer treat-ment (Sliva et al.2002,2003; Sliva2003).

Five novel antibiotics described as irpexans (1, 2, 3a, 3b,4) were isolated from fermentations of anIrpex species inthe course of screening for new inhibitors of AP-1- and

NF-B-mediated signal transduction pathways in COS-7cells, using SEAP as a reporter gene. The expression of anAP-1- and NF-B-driven SEAP reporter gene was inhibitedin a dose-dependent manner with 14-acetoxy-15-hydroxyir-

pexan (3b) being the most potent compound, followed by

14,15-irpexanoxide (2), 14,15-dihydroxyirpexan (3a), and14-acetoxy-22,23-dihydro-15,23-dihydroxyirpexan (4). Ir-

pexan (1) exhibited no activity. All five compounds wereterpenoids with a mannose moiety; and their structures wereelucidated by spectroscopic methods (Silberborth et al.2000).

Protein kinase inhibitors

Protein kinases have emerged as key regulators of allaspects of neoplasia including proliferation, invasion, an-giogenesis, and metastasis. Not surprisingly, sequencing ofthe human genome has revealed at least 500 distinctkinases, which can be grouped into some 20 known familieson the basis of structural relatedness. Compounds that pos-sess inhibitory activity for protein tyrosine kinases wereinitially isolated from natural sources in the early to mid-1980s. These compounds include the flavonoid quercetin,the isoflavonoid genistein (detected initially in culturemedia ofPseudomonasspp in the search for quercetin-like

flavonoids), the benzoquinoid ansamycin antibiotic herbi-mycin A, and erbstatin. Genisteins capacity to inhibit themitogen-stimulated growth of mammalian cells in culturewas presumed by most investigators to be due to the inhi-

bition of tyrosine kinase activities associated with criticalgrowth factor receptors. However, experimental data fromhuman breast cells (Peterson and Barnes1996) and prostatecancer cells (Peterson and Barnes1993) strongly suggestedthat genistein affects these cells (and therefore other sys-tems) by mechanisms other than inhibition of protein tyro-sine kinase activity.

Using a screening approach based on a comparative eval-uation of antiproliferative effects in a panel of tumor cells

withdifferential expression of protein tyrosinekinases, threebenzoquinoid macrolidic fungal metabolites produced byClitocybe clavipes, clavilactones A, B, and D (respectivelyCA, CB, CD) and two semisynthetic derivatives of these

products, diacetyl-CA and dimethyl-CA, were identified asinhibitors of protein tyrosine kinases. Naturally occurringCA, CB, and CD showed inhibitory activity in kinase assaysagainst the Ret/ptc1 and epidermal growth factor receptor(EGF-R) tyrosine kinases, while being less effective againstthe v-Abl tyrosine kinase and p34 (Cdc2) serine/threoninekinase (IC50 values of 2.8, 5.5, 81.3, 128.0 M, respec-tively, for the most potent compound CD). CB was shownto be a non-competitive inhibitor of EGF-R with respect

to ATP or poly(Glu(6)Ala(3)Tyr). CD also preferentiallyinhibited the growth of A431 cells, which overexpress aconstitutively active EGF-R, as opposed to IGROV-1 andSKOV-3 cells, which express low levels of the receptor.Furthermore, EGF-R was shown to be a target for clav-ilactones in A431 cells, since EGF-induced receptor auto-

phosphorylation was inhibited in the presence of CB, CD,and diacetyl-CA. Both CD and diacetyl-CA displayedweak activity when administered daily (intraperitoneally)to mice bearing the ascitic A431 tumor (Cassinelli et al.2000).

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Protein- and DNA-alkylating agents

The sesquiterpenes illudin S and illudin M are toxic com-pounds obtained from the mushroomsOmphalotus illudensandLampteromyces japonicus. They possess antibacterialand antitumor properties, but have an unfavorable toxicity

profile when tested in animals (Kelner et al. 1987). De-rivatives of illudins have been prepared and display in-

creased histiospecific toxicity toward malignant cells versusnormal cells. Among these are dehydroilludin M and acyl-fulvene formed by the treatment of illudin S with dilutesulfuric acid (McMorris et al.1996a). The most efficaciousderivative is irofulven (MGI 114, or hydroxymethyl-acylfulvene) prepared by the reaction of acylfulvene with

paraformaldehyde in dilute sulfuric acid (McMorris et al.1996b). Studies of the mechanism of toxicity of illudinsindicate that they may behave as alkylating agents of pro-tein and DNA (McMorris et al. 2004).

Illudin S reacts spontaneously at room temperature withthiols, such as cysteine or glutathione, at an optimum pH ofabout 6. Toxicity to HL-60 myeloid leukemia cells can be

modulated by altering glutathione levels in cells (McMorriset al.1990). Michael-type addition to the ,-unsaturatedketone gives a cyclohexadiene intermediate, an extremelyreactive alkylating agent, which is rapidly converted to astable aromatic product (McMorris et al. 1999).

Cytotoxicity of MGI 114, like that of illudin S, is be-lieved to also involve enzymatic reduction by a cytosolic

NADPH-dependent enzyme. Illudin S undergoes biore-ductive activation with NADPH in a rat liver cytosol

preparation. The addition of hydride to the ,-unsaturatedketone produces a highly unstable intermediate, as in thereaction with thiols. This intermediate is a potent alkylatingagent (McMorris et al.1999). Aromatic products were iso-

lated from the enzyme-catalyzed reaction.Irofulven demonstrated antitumor activity against human

pancreatic carcinoma cell lines in vitro and in vivo (van Laaret al. 2004), HT-29 and HCT-116 colorectal and A2780ovarian carcinoma cells (Poindessous et al. 2003a), head andneck, non-small-cell lung, malignant glioma, colon, ovary,

poorly differentiated androgen-independent prostate cancercarcinoma cells, and to a lesser extent, sarcoma and leu-kemia cell lines (Poindessous et al.2003b), and human pan-creatic cancer cells (Wang et al. 2002).

Modulators of G1/S and G2/M checkpoints

When normal mammalian cells are subjected to stress sig-nals (e.g., hypoxia, radiation, DNA damage, etc.) p53 isactivated. In addition to its activation, ubiquitin-dependentdegradation of the p53 protein is blocked, and conse-quently, the p53 half-life increases significantly (Soussi2000), leading to the accumulation of p53 and transcrip-tion of target genes involved in cell cycle control, such as

p21WAF1/CIP1 (encoding a E-Cdk2 inhibitor; Fei et al.2002), and target genes involved in apoptosis-like BAX(Chipuk et al. 2004) andApaf-1 (Hickman and Helin 2002).The resulting increase in p53-dependent gene transcription

leads to the p53-mediated induction of apoptosis and/orcell cycle arrest.

Cancer cells, through a variety of strategies, can acquireresistance to apoptosis. The most commonly occurring lossof a pro-apoptotic regulator involves the p53 tumor sup-

pressor gene. The resulting functional inactivation of itsproduct, the p53 protein, is seen in more than 50% of humancancers and results in the removal of a key component of the

DNA-damage sensor that can induce the apoptotic effectorcascade (Oren1999).

Genistein is the aglycone of genistin. The isoflavone isfound naturally as theglycoside genistin and as the glycosides6-O-malonylgenistin and 6-O-acetylgenistin. Genistein andits glycosides are mainly found in legumes, such as soy-

beans and chickpeas, and recently have been discovered inthe mushroomF. velutipes (Kang et al.2003).

Rao et al. (2004) demonstrated that 1,25(OH)(2)D(3) andgenistein cooperate to up-regulate the vitamin D receptor

protein by increasing the stability of the vitamin D re-ceptor. Genistein and 1,25(OH)(2)D(3) also cooperate toup-regulate the levels of p21WAF1/CIP1. Small interfering

RNA-mediated knockdown of p21WAF1/CIP1

expressionshowed thatp21WAF1/CIP1 is essential for significant growthinhibition of LNCaP cells in response to either compoundor their combination.

In human hepatocellular carcinoma (HepG2) cells, ge-nistein modulates Cdc2 kinase activity and leads to G2/Marrest. It has been shown that genistein caused an increasein both Cdc2 phosphorylation and expression of the Cdc2-active kinase, Wee1. It also enhanced the expression ofthe cell cycle inhibitor,p21WAF1/CIP1, which interacts withCdc2. Furthermore, phosphorylation/inactivation of Cdc25C

phosphatase, which dephosphorylates/activates Cdc2, wasincreased. Genistein enhanced the activity of the check-

point kinase, Chk2, which phosphorylates/inactivatesCdc25C, induced accumulation ofp53, and activated theataxia-telangiectasia mutated (ATM) gene. Caffeine, anATM kinase inhibitor, inhibited these effects of genisteinon Chk2, p53, and p21WAF1/CIP1 (Chang et al. 2004).

Genistein inhibited in a time- and dose-dependent pro-liferation of the SKOV-3 human ovarian carcinoma cellline. The characteristic morphological changes of apopto-sis were observed by staining and electron microscopy inSKOV-3 exposed to genistein. Its inhibitory effect appearsto be due to the up-regulation ofp21WAF1/CIP1 mRNA and

protein expression and the down-regulation of PCNA andcyclin B1 protein. The onset of apoptosis in ovarian car-

cinoma cell is related to the up-regulation of Bax anddown-regulation of Bcl-2 in mRNA and protein levelinduced by genistein (Li and Mi2003).

Genistein suppressed the proliferation of p53-nullhuman prostate carcinoma cells. Genistein significantly in-hibited the cell growth (this effect being reversible) andinduced a dendrite-like structure. The inhibitory effects ofgenistein on cell growth proliferation were associatedwith a G2/M arrest in cell cycle progression concomitantwith a marked inhibition of cyclin B1 and an induction ofCdk inhibitor p21WAF1/CIP1 in a p53-independent manner.Following genistein-treatment of cells, an increased binding

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of p21 with Cdk2 and Cdc2 paralleled a significant decreasein Cdc2 and Cdk2 kinase activity with no change in Cdk2and Cdc2 expression (Choi et al.2000).

The antitumor effect of an alcohol extract ofG. lucidumwas investigated using MCF-7 breast cancer cells. Alcoholextract ofG. luciduminhibited cell proliferation in a dose-and time-dependent manner, which might be mediatedthrough up-regulation of p21WAF1/CIP1 and down-regula-

tion of cyclin D1. Furthermore, this compound coulddirectly induce apoptosis in MCF-7 cells, which might bemediated through up-regulation of a pro-apoptotic Bax

protein (Hu et al. 2002).PSP extracted from Trametes versicolor significantly

reduced proliferation of MDA-MB-231 breast cancer cells,as compared with controls. Immunostaining showed thatPSP increasedp21WAF1/CIP1 expression and decreased cyclinD1expression (Chow et al.2003).

Inhibitors of MAPK protein kinase signaling pathways

The mitogen activated protein kinases (MAPKs) are anevolutionary conserved family of proline-directed serine/threonine kinases. Three major MAPK cascades have beenidentified so far in mammalian cells: the extracellular signal-regulated kinase (ERK1/2) cascade, the stress-activated

protein kinase/c-Jun N-terminal kinase (SAPK1/JNK) cas-cade, and the p38 MAPK. Following stimulation by anupstream component, a serine/threonine kinase (a MAPKkinase kinase; MEKK) phosphorylates a MEK, which inturn phosphorylates a MAPK on threonine and tyrosineresidues, resulting in its activation. Activated MAPKs phos-

phorylate a wide gamut of substrates including other ki-nases, phosphatases, hormone and growth factor receptors,

cytoskeletal proteins, and transcription factors. These ki-nases serve as the integrators of signals emanating fromvarious receptor tyrosine kinases (RTKs), nonreceptortyrosine kinases, G protein-coupled receptors, cytokine re-ceptors and TGF- family receptors. Nearly all cell-surfacereceptors utilize one or more MAPK cascades, mediat-ing cell proliferation, differentiation, and transformation(ERKs), or stress responses, apoptosis, and inflammation(JNKs/RKs; Fu et al. 2004; Wada and Penninger2004). The

biological effects of MAPKs are modulated by cellularcontext and by cross-talk with other signaling pathways. Forexample, one of the signals that regulate the activation ofMAPKs is cAMP, which operates by activation of PKA,

which in turn activates B-Raf and inhibits Ras and c-Raf-1(Liebmann 2001). The concept that activation of the MAPK

pathway plays a pivotal role in oncogenesis is supported bythe fact that several oncoproteins are mutationally activatedforms of enzymes that act upstream of the MAPK cascade,including ErbB-2 and Scr RTKs, Ras and Raf (Hilger et al.2002).

The pathways that mediate intracellular signal transduc-tion and cell-to-cell signaling interactions are replete with a

plethora of potential therapeutic targets, rendering cell sig-

naling-directed cancer therapy a very promising therapeuticapproach. The positioning of multiple onco-proteins andtumor-suppressive proteins in the same pathways under-scores the importance of some of these signaling routes inmalignant transformation. Therefore, identification of thecritical components in these pathways provides tremen-dous opportunities for the creation of anticancer agents thattarget the fundamental molecular processes responsible for

cancer development, bypassing the obstacle of killingcancer cells resistant to conventional chemotherapy andradiation.

The triterpene-enriched fraction, WEES-G6, was pre-pared from mycelia ofG. lucidum by sequential hot waterextraction, removal of ethanol-insoluble polysaccharides,and then gel-filtration chromatography. WEES-G6 selec-tively inhibited growth of Huh-7 human hepatoma cells, butnot Chang liver cells, a normal human liver cell line. Treat-ment with WEES-G6 caused a rapid decrease in the activityof cell growth regulative protein PKC and the activation ofJNK and p38 MAPKs. The changes in these moleculesresulted in a prolonged G2 cell cycle phase and strong

growth inhibition. None of these effects were seen in normalliver cells (Lin et al.2003).

Modulation of gap junctional intercellular communica-tion (GJIC) is a known cellular event associated with tumor

promotion. The mushroomPhellinus linteusextract (PL; at5 g/ml, 25 g/ml) prevented the inhibition of GJIC and

blocked the hyper-phosphorylation of connexin 43 byH2O2. Furthermore, PL was able to inactivate both ERK1/2and p38 MAPKs. However, PL did not affect the JNK path-way (Cho et al.2002).

Ganoderma lucidumextract induced the neuronal differ-entiation of PC12 cells and prevented nerve growth factor-dependent PC12 neurons from apoptosis. Moreover, these

effects ofG. lucidum might be mediated via the Ras/ERKand CREB signaling pathways, as demonstrated by the

phosphorylation of ERK1, ERK2, and CREB (Cheung et al.2000).

Ethanolic extracts (at 70%) of YZ, a proprietary dietarysupplement prepared from extracts of Trametes versicolor,significantly reduced androgen-dependent LNCaP cellgrowth and down-regulated the levels of secreted PSA,

but had less effect on the expression of intracellular PSAand did not affect levels of the androgen receptor (Hsiehand Wu 2001). YZ had a much less pronounced sup-

pressive effect on the proliferation of PC-3 and DU-145androgen-independent prostate cancer cells, compared with

LNCaP. Western blot analyses showed that the expressionof Rb and PCNA, integrally involved in mammalian cellDNA replication, were significantly reduced by treatmentwith YZ in PC-3 and DU-145 cells, respectively. Furtheranalysis showed that YZ increased the levels of signaltransducers and the activator family of transcription factorsSTAT 1 and STAT 3 in JCA-1 androgen-independent

prostate cancer cells and not LNCaP cells (Hsieh and Wu2001; Hsieh et al. 2002b).

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Aromatase and sulfatase inhibitors

Sulfation and desulfation are important reactions in themetabolism of many steroid hormones. Estrone, estradiol,and dehydro-epiandrosterone circulate predominantly in thesulfated form and, as such, are not biologically active (i.e.,do not bind target receptors). Furthermore, the sulfatedforms of many steroid hormones exhibit half-lives up to

10-fold higher than the desulfated form. Estrogen metabo-lism is an important factor in the proliferation of estrogenreceptor (ER)-positive tumors. Anti-estrogens represent firstline therapy against breast cancer. Sulfation and desulfationof estrone and17-estradiol clearly occur in both normal andmalignant breast tissues. In benign breast tumors, estrogensare biosynthesized from steroid precursors via the aromati-zation of androstenedione to estrone by aromatase in adiposetissue or via the hydrolysis of estrone sulfate by arylsulfataseC (ARSC) in epithelial tissue. Aromatase inhibitors are usedwidely as second-line therapy in breast cancer; and there isnow evidence for a chemopreventive role for these agents(Masamura et al. 1995). However, in many ER-positive

breast tumors, ARSC activity is 30- to 150-fold higher thanaromatase activity, and, therefore, the ARSC pathway repre-sents the major route for intra-tumoral estrogen synthesis.The realization of the importance of the ARSC pathway forintra-tumoral estrogen production has made the inhibition ofthis enzyme a target for the therapeutic control of breast tu-mors. Potent third-generation steroid- and non-steroid-de-rived sulfatase inhibitors are now being developed andtargeted for breast cancer therapy (Ahmed et al.2002).

Aromatase and sulfatase inhibitors were isolated from anedible mushroom, Lepiota americana. 2-Aminophenoxa-zin-3-one inhibited aromatase at IC50=5.7 M, and 3-hydroxy-5,8-epidioxyergosta-6,22-dieneinhibitedsulfatase

at IC50=0.9 M. It was found that 2-aminophenoxazin-3-one was not active against sulfatase, and 3-hydroxy-5,8-epidioxyergosta-6,22-diene was not active against aromatase(Kim et al.2000).

Interestingly, actinomycin D is one of the most exhaus-tively studied DNA-binding ligands; and its widespreadinterest stems from its function as a model sequence-specificantitumor antibiotic. It consists of a planar 2-aminophenox-azin-3-one chromophore with twobulkycyclic pentapeptidelactones. This drug is used clinically for the treatment ofmalignant tumors, such as pancreatic cancer (Kleeff et al.2000), and is also used in combination with other antitumoragents to treat high-risk tumors (Matsui et al.2002).

Agaricus bisporus extracts suppress aromatase activitydose-dependently. Enzyme kinetics demonstrated a mixedinhibition, suggesting the presence of multiple inhibitorsor more than one inhibitory mechanism. In cell aromataseactivity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phy-tochemicals in the mushroom aqueous extract inhibitedaromatase activity and the proliferation of MCF-7aro cells(Grube et al.2001).

Matrix metalloproteinases inhibitors

The matrix metalloproteinases (MMPs) are a family of atleast 15 secreted and membrane-bound zinc-endopepti-dases. Collectively, these enzymes can degrade all of thecomponents of the extracellular matrix, including fibrillarand non-fibrillar collagens, fibronectin, laminin, and base-ment membrane glycoproteins. MMPs are thought to be

essential for the diverse invasive processes of angiogenesisand tumor metastasis. Numerous studies have shown thatthere is a close association between expression of variousmembers of the MMP family by tumors and their pro-liferative and invasive behavior and metastatic potential.In some human cancers, a positive correlation has also beendemonstrated between the intensity of new blood vesselgrowth (angiogenesis) and the likelihood of developingmetastases (Klein et al. 2004). Thus, control of MMP ac-tivity in these two different contexts has generated con-siderable interest as a possible therapeutic target.

The novel hydroquinone, (E)-2-(4-hydroxy-3-methyl-2-butenyl)-hydroquinone, and a known compound, poly-

porenic acid C, were isolated from Piptoporus betulinus(Kawagishi et al. 2002). Polyporenic acid C, a lanostane-type triterpene, was also isolated from Daedalea dickinsii(Kawagishi et al.1997). Both compounds have been shownto inhibit collagenase (MMP-1), which cleaves all three-chains of native interstitial collagens (Kawagishi et al.1997). The hydroquinone derivative has been shown toinhibit stromelysin (MMP-3), which degrades a wide va-riety of protein substrates, including gelatin, fibronectin,and laminin and the core protein of cartilage proteoglycans.It also inhibits a 92-kDa gelatinase (MMP-9), which cleavesgelatin and the basement membrane (Kawagishi et al. 2002).

Cyclooxygenase inhibitors

It is now well established that the inducible isoform of cy-clooxygenase, COX-2, is commonly over-expressed inmany solid tumors (Taketo 1998a,b). Epidemiological stud-ies and clinical trials employing selective and nonselectiveCOX-2 inhibitors indicate that COX-2 is mechanisticallyinvolved in colorectal carcinogenesis, gastric carcinoma,

breast cancer, and prostate cancer.One chemopreventive approach that has received atten-

tion is the regular use of nonsteroidal anti-inflammatorydrugs (NSAIDs). It is now generally accepted that NSAIDs

prevent the development of colorectal cancer, and there issome evidence for a protective effect against breast cancerand esophageal cancer. Proposed mechanisms for theseeffects include the induction of apoptosis, inhibition of an-giogenesis, and direct inhibition of cellular growth, all oc-curring, at least partly, through the inhibition of the COXenzymes involved in prostaglandin synthesis. Over-expres-sion of COX-2 has been observed in human prostate cancercells (Hussain et al. 2003), and higher levels of prosta-glandins have been detected in malignant (as compared with

benign) prostate tissues (Kirschenbaum et al. 2001). Fur-thermore, NSAIDs have been shown to inhibit prostate

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cancer cell proliferation and induce apoptosis in vitro (Hsuet al.2000). Thus, the COX enzymes and the synthesis of

prostaglandins may represent new targets for both chemo-prevention and antitumor therapy (Lieberman2002).

The bioassay-guided isolation and purification of thehexane extract of cultured mycelia ofGrifola frondosa led tothe characterization of a fatty acid fraction and three com-

pounds: (1) ergosterol, (2) ergostra-4,6,8(14),22-tetraen-3-

one, and (3) 1-oleoyl-2-linoleoyl-3-palmitoylglycerol. Thefatty acid fraction and compounds 13 showed COX en-zyme inhibition. The inhibition of COX-1 enzyme by thefatty acid fraction and compounds 13 at 250 g/ml were98, 37, 55, and 67%, respectively. Similarly, the fatty acidfraction and compounds 13 at 250g/ml reduced COX-2enzyme activity by 99, 37, 70, and 4%, respectively (Zhanget al.2002).

A methanol extract ofCordyceps pruinosa inhibited in-flammatory mediators in a LPS-stimulated RAW264.7 mu-rine macrophage cell line and primary macrophages, bysuppressing the gene expression of IL-1, TNF-, induc-ible nitric oxide synthase (iNOS), and COX-2. Moreover, the

extract suppressed NF-B activation in LPS-stimulatedRAW264.7 cells. Administration of the extract significantlydecreased the plasma levels of these inflammatory mediatorsin LPS-injected mice (Kim et al.2003).

The gerronemins A-F (16) were isolated as cytotoxiccomponents of an extract of a Gerronema species. Theirstructures were elucidated by spectroscopic techniques; andthey were composed of a C12C16 alkane or alkene sub-stituted at both ends by 2,3-dihydroxyphenyl groups. Thegerronemins blocked the inducible expression of a humancyclo-oxygenase 2 (hCOX-2) and iNOS promoter-drivenreporter gene with IC50 values of 15 g/ml (Silberborthet al. 2002).

DNA topoisomerases and DNA polymerase inhibitors

The DNA topoisomerases and DNA polymerases recentlyemerged as important cellular targets for chemical inter-vention to treat cancer. DNA topoisomerases are a group ofenzymes that alter the topological structure of DNA by

briefly creating and resealing DNA strand breaks to facil-itate the passage of other DNA strands. Type I topoisom-erases (topo I) introduce transient single-strand breaks intoDNA. Topo II is required to relieve torsional strain in theDNA helix during replication and to mediate the breakage

of double-stranded DNA and re-ligation of the breaks.Both topo I and II can resolve positively or negatively su-

percoiled DNA domains during DNA replication. Topo IIis increased in rapidly proliferating cells, reaching a peakduring the G2/M phase. Topo II is found in fairly constantlevels throughout the cell cycle. Some anti-topo drugs havethe inhibition of enzymatic activity as their primary modeof action. Other drugs targeting the topos interfere with theenzymes cleaving and rejoining activities and, by doingso, they increase the half-life of the transient topo-catalyzedDNA break. Cancer chemotherapeutic drugs that interferewith topo function, such as doxorubicin, daunorubicin, mi-

toxanthrone, camptothecin, amsacrine, etoposide, and teni-poside are used to treat leukemias, lymphomas, breast, lung,and testicular cancers (Topcu2001; Holden2001).

DNA polymerases are eukaryotic enzymes with differentinteractive roles in nuclear DNA synthesis. There are severalsubtypes of mammalian DNA polymerases and their local-ization and function have been clarified. DNA polymerases, , and have been implicated as being responsible for

DNA replication, whereas DNA polymerases , , and have been suggested as working in DNA repair. DNA

polymerase is encoded in the nucleus, but localizes in themitochondria and is responsible for mitochondrial DNAreplication. DNA polymerase is one of the critical mam-malian S-phase enzymes, necessary for the initiation ofDNA synthesis in cells traversing the G1/S phase rep-lication block associated with Rb and p53 and other cel-lular oncoproteins. After the initiation of DNA synthesis,nucleotides are added to the 3 OH primer end generated

by DNA polymerase. DNA polymerase is one of the mostimportant target molecules of antitumor agents, especiallyfor antimetabolite nucleosides that include 1--D-arabi-

nofuranosyl cytosine, 2-deoxy-2,2-difluorocytidine, and1-(2-deoxy-2-fluoro-4-thio--D-arabinofuranosyl) cytosine(Miura and Izuta2004).

Nine lanostane-type triterpene acids were found in scle-rotia of Poria cocos. Among the nine compounds, onlydehydroebriconic acid could potently inhibit DNA topo IIactivity (IC50=4.6 M), while the compound moderatelyinhibited the activities of DNA polymerases , ,,, ,, , , and only from mammals, to similar extents.Another compound, dehydrotrametenonic acid, also showedmoderate inhibitory effects against topo II (IC50=37.5M)and weak effects against all the polymerases tested. Bothcompounds showed no inhibitory effect against topo I,

higher plant (cauliflower) DNA polymerase I (-like poly-merase) or II (-like polymerase), calf thymus terminaldeoxynucleotidyl transferase, human immunodeficiencyvirus type-1 reverse transcriptase, prokaryotic DNA poly-merases such as the Klenow fragment ofEscherichia coliDNA polymerase I, Taq DNA polymerase, and T4 DNA

polymerase, or DNA metabolic enzymes such as T 7 RNApolymerase, T4 polynucleotide kinase, and bovine deoxy-ribonuclease I. These findings suggest that dehydro-ebriconic acid and dehydrotrametenonic acid should bedesignated as topo II-preferential inhibitors although theyalso moderately inhibited all the mammalian DNA poly-merases tested. Both dehydrotrametenonic acid and de-

hydroebriconic acid could prevent the growth of humangastric cancer cells and their LD50 values were 63.6M and38.4M, respectively. The cells were halted at the G1 phasein the cell cycle (Mizushina et al. 2004; Akihisa et al. 2004).

Mizushina et al. (1998b) and Tanaka etal. (1998) isolatednew triterpenoid compounds, designated fomitellic acids Aand B from the basidiomycete Fomitella fraxinea, whichselectively inhibit the activities of mammalian DNA poly-merasesand. Recently, Mizushina et al. (2000) reportedthat these triterpenoids were potent inhibitors of calf DNA

polymerase, rat DNA polymerase , and human DNAtopo I and II, and showed moderate inhibitory effects on

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plant DNA polymerase II and human immunodeficiencyvirus reverse transcriptase. However, these compounds didnot influence the activities of prokaryotic DNA polymer-ases, such as E. coli DNA polymerase I, or other DNAmetabolic enzymes, such as human telomerase, T7 RNA

polymerase, and bovine deoxyribonuclease I. These triter-penoids were not only mammalian DNA polymerase in-hibitors, but also inhibitors of DNA topo I and II, even

though the enzymic characteristics of DNA polymerasesand DNA topos differed markedly, including their modesof action, amino acid sequences, and three-dimensionalstructures. These triterpenoids did not bind to DNA, sug-gesting that they act directly on these enzymes. Fomitellicacid A prevented the growth of NUGC gastric cancer cells,with LD50 values of 38 M and 30 M, respectively(Mizushina et al.2000).

Terpenoids 1, 2 and 3, which selectively inhibit eukary-otic DNA polymerase activities, were isolated from thefruiting body of the basidiomycete Ganoderma lucidum,and their structures were determined by spectroscopic anal-ysis. New terpenes, lucidenic acid O and lucidenic lactone,

prevented not only the activities of calf DNA polymerase and rat DNA polymerase , but also those of humanimmunodeficiency virus type 1 reverse transcriptase. Athird new terpene, cerevisterol, which was reported to bea cytotoxic steroid, inhibited only the activity of DNA

polymerase(Mizushina et al. 1999).Two other compounds from G. lucidum that inhibit eu-

karyotic DNA polymerase were identified as cerebrosides:(4E,8E)-N-D-2-hydroxypalmitoyl-1-O--D-glucopyrano-syl-9-methyl-4,8-sphingadienine and (4E,8E)-N-D-2-hydro-xystearoyl-1-O--D-glucopyranosyl-9-methyl-4,8-sphinga-dienine. Thesecerebrosidesselectively inhibited the activitiesof replicative DNA polymerases, especially the-type, from

phylogenetically broad eukaryotic species, whereas theyhardly influenced the activities of DNA polymerase ,

prokaryotic DNA polymerases, terminal deoxynucleotidyltransferase, HIV reverse transcriptase, RNA polymerase,deoxyribonuclease I, and ATPase. The inhibition of anotherreplicative polymerase, the-type, was moderate. The inhi-

bitions of the replicative polymerases were dose-dependent,andtheIC50 foranimalor mushroom DNApolymerasewasachieved at approximately 12 g/ml (16.2M) and that foranimal DNA polymerase at57g/ml (77.2M; Mizushinaet al.1998a).

The 490 quinone, a natural sulfhydrylarylating reagentfrom the mushroomAgaricus bisporus, markedly inhibited

L1210 murine leukemia DNA polymerase while givinglittle inhibition of DNA polymerase from this source.This quinone was more strongly inhibitory than p-chloro-mercuribenzoate orN-ethylmaleimide and was less readilyneutralized by sulfhydryl-containingmolecules,such as dithio-erythritol. Preliminary experiments indicated that DNA

protects DNA polymerasefrom inhibition by the 490 qui-none. The inhibition of DNA synthesis by the 490 quinonemay contribute significantly to the cytotoxicity of this com-

pound and to the potential of -L-glutaminyl-4-hydroxy-benzene as an antitumor agent (Graham et al. 1977).

Anti-angiogenic substances

Tumor growth and metastasis depend upon the developmentof a new vasculature in and around the tumor. Angiogene-sis is the process of new blood vessel formation from pre-existing ones. Angiogenesis facilitates progressive tumorgrowth by providing adequate oxygenation to the tumorthrough a series of interrelated steps, including endothelial

cell proliferation, motility of endothelial cells through theextracellular matrix toward angiogenic stimuli, and capil-lary differentiation. The balance between stimulatory andinhibitory factors released by the tumor and its microen-vironment regulates the process (Beecken et al. 2000).Some important mediators that have been studied includeacidic and basic fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF), transforming growthfactors (TGFs) and , platelet-derived growth factor(PDGF), angiogenins, IL-8, and TNF-. Because tumorgrowth and metastasis depend on angiogenesis, a great dealof attention has been focused on therapy that can interruptthis process. Anti-angiogenic therapy can target endothe-

lial cells directly and inhibit the production or action, orboth, of pro-angiogenic peptides by the tumor cells orhost, or enhance the expression of angiogenesis inhibitorswithin the tumor (Hayes et al. 1999). Anti-angiogenictherapy that targets endothelial cells, rather than tumorcells directly, has been evaluated as a novel therapeuticstrategy for malignant diseases. The theoretical advantageof this therapy is that endothelial cells are unlikely to ac-quire mutations that lead to drug resistance. Endothelialcells are also readily exposed to blood-borne agents, cir-cumventing the problem of drug delivery (Scappaticci2002). Although it was widely assumed that anti-angio-genic therapy was antiproliferative, it was recently found

that anti-angiogenic therapy can induce apoptosis andtumor regression (Gingras et al. 2003).

An antitumorsubstancewasisolated from thelipid fractionof Agaricus brasiliensis. The substance was identified asergosterol. Tumor growth was retarded by the oral adminis-tration of thelipid fraction extractedfromA. brasiliensis. Oraladministration of ergosterol to Sarcoma 180-bearing miceat doses of 400 mg/kg and 800 mg/kg administered for20 days significantly reduced tumor growth without side-effects. Ergosterol had no cytotoxicity against tumor cells.Two in vivo models have been used to clarify the antitu-mor activity of ergosterol. Intraperitoneal administration ofergosterol at doses of 5, 10, and 20 mg/kg for five con-

secutive days inhibited the neovascularization induced byLewis lung carcinoma cell-packed chambers, suggestingthat either ergosterol or its metabolites may be involvedin the inhibition of tumor-induced neovascularization. Inthe second model, the inhibitory effects of ergosterol weretested on Matrigel-induced neovascularization. FemaleC57BL/6 mice were subcutaneously inoculated with Matri-gel containing acidic FGF and heparin with or withoutergosterol. Ergosterol inhibited the Matrigel-induced neo-vascularization. From these results, it seems likely thatthe antitumor activity of ergosterol might be due to direct

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inhibition of angiogenesis induced by solid tumors (Takakuet al.2001; Didukh et al.2004).

Human umbilical vein endothelial cells (HUVECs),HT1080, and B16-F10 cells were used to evaluate the ef-fects ofCordyceps militaris extract (CME) on angiogene-sis and tumor growth. DNA fragments, angiogenic relatedgene expressions, capillary tube formation, wound heal-ing in vitro, and tumor growth in vivo were measured. CME

inhibited growth of HUVECs and HT1080. CME at 100mg/l and 200 mg/l reduced 72-kDa gelatinase (MMP-2)gene expression in HT1080 cells by 6.0% and 22.9% after3 h and by 14.9% and 32.8% after 6 h treatment. CMEdid not affect 92-kDa gelatinase (MMP-9) gene expres-sion in B16-F10 melanoma cells. CME at 100 mg/l and200 mg/l also reduced bFGF gene expression in HUVECs

by 22.2% and 41.3%. CME inhibited tube formation ofendothelial cells in vitro and in vivo. CME repressed thegrowth of B16-F10 melanoma cells in mice, comparedwith the control group (Yoo et al. 2004).

Ethanol extract (70%) ofPhellinus linteus (PL) showedstrong anti-angiogenic activity, which was detected using

the chick embryo chorioallantoic membrane assay. PL wasalso activeas an antioxidant,and its activity wascomparablewith vitamin C in scavenging the stable free radical1,1-diphenyl-2-picrylhyrazyl. It also inhibited lipid perox-idation in a concentration-dependent manner, arguing thatantioxidant and anti-angiogenic activities ofP. linteus could

be partly responsible for its antitumor effect (Song et al.2003).

The protein kinase C (PKC) isoform plays an im-portant role in angiogenesis (Berns et al.2000). Yoshiji etal. (1999) demonstrated that PKClies on the signal trans-duction pathway by which VEGF augments developmentand angiogenesis, not only at the initial stage, but also after

the tumor is fully established. Naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) was iso-lated from the culture broth ofP. linteus(Park et al.2004),Gymnopilus marginatus,G. patriae,G. parvisporus (Reesand Ye 1999), and Inonotus hispidus (Awadh Ali et al.2003). Hispidin is a potent inhibitor of PKC(IC50=2 M).Hispidin synthesized by Gonindard et al. (1997) wasshown to be cytotoxic (between 1 mmol/l and 0.1 mol/l)toward normal MRC-5 human fibroblasts, the SCL-1human cancerous keratinocytes cell line, and the Capan-1human cancerous pancreatic duct cell line. Interestingly,the addition of hispidin in three successive doses (between10 mol/l and 0.1 mol/l) led to a 100-fold increase in

activity with an enhanced activity on cancer cells comparedwith normal cells (Gonindard et al.1997).

The triterpenoid fraction (100 mg/kg, 200 mg/kg) of thefruiting bodies of Ganoderma lucidum inhibited primarysolid-tumor growth in the spleen, liver metastasis, and sec-ondary metastatic tumor growth in the liver in intrasplenicLewis lung carcinoma-implanted mice. In addition, the tri-terpenoid fraction (800 g/ml) inhibited angiogenesis in-duced by Matrigel (a soluble basement membrane extractof the EngelbrethHolmSwam tumor) supplemented withVEGF and heparin in an in vivo model. This suggested thatthe antitumor and antimetastatic activities of the triterpe-

noid fraction ofG. lucidum might be due to the inhibitionof tumor-induced angiogenesis. The acidic fraction of thetriterpenoid fraction inhibited the Matrigel-induced angio-genesis. Compound I was isolated from the acidic fractionas an active substance that inhibited Martigel-inducedangiogenesis. Compound I was identified as ganoderic acidF, based on the data of IR, 1H-NMR, 13C-NMR, and MSanalyses (Kimura et al.2002).

Human clinical evaluations

A number of mushroom high-molecular-weight polysac-charides have proceeded through phase I, II, and III clin-ical trials. Lentinan (Lentinus edodes), PSK, and PSP (T.versicolor) have been used in clinical trials with hundredsof cancer patients. However, other compounds have only

been assessed with small numbers of patients.There have been numerous clinical trials of lentinan in

Japan although none have been placebo-controlled anddouble-blinded. However, lentinan has been approved for

clinical use in Japan for many years and is manufactured byseveral pharmaceutical companies. Wasser and Weis (1999)reviewed the accumulated information on antitumor activity,

prevention of metastasis, and suppression of chemical andviral oncogenesis in animal models by lentinan. Lentinanhas proved successful in prolonging the overall survival ofcancer patients, especially those with gastric and colorectalcarcinoma (Furue and Kitoh 1981; Taguchi et al. 1985;Hobbs2001).

There have been several decades of successful clinicaltrials using PSK to treat head and neck, upper gastro-in-testinal, colorectal, and lung cancers with some reportedsuccess in treating breast cancer as well. Clinical trials with

PSK were recently extensively reviewed by Kidd (2000).Remarkably, by 1987, PSK accounted for more than 25% oftotal national expenditure for anticancer agents in Japan.

While PSK has been almost exclusively developed andtested in Japan, PSP, in contrast, is a product of China andcontinues to be assessed for efficacy safety by their sci-entists and oncologists. Many Phase III clinical trials ofPSP combined with conventional therapies have demon-strated significant benefits against cancers of the stomachand lung (Jong and Yang1999; Yang1999; Yao1999).

The maitake D-fraction is a relatively new compound,and there are a number of clinical trials in breast, prostate,lung, liver, and gastric cancers underway in the United

States and Japan. Most of these are at an early clinical stage(phase I/II). In early 1998, for example, Maitake Productsreceived FDA approval for an Investigational new drugapplication to conduct a phase II pilot study on the efficacyof a maitake D-fraction in treating advanced breast and

prostate cancer patients (Zhuang and Wasser2004).Despite the fact that schizophyllan has been approved for

clinical use in Japan, not many clinical trials have beencarried out; and many are not blinded. Examples includeclinical trials evaluating schizophyllan as an assistant im-munotherapy in the treatment of head and neck (Kimuraet al.1994) and gastric cancer (Fujimoto et al.1991).

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As for low-molecular-weight mushroom compounds,only a minute fraction of the many newly discovered com-

pounds have proceeded to a higher level of clinical eva-luation. To our knowledge, irofulven is the most extensivelystudied compound in this group. Phase II clinical trials were

performed in advanced melanoma (Pierson et al. 2002),advanced renal cell carcinoma (Berg et al.2001), relapsedor refractory non-small cell lung cancer (Sherman et al.

2004), metastatic colorectal cancer (Nasta et al.2003), andrecurrent or persistent endometrial carcinoma (Schilder et al.2004). Unfortunately, irofulven demonstrated minimal tono significant antitumor activity in these trials. Despite ev-idence of irofulven activity in pancreatic cancer, MGIPHARMA (April 2002) stopped a phase III irofulven clin-ical trial for refractory pancreatic cancer, based on prelim-inary data analysis. There are still ongoing phase II clinicaltrials in ovarian, prostate, and hepatocellular cancers.

Conclusions

Natural products have been major molecular structural re-sources for drug discovery. Antibiotics, penicillin, the anal-gesic and antipyretic drug aspirin, the anticancer drug taxol,the anti-malarial drug artemisinin, and the anti-Alzheimersdisease drug huperzine A are typical, successful examples.The reservoir of natural products contains an abundance ofchemical novelty and diversity: about 40% of the chemicalscaffolds of the published natural products are unique andhave not been made by synthetic chemistry. Another ad-vantage is their chiral center. Most natural products arechiral and occur as single enantiomers; and many moderndrugs are chiral and have their biological activity associatedwith only one of the enantiomers. Therefore, using natural

products as enantiomerically pure starting materials is a goodsolution to this problem. In addition, statistical data indicatethe advantage and potential of natural products for discover-ing new drugs or leads. Among the 520 new drugs approvedin the United States between 1983 and 1994, 157 were thenatural products or derived from natural products and morethan 60% of antibacterials and anticancer drugs originatedfrom natural products.

Drug discovery in the past 100 years has involved ap-proximately 500 targets, while in the same period about200,000 natural products have been isolated from differentnatural species. In addition, the completion of the humangenome suggests there are 30,00040,000 genes and at least

as many proteins. Many of these proteins are potential tar-gets for drug intervention to control human diseases.

Today, it becomes increasingly difficult to synthesize ordiscover new and interesting lead compounds. But, with theaid of techniques like high-throughput screening assays, 3-D

proteinligand models, virtual screening, and computer-assisted rational drug design and the expanding knowledgeof the molecular basis of tumorigenesis and metastasis, itseems feasible to harness the natural pool and discovernovel compounds that rationally target the abnormal mo-lecular and biochemical signals leading to cancer.

Acknowledgements We thank Prof. N. Ohno (Japan) for criticalreading of the manuscript, and R. Permut for English editing. Thisresearch was supported by the Ministry of Science and Technologyof Israel, grant No. 3-0561.

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