Nutrition &Cancer, January 2009 by Ben-Zion Zaidman, Solomon P. Wasser, Roumyana D. Petrova, Jamal Mahajna, Nesly Dotan

Summary:

Prostate cancer (PCa) is the most common male malignancy in many Western countries. Primary PCa is hormone dependent and is manageable by hormonal therapy. However, it rapidly develops to hormone-refractory tumors due to the accumulation of mutations in the androgen receptor and/or the acquisition of alternative cellular pathways that support proliferation and inhibit apoptosis of prostate cancer. To date, no effective therapy is available for clinically hormone-insensitive or hormone-refractory stages of prostate cancer.ABSTRACT FROM AUTHORCopyright of Nutrition &Cancer is the property of Lawrence Erlbaum Associates and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder’s express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.

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Nutrition and Cancer, 61(1), 16?26 Copyright ? 2009, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635580802379323 Pharmacological Values of Medicinal Mushrooms for Prostate Cancer Therapy: The Case of Ganoderma Lucidum Jamal Mahajna Cancer Drug Discovery Program, Migal-Galilee Technology Center, Kiryat Shmona, Israel Nesly Dotan and Ben-Zion Zaidman Cancer Drug Discovery Program, Migal-Galilee Technology Center, Kiryat Shmona, Israel and the Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Israel Roumyana D. Petrova Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria Solomon P. Wasser Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Israel Prostate cancer (PCa) is the most common male malignancy in many Western countries. Primary PCa is hormone dependent and is manageable by hormonal therapy. However, it rapidly develops to hormone-refractory tumors due to the accumulation of muta- tions in the androgen receptor and/or the acquisition of alternative cellular pathways that support proliferation and inhibit apopto- sis of prostate cancer. To date, no effective therapy is available for clinically hormone-insensitive or hormone-refractory stages of prostate cancer. Whereas prostate cancer is very common in Western coun- tries, its levels are very low in Asia, providing evidence for a potential link between diet, environmental factors, and can- cer incident. Natural products have been used as a source of new pharmaceuticals including anticancer drugs. The medici- nal properties of mushrooms have been well known in Eastern Asia for thousands of years. Of special interest is the impli- cation of several mushrooms in prostate cancer prevention in- cluding the popular mushroom Ganoderma lucidum (Ling Zhi or Reishi) that has been widely used for the general promotion of health and longevity in Asia. Dried powder of G. lucidum was popular as a cancer chemotherapy agent in ancient China. The pharmaceutical activities of G. lucidum substances targeting Submitted 27 November 2007; accepted in final form 13 April 2008. Address correspondence to Jamal Mahajna, Cancer Drug Dis- covery Program, Migal, P.O. Box 831, Kiryat Shmona 11016, Israel. Phone: +972-4-6953537. Fax: +972-4-6944980. E-mail: jamalm@migal.org.il signal transduction pathways or molecular targets implicated in prostate carcinogenesis are reviewed. OVERVIEW For thousands of years, natural products have played an im- portant role throughout the world in treating and preventing hu- man diseases. Natural product medicines have originated from various source materials including plants, fungi, microorgan- isms, and animals. Such natural bioactive substances possess an enormous structural and chemical diversity, unsurpassable by any synthetic library; they are evolutionally optimized as drug-like molecules and might be considered biologically val- idated. Moreover, these molecules can serve as templates for semisynthetic and fully synthetic modifications. An analysis of the origin of drugs developed between 1981 and 2002 showed that natural products or natural product- derived drugs comprised 28% of all novel chemical entities (NCEs) launched onto the market (1). In addition, 24% of these NCEs were synthetic or natural mimic compounds based on a study of pharmacophores related to natural products. This com- bined percentage (52% of all NCEs) suggests that natural prod- ucts are important sources for new drugs and are also good lead compounds suitable for further modification during drug devel- opment (2). In the case of anticancer agents, natural products have made significant contributions as either direct treatments or templates for synthetic modification. In this category, there are some 140 anticancer agents available to Western countries and Japan; 62% of them are nonsynthetic agents. It is estimated that there are approximately 1.5 million species of fungi in the world, of which approximately 82,000 are 16 À; MEDICINAL MUSHROOMS FOR PROSTATE CANCER THERAPY 17 described (3). About 20,000 of the known species belong to macrofungi, of which about 5,000 are edible and over 2,000 safe (4). Fungi from the Basidiomycota division are of great in- terest due to the large number of biologically active compounds they contain (5?13). Fungal fruiting bodies, mycelium, or the culture broth in which the mycelium has been cultivated are all explored for biological activities. Consequently, approximately 650 species of higher Basidiomycetes have been found to pos- sess antitumor activities (7). Prostate Cancer Prostate cancer is the most common male malignancy in many Western countries and the third leading cause of can- cer deaths in men worldwide. The age-standardized incidence rate of prostate cancer is highest in the United States (137 per 100,000 in Black men), lower in European countries (28 and 31 per 100,000 in England and Denmark, respectively), and lowest in Asia (10 and 2.3 per 100,000 in Japan and China, respectively) (15). The epithelium of the prostate gland is under the hormonal control of androgens, the production of which depends on the hypothalamic-pituitary-testicular axis. The Leydig cells of the testes produce 95% of total androgens, and the adrenal glands produce the remaining 5%. In the prostate, free testosterone diffuses directly into the epithelial or stromal cells where it is converted into the functionally active androgen, dihydrotestos- terone (DHT), by the action of 5- reductase enzyme system located on the nuclear membrane. Dihydrotestosterone action is mediated by the androgen receptor (AR) (16), which functions to preserve the normal function and structure of the prostate. The androgen receptor is a structurally conserved member of the nu- clear receptor superfamily of ligand-activated transcription fac- tors. Androgen ablation therapy has been shown to produce the most beneficial responses in patients with hormone-responsive prostate cancer (19). Castration, either surgically or chemically, remains the standard treatment option for most patients. Com- bined with antiandrogens that interfere with androgen receptor function, these methods significantly prolong the survival of prostate cancer patients (20). Androgen deprivation therapy is initially effective in repressing primary prostate tumors. How- ever, progression to the clinically hormone-insensitive stage is usually inevitable. To date, no effective therapy is available for hormone-insensitive or hormone-refractory stages of prostate cancer (21). Prostate cancer has a complex etiology; currently age, ethnic- ity, obesity, and family history are the most consistently reported risk factors associated with the disease. Other potential risk fac- tors, such as environmental (17) and dietary (18) factors, have also been suggested. Conversion ofa normal cell into the malig- nant state is often linked to genetic alterations of the cell (22,23). The known tumor-suppressor genes, Retinoblastoma (Rb) and p53, were reported to play an important role in the progression of prostate cancer (24). Both genes appear to be early events in prostatic carcinogenesis (25,26). In addition, mutations or dele- tions of pTEN, a tumor suppressor gene, have been found in 30% of primary prostate cancers and 63% of malignant cases, rank- ing it as one of the most common determinants of prostate tumor progression (27,28,57). The abnormal expression of growth fac- tors and their receptors including the epidermal growth factor (EGF) (29), the transforming growth factor- (TGF-) (30), the transforming growth factor- (TGF-) (31), HER-2/neu, and c-erbB-3 oncogenes (32) may also contribute to the growth and development of both local and metastatic prostate cancer. The progression of prostate tumors to a hormone-refractory state is frequently associated with the increased expression of the antiapoptotic gene Bcl-2 (33), and the mutation of pTEN (34). Androgen Receptor in Prostate Cancer Like all steroid hormone receptors, the androgen receptor consists of 4 distinct regions in the expressed receptor that have specific functions (35), including the DNA-binding do- main, which is responsible for the interaction with hormone- responsive elements of the target gene promoters (36), and the ligand binding region, which is located at the carboxyl termi- nus of the receptor and contains the activation function 2 (AF2) domain and regulates ligand-dependent receptor function. Fur- thermore, the ligand-binding domain is thought to be important in the function of coactivator proteins (37) and in regulating transcription (38). The androgen receptor remains cytoplasmic until ligand binding occurs, and the dissociation of heat-shock proteins (HSPs) is thought to allow conformational change in the androgen receptor and mediate translocation to the nucleus. In the absence of ligand, the androgen receptor in the cytoplasm is rapidly degraded. In the presence of ligand, the androgen re- ceptor dimerizes, translocates to the nucleus, and initiates gene transcription by binding to specific androgen responsive ele- ments (AREs) found in androgen-responsive promoters (39). During androgen-independent progression, prostate cancer re- lies on various cellular pathways, some involving the androgen receptor and others bypassing it. In the former type of path- ways, a mutated androgen receptor may be activated by various ligands. In addition, deregulated growth factors and cytokines can activate the androgen receptor, usually with the help of androgen-receptor coactivators. Most of these growth factors, including EGF, insulin-like growth factor (IGF) and fibroblast growth factor (FGF), are potent mitogens and are upregulated by androgens, although the exact mechanisms are unknown. In contrast, TGF- activity is downregulated by androgens (49). In the pathways that bypass the androgen receptor, the loss of pTEN reverses the inhibition of the PI3K/Akt pathway, permit- ting activated Akt to phosphorylate Bad. This activation results in the release of Bcl-2, which eventually leads to cell survival. In addition, androgen-independent cells may overexpress Bcl- 2 (50). Recently, increasing evidence implicated the canoni- cal Wnt signaling pathway in modulating the androgen signal- ing at multiple levels (51,52). -catenin protein, a particularly À; 18 J. MAHAJNA ET AL. critical molecular component of canonical Wnt signaling, is now known to promote androgen signaling through its ability to bind to the androgen receptor protein in a ligand-dependent fash- ion and to enhance the ability of liganded androgen receptor to activate transcription of androgen-regulated genes (53). Further- more, other components of the Wnt signaling pathway also aug- ment androgen receptor function through diverse mechanisms (51?55). Historical and Contemporary Uses of Medicinal Mushrooms The medicinal use of fungi dates back thousands of years and is recorded in the histories of both traditional Western medicine (TWM) and traditional Chinese medicine (TCM) (72). Although fungi have often been marginalized in traditional Western medicine and have not been widely incorporated into Western diets, the use of fungi has been central to the cultures of most Asian countries since antiquity (73). In traditional Chi- nese medicine, fungi are used in their entirety, fresh or dried, to treat the patient (and the patient’s ailments) as a whole (72). In addition, certain fungi have been used in both traditional Western medicine and in traditional Chinese medicine to target specific conditions. Such is the case with Fomitopsis officinalis (Vill.) Bond. et Singer, called Agaricum by Dioscorides in his collection De Materia Medica ( 60 A.D.), where it is described as a powerful panacea, especially suited to treat intestinal ail- ments (73). Other medicinal fungi are described in the “Herbal Classic” of traditional Chinese medicine, a compilation of two ancient bodies of writing collected between 200?264 A.D., the Sien nung Pen ts’ao king (or Pen king) and Ming I pie lu (or Pie lu) (73). A number of ancient medicinal fungi discussed are still in use. These include Ganderma lucidum, Poria cocos (Schwein.) F.A. Wolf, Grifola umbellata (Pers.) Pil?at, Calvatia lilacina (Berk et Mont.) Lloyd, and Tremella fuciformis Berk. Special attention is given to “chi” or “Ling chi (zhi)” varieties of G. lucidum, said to promote well-being and immortality (73). In Asia, this fungus has traditionally been used to treat age-related ailments such as coronary disease, hypertension, bronchial prob- lems, and cancer (74). Fungal Secondary Metabolites Secondary metabolite production in fungi is a complex pro- cess coupled with morphological development (75). In most cases, the function of secondary metabolites for producing fun- gus is unknown but is inferred from several studies using mu- tants or enzyme inhibitors. These substances have their origins as derivatives from many intermediates in primary metabolism, but most can be classified according to 5 main metabolic sources: 1) amino acid-derived pathways, 2) the shikimic acid pathway for the biosynthesis of aromatic amino acids, 3) the acetate- malonate pathway from acetyl coenzyme A, 4) the mevalonic acid pathway from acetyl coenzyme A that functions in primary metabolism for the synthesis of sterols, and 5) polysaccharides and peptidopolysaccharides. The polyketide and mevalonic acid pathways are most often involved, and they produce a greater variety of compounds than the other pathways (11). Some of these compounds have tremendous importance to humankind in that they display a broad range of useful antibacterial, antivi- ral, and pharmaceutical activities as well as less desirable toxic activities. Two major groups of secondary metabolites are responsible for toxic activities (the division is rather arbitrary): mycotoxins and mushroom poisons (76). All mycotoxins are low-molecular- weight natural products (i.e., small molecules) produced as sec- ondary metabolites by filamentous microfungi, whereas mush- rooms and other macroscopic fungi produce mushroom poisons. Depending on the definition used, and recognizing that most fun- gal toxins occur in families of chemically related metabolites, some 300 to 400 compounds are now recognized as mycotoxins (77). The second group of toxic metabolites is mushroom poi- sons. About 300 species of mushrooms are poisonous to humans. These species produce a wide spectrum of poisons that have been divided into the following 7 main categories (in brackets, an ex- ample of producing fungi): amanitin (Amanita phalloides [Vaill. : Fr.] Link, Galerina autumnalis [Peck] A.H. Sm. et Singer), orellanine (Cortinarius orellanus Fr.), gyromitrin (Gyromitra esculenta [Pers.] Fr.), muscarine (Clitocybe dealbata [Sowerby] Gillet), ibotenic acid (Amanita cothurnata G.F. Atk.), psilo- cybin (Psilocybe baeocystis Singer et A.H. Sm., Panaeolus castaneifolius [Murrill] A.H. Sm., Conocybe cyanopus [G.F. Atk.] K?uhner) and coprine (Coprinus atramentarius [Bull.] Fr.) (78). Among the fungal secondary metabolites are lectins, lac- tones, terpenoids, alkaloids, antibiotics, and metal chelating agents (6). Fungi also contain a number of enzymes such as lac- case, superoxide dismutase, glucose oxidase, and peroxidases. It has been shown that such an enzyme therapy can also play an important role in cancer treatment preventing oxidative stress and inhibiting cell growth (79). Anticancer Activity of Fungal Substances It has been demonstrated that fungal metabolites can be used as inhibitors of molecular targets in malignant cells in order to combat certain cancers. Fungal anticancer substances can be roughly divided into two groups of high- and low- molecular-weight molecules. The major difference between these two groups is their mechanism of action. Most of the high- molecular-weight compounds are polysaccharides or protein- bound polysaccharides (80). It appears that these compounds are capable of interacting nonspecifically with the immune sys- tem to upregulate or downregulate many aspects of the host re- sponse (81). The second group comprises low-molecular-weight secondary compounds that can penetrate the cell membrane and act on specific signal-transduction pathways (11). These include mainly sesquiterpenes (which are the predominant secondary metabolites of Basidiomycetes), triterpenes, steroids and sterols, À; MEDICINAL MUSHROOMS FOR PROSTATE CANCER THERAPY 19 and a few polyketides (abundantly produced by Actinomycetes). Anticancer Activity of Ganoderma Lucidum Ganoderma is the most popular and intensely investigated genus among the medically active mushrooms. Plenty of its species are famous for their antiviral, antibacterial, antifun- gal, anticancer, and immunostimulating activities and have been used traditionally in the folk medicine of Eastern countries for centuries. These activities were due to the production of various metabolites such as proteins, terpenes, sterols, and so forth. The genus Ganoderma belongs to the class of Hymeno- mycetes. Within the genus Ganoderma, over 250 taxonomic names have been reported worldwide (82) including G. adsper- sum, G. applanatum, G. australe, G. lucidum, and G. tsugae, to name a few. However, the majority of reports in the literature appear to refer to one species, G. lucidum (83). Ganoderma lucidum (W. Curti: Fr.) P. Karst. (Ling Zhi or Reishi), an oriental medical mushroom, has been used widely in Asian countries for centuries to prevent or treat different diseases including cancer (Fig. 1). Dried powder of G. lucidum, which was recommended as a cancer chemotherapy agent, is currently used popularly worldwide in the form of dietary supplements. G. lucidum extracts were reported to possess cytotoxic ac- tivity against various cancer cell lines including leukemia, lymphoma, multiple myeloma (84,112), human hepatoma PLC/PRF/5 and KB, human breast cancer MDA-MB-231 (85), human prostate cancer PC-3 (86), human breast cancer MCF- 7 (87), human cervix uteri tumor HeLa (88), and low-grade bladder cancer MTC-11 (89) cell lines. The cytotoxic effects of G. lucidum as demonstrated by the studies of Jiang et al. (85,86) and Zhu et al. (88) were concentration dependent. This activity of G. lucidum can be attributed directly to specific compounds from experiments employing isolated and purified molecules. However, the molecular mechanism(s) responsible for the in- hibitory effects have not been fully elucidated. Ganoderma Lucidum Inhibits Proliferation of Prostate Cancer Cells Proliferation is the multiplication or reproduction of cells resulting in the rapid expansion of a cell population. Cell pro- liferation is controlled by cell cycle regulatory elements. Hsieh and Wu (92) tested the ability of extracts from indi- vidual herbs containing the herbal mixture PC-SPES, of which Ganoderma lucidum is one of its components, using amounts estimated to be equivalent to that present in the herbal mixture to suppress LNCaP, an androgen-dependent prostate cancer cell line, growth and/or lower prostate-specific androgen (PSA) ex- pression, compared to cells treated with PC-SPES. Treatment of LNCaP cells with 5 microl/ml ethanol extracts of Ganoderma lucidum showed a 63.5% reduction in cell growth and exhibited a similar decrease in cell viability. Additional studies demon- strated the ability of Ganoderma lucidum extracts to also inhibit cell proliferation of AR-independent cancer cell lines such as PC-3 in a dose- and time-dependent manner (86). Growth in- hibition of PC-3 cells by Ganoderma lucidum was mediated by the downregulation of expression of cyclin B and Cdc2 and by the upregulation of p21 expression. The inhibition of cell growth was also demonstrated by cell cycle arrest at the G2/M FIG. 1. Ganoderma lucidum fruit bodies. À; 20 J. MAHAJNA ET AL. phase (86). Liu et al. (62) reported that the LNCaP growth in- hibitory activity of G. lucidum extract is mediated by Ganoderol B isolated from G. lucidum fruiting body extract. Liu et al. (62) reported that Ganoderol B inhibits 5- reductase activity and thereby causes inhibition of the proliferation of the androgen- dependent LNCaP cell line. Ganoderma Lucidum Induces Apoptosis in Prostate Cancer Cells Apoptosis is the predominant mechanism by which can- cer cells die when subjected to chemotherapy or irradiation. However, cancer cells develop resistance to these therapies that may be due, at least in part, to the development of effective antiapoptotic mechanisms (103). Another mechanism allow- ing escape from apoptosis is the activation of survival signal transduction pathways, including Akt-dependent (104) and Akt- independent mechanisms (105). An Akt-independent example includes EGF-induced survival mechanisms (31). When LNCaP cells are treated with PI3K inhibitors and deprived of survival factors, they spontaneously undergo apoptosis. However, treat- ment with EGF or androgen can protect cells from apoptosis, although Akt activity remains inhibited. It was found that EGF can protect LNCaP cells from apoptosis induced via the mi- tochondrial pathway but not from apoptosis induced via the death-receptor pathway (106,107). A large portion of prostate cancer cells contain deregulated Akt. For example, in LNCaP prostate cancer cell line, Akt is constitutively active as a result of a frame-shift mutation in the pTEN tumor suppressor gene, which encodes a phosphatase that inactivates the lipid products of PI3K. As a result, the lack of the pTEN protein in these cells resulted in a constitutively activated antiapoptotic NF-B pathway (108). Thus, these cells are less sensitive to anticancer drugs whose mechanism of action is based on the induction of apoptosis (109). Ganoderma lucidum induced apoptosis of PC-3 cells with a slight decrease in the expression of NF-B-regulated Bcl-2 and Bcl-xl and the upregulation of the proapoptotic Bax pro- tein, resulting in the enhancement of the ratios of Bax/Bcl-2 and Bax/Bcl-xl (86). Bemis et al. (110) studied the bioactivity of a unique preparation of concentrated soybean isoflavones fermented with G. lucidum mycelia named genistein com- bined polysaccharide (GCP). During fermentation, a concen- trated mixture of aglycone isoflavones was produced due to the hydrolytic cleavage of the sugar moiety from the isoflavone via G. lucidum-derived -glycosidase. The potential utility of GCP as a prostate cancer chemopreventative agent was ana- lyzed in vitro and in vivo…