Clin Exp Metastasis (2010) 27:361369 DOI 10.

1007/s10585-010-9334-z

REVIEW

The in vitro and in vivo experimental evidences disclose the chemopreventive effects of Ganoderma lucidum on cancer invasion and metastasis
Chia-Jui Weng Gow-Chin Yen

Received: 16 March 2010 / Accepted: 29 April 2010 / Published online: 12 May 2010 Springer Science+Business Media B.V. 2010

Abstract The Ganoderma lucidum (Leyss. ex Fr.) Karst, an edible mushroom, has been utilized for centuries in East Asia to prevent or treat various diseases and to reduce the likelihood of cancer invasion and metastasis. The primary bioactive compounds are commonly considered to be polysaccharides and triterpenoids. Evidence that G. lucidum extract and its bioactive compounds may have a potential inhibitory effect on cancer invasion and metastasis is increasingly being reported in the scientic literature. This review assembles and summarizes past publications on the in vitro and in vivo effects of G. lucidum on cancer invasion and metastasis, and concludes that these effects occur through modulation of the phosphorylation of extracellular signal-regulated kinase (ERK1/2), phosphatidylinositol 3-kinase (PI 3-kinase) or Akt kinase (protein kinase B). Activation of these kinases subsequently inhibits the activity or expression of activator protein-1(AP-1) and nuclear factor-kappa B (NF-jB), resulting in the downregulation of urokinase plaminogen activator (uPA), uPA receptor (uPAR), matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-b1, interleukin (IL)-8, inducible nitric oxide (NO) and b1-integrin as shown in various cell lines or animal models. G. lucidum may be an effective nutraceutical used in the prevention of cancer metastasis. To further elucidate the bioactive components present in G. lucidum and the anti-

metastatic mechanisms underlying these compounds, more in vitro and in vivo tests as well as clinical trials are necessary. Keywords Anti-invasion Anti-metastasis Ganoderma lucidum Polysaccharides Triterpenoids Abbreviations Akt kinase Protein kinase B AP-1 Activator protein-1 CAM Chick chorioallantoic membrane assay CDK Cyclin-dependent kinase ECM Extracellular matrix ERK1/2 Extracellular signal regulated kinase GLE G. lucidum extract GLPP G. lucidum polysaccharides peptide GTE Green tea extract IL Interleukin LLC Lewis lung carcinoma MMP Matrix metalloproteinase MT-1 MMP Membrane type 1-MMP NF-jB Nuclear factor-kappa B NO Nitric oxide PI 3-kinase Phosphatidylinositol 3-kinase PMA Phorbol-12-myristate-13-acetate TGF-b1 Transforming growth factor-b1 Th1 T-helper type 1 uPA Urokinase plaminogen activator uPAR uPA receptor VEGF Vascular endothelial growth factor Introduction Cancer metastasis refers to the spread of cancer cells from the primary neoplasm to distant sites where secondary

C.-J. Weng Graduate Institute of Applied Science of Living, Tainan University of Technology, 529 Jhongjheng Road, Yongkang, Tainan 71002, Taiwan G.-C. Yen (&) Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan e-mail: gcyen@nchu.edu.tw

123

362

Clin Exp Metastasis (2010) 27:361369

tumors are formed. Metastasis occurs through a complex multistep process consisting of entrance into the circulation from the primary tumor, migration to distant organs, adhesion to endothelial cells lining the blood vessel, and inltration into the underlying tissue. Metastasis is responsible for the majority of failures in cancer treatment, and is the major cause of death from cancer [1]. Upon cancer diagnosis, additional chemotherapy may be recommended to prevent local recurrence of the primary tumor and the spread of tumor cells. However, severe side effects may be induced at the effective dose of the chemotherapeutic. Therefore, in addition to minimizing the growth of the existing tumor, treatments that limit its spread to new sites and block invasion have been shown to enhance the survival of cancer patients [2, 3]. Experimental studies have demonstrated that phytochemicals derived from some types of plants may prevent cancer metastasis [4]. Ganoderma lucidum (Leyss. ex Fr.) Karst, also called Lingzhi, has been used in traditional Chinese medicine to improve health and longevity. Lingzhi has been used for thousands of years in the treatment of various diseases, including neurasthenia, hypertension, hepatopathy and carcinoma [513]. Currently, the dried powder and aqueous/ethanol extracts of G. lucidum are used worldwide as dietary supplements [14]. The mechanisms of action of G. lucidum include the induction of cell apoptosis, inhibition of cell proliferation, and suppression of the motility of highly invasive breast and prostate cancer cells [15]. Additionally, the dried powder and aqueous/ ethanol extracts of G. lucidum have been shown to reduce the likelihood of invasion and metastasis, and prevent occurrence or reoccurrence of various types of cancer [16, 17]. Although there are different compounds with various pharmacological activities extracted from mycelia, the fruiting bodies or spores of G. lucidum, the anti-cancer and anti-metastatic activities of this edible mushroom are almost completely due to its polysaccharide and triterpenoid components [17, 18]. Increasing evidence suggests that the anti-cancer or anti-metastatic effect of the polysaccharides and polysaccharopeptides in these mushrooms may be associated with their immunostimulating activities [1922]. Many triterpenoid compounds such as ganoderic acids, ganolucidic acids, ganolactone, lucidenic acids, methyl lucidenate and hydroxylucidenic acid also found in Ganoderma species have shown some therapeutic effects [17, 18, 2326]. One of these effects is likely to be the observed anti-invasive activity characteristic of G. lucidum. However, efforts to identify the bioactive components of triterpenoids responsible for the observed anti-invasive activities characterized by G. lucidum are incomplete. Further research is needed to uncover the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on cancer cells.

Reviews on the anti-cancer potential of G. lucidum are available [10, 11, 2730], the majority of which are focused on the investigations of the anti-cancer effect [10, 11, 27, 28], the regulation of the cell cycle and cell signaling [11, 28] and the pharmacological effects of bioactive components [10, 27, 28, 30]. The current review presents evidence for anti-invasive and anti-metastatic effects of G. lucidum and the underlying molecular signaling mechanisms. The anti-invasive and anti-metastatic effects of G. lucidum The anti-invasive and anti-metastatic effects of G. lucidum in vitro and in vivo have been well documented. The observation that treatment with G. lucidum inhibited metastasis of tumors to the lung and resulted in a prolonged lifespan of tumor-transplanted mice was observed for the rst time more than 15 years ago [31]. In recent years, it has also been shown that pre-feeding with the G. lucidum antlered form (AF)-containing diet could suppress lung metastasis in Cyclophosphamide (CY)-induced C57BL/6 mice injected with Lewis lung carcinoma (LLC). In particular, the number of nodules in the G. lucidum AF-fed group injected with LLC 7 days after CY administration was signicantly lower compared to the control group receiving no G. lucidum [32]. Although CY is one of the most commonly-used chemotherapeutic agents [33], it exhibits adverse effects such as increasing the risk of cancer metastasis. Another report also demonstrated that the adhesion and migration of the highly invasive MDAMB-231 breast cancer cell line and PC-3 prostate cancer cell line were both inhibited upon exposure to G. lucidum extract (GLE) in vitro [3436]. The inhibitory effect of GLE on MDA-MB-231 cells may be due to the suppression of phosphatidylinositol 3-kinase (PI 3-kinase) signaling [15]. In addition, GLE (0500 lg/ml) and green tea extract (GTE, 060 lg/ml) both suppress the adhesion of MDAMB-231 cells to the extracellular matrix protein vitronectin in a dose-dependent manner. Furthermore, GLE (125, 250 and 500 lg/ml) and GTE (30, 60 and 125 lg/ml) inhibit the migration of MDA-MB-231 cells by 48, 63 and 74%, and by 13, 25 and 38%, respectively. The combination of GLE and GTE synergistically inhibits the adhesion and migration of invasive breast cancer cells [37]. The extract of G. lucidum was found to inhibit oxidative stress-induced invasion of MCF-7 breast cancer cells through suppression of interleukin (IL)-8 secretion [38]. The ethanol extract of the G. lucidum fruiting body was also shown to have strong anti-angiogenic activity in a dose-dependent manner, which is supported by the inhibition of inducible NO (nitric oxide) production [39]. From the reports mentioned above, it is clear that G. lucidum possesses anti-metastatic and

123

Clin Exp Metastasis (2010) 27:361369

363

anti-invasive activities. The inhibition of invasion and/or metastasis in vitro or in vivo following exposure to G. lucidum extract is summarized in Table 1. The bioactive components in G. lucidum with anti-invasive and/or antimetastatic activities are further discussed in the following section. The bioactive components on anti-invasion and anti-metastasis in G. lucidum G. lucidum is a species of Aphyllophorales (Polypore) fungi. There have been numerous publications describing various compounds from these fungi that have shown antimicrobial, antiviral, cytotoxic, antineoplastic, and/or anti-invasive activities [40]. Over the last few decades, investigations into the composition of G. lucidum have revealed that it contains a variety of bioactive components

such as triterpenes, polysaccharides, nucleosides, steroids, fatty acids, alkaloids, proteins, peptides, amino acids, and inorganic elements [4143]. It is believed that the anticancer and anti-metastatic properties of these mushrooms are derived from its diversied chemical constituents, although it is comprised primarily of two types of compounds: polysaccharides and triterpenes [6, 8, 28, 29, 4450]. Polysaccharides are the main components in the water-soluble extract of G. lucidum. For the past 30 years, G. lucidum polysaccharides have been clearly demonstrated to have hypoglycemic, immuno-modulating, antitumor and anti-metastatic activities [8, 12, 20, 51]. However, fractionates of polysaccharides were not as effective as their equivalent dose of the crude extract of the whole mushroom. Hence, the bioactivity of G. lucidum was presumed to be a synergetic effect of multiple compounds in the mushroom [52]. Analysis of constituents from

Table 1 Inhibition of invasion and/or metastasis in vitro or in vivo following treatment with G. lucidum extract Cell or animal model Tumor-transplanted mice MDA-MB-231 breast cancer cells 0.52.5 mg/ml (4 h) Dose (Duration) Effects Inhibition of lung metastasis Inhibition of 1. uPA and uPAR 2. adhesion and migration PC-3 prostate cancer cells 0.52.5 mg/ml (24 h) Inhibition of 1. uPA and uPAR 2. adhesion and migration PC-3 prostate cancer cells 0.1250.5 mg/ml (24 h) Inhibition of 1. secretion of VEGF TGF-b1 2. migration CAM assay 1.2510 mg/ml (2 days) 0.250.5 mg/ml (24 h) 3. angiogenesis Inhibition of 1. inducible NO 2. angiogenesis MCF-7 breast cancer cells Inhibition of 1. secretion of IL-8 2. oxidative stress-induced migration CY-induced C57BL/6 mice injected with 2.5% (7 days) LLC Enhancement of 1. NK activity 2. T cell function Inhibition of lung metastasis HepG2 human hepatoma cells 0.51 mg/ml (24 h) Inhibition of 1. invasion 2. MMP-9 expression HepG2 implanted ICR-nu/nu nude mice 200800 mg/kg (68 days) Inhibition of 1. lung metastasis 2. MMP-2/9 activities ND Not determined ERK1/2 Akt kinase N.D. AP-1 NF-jB N.D. [79] N.D. N.D. [32] ERK1/2 AP-1 NF-jB [38] ERK1/2 Akt kinase N.D. AP-1 NF-jB [14, 77] N.D. AP-1 NF-jB [34, 35] Signalings Transcription factors N.D. PI 3kinase N.D. AP-1 NF-jB References [31] [15, 34 36]

N.D.

[39]

[79]

123

364

Clin Exp Metastasis (2010) 27:361369

G. lucidum species revealed more than 130 different types of triterpenoids [5355]. The triterpenoid fraction of G. lucidum has also been shown to have anti-cancer, antitumor and anti-metastatic effects in various in vivo and in vitro models [10, 56]. Metastasis occurs through a complex, multistep process consisting of the invasion of cells from a primary tumor into the circulation, migration of these cells to distant organs, adhesion to endothelial cells that line the blood vessel, inltration into the underlying tissue and subsequent recruitment of a blood supply through the formation of new capillaries (angiogenesis). However, the antimetastatic activities of G. lucidum are not the result of a single mechanism of action. Invasion and metastasis of cancer cells involves the degradation of environmental barriers such as the extracellular matrix (ECM) and the basement membrane by various proteolytic enzymes, including serine proteinase, matrix metalloproteinases (MMPs), membrane type-1 MMP (MT-1 MMP), cathepsins, and plasminogen activator. Degradation of the basement membrane results in promoted mobility of cancer cells [5759]. Among these proteases, MMP-2 and MMP-9 expression is high in various malignant tumors, and is closely related to the ability of these cells to invade and metastasize [6062]. Activation of MMPs is initiated by the urokinase plasminogen activator (uPA) receptor (uPAR) that binds to and activates uPA. uPA is a strong plasminogen activator that specically cleaves the proenzyme/zymogen plasminogen to form the active enzyme plasmin, which then activates pro-MMP enzymes. Both uPA and uPAR are overexpressed in various tumors. Thus, the uPARuPA interaction at the surface of cancer cells is highly involved in the invasion and metastasis of cancer cells [34, 63]. The mechanisms of anti-invasive and anti-metastatic effects vary between the polysaccharide and triterpenoid fractions. G. lucidum polysaccharides prevent tumor metastasis indirectly via the activation of an immune response from the host organism [20], whereas triterpenes in G. lucidum suppress invasive behavior of cancer cells directly [45, 51]. Next, we will present scientic evidence of anti-invasive and anti-metastatic effects or mechanisms of polysaccharides and triterpenes in G. lucidum. The polysaccharides fraction Ikekawa et al. [64] initially reported striking host-mediated antitumor activity of hot water extracts of various polypores on Swiss albino mice transplanted with Sarcoma 180. Since that time, numerous reports concerning the isolation and characterization of anticancer polysaccharides have been published. Based on these reports, b-D-glucans are considered to be the major component of the

polysaccharides in polypores, and b-(1 ? 3)-D-glucopyrans having a mean molecular weight of 500,000 2,000,000 Dalton and characteristic b-(1 ? 6)-D-glucosyl branches (Fig. 1) could be responsible for the antitumor bD-glucans isolated from polypores [24, 40]. b-D-glucans with the greatest antitumor activity were observed to have higher molecular weights, lower levels of branching, and greater water solubility compared to other polysaccharides [24]. Tumor growth and metastasis is angiogenesis dependent. Several lines of direct evidence have shown that angiogenesis is essential for tumor growth and metastasis [6567]. The chick chorioallantoic membrane (CAM) assay is commonly employed to examine the anti-angiogenic activity of sample. A G. lucidum polysaccharides peptide (GLPP) was extracted and tested for its antiangiogenic properties by CAM assay. The microvessels around the disc were obviously reduced after treatment with 80 lg GLPP per disc for 48 h. These results indicate that GLPP might be a potent inhibitor of angiogenesis and subsequent metastasis [47]. A polysaccharide mixture containing isoavone aglycons produced from cultured G. lucidum mycelia inhibited angiogenesis in the CAM assay as well as in the mouse dorsal air-sac model following implantation of colon-26 tumor cells [68]. Wu et al. [69] have demonstrated that polysaccharides prepared from different sources of G. lucidum, including the fruiting body grown in wood logs, mycelia, as well as the wild-type fruiting body, are the major contributing fraction in reducing adhesion of human breast carcinoma MT-1 cells. This anti-adhesive effect of polysaccharides in G. lucidum on MT-1 cells may be mediated via the integrin pathway. Cao et al. [70] employed human lung carcinoma PG cells as an invasive model, and found that the adhesion, motility and MMP-9 activities were all inhibited following treatment with 100 lg/ml polysaccharide peptide from G. lucidum. According to the reports, polysaccharides from G. lucidum are suggested to be a potent candidate for antiinvasion or anti-metastasis of cancer cells. However, the observed negative effects on invasion and metastasis are almost completely based on angiogenesis in vitro and in vivo, and adhesion or migration in vitro. No in vivo evidence validating metastasis inhibition in an animal model has been published. The inhibition of invasion and/or metastasis in vivo or in vitro following treatment with the polysaccharide components of G. lucidum are summarized in Table 2. The triterpenoids fraction Numerous triterpenoids have been isolated from various species of the Polyporaceae. After over 130 pharmacologically-active triterpenoids were initially discovered, additional triterpenes have been isolated from this polypore

123

Clin Exp Metastasis (2010) 27:361369 Fig. 1 Structure of b-D-glucans [(1-6)-D-glucosyl branched b(1-3)-D-glucopyran]

365

Table 2 Inhibition of invasion and/or metastasis in vitro or in vivo following treatment with the polysaccharide components of G. lucidum Cell or animal model Genistein combined polysaccharide CAM assay Colon-26-implanted BALB/c mice Polysaccharides peptide CAM assay Polysaccharides MT-1 human breast carcinoma cells 0.5 mg/ml (12 h) Inhibition of 1. b1-integrin 2. adhesion Polysaccharides peptide PG human lung carcinoma cells 100 lg/ml Inhibition of 1. adhesion 2. migration 3. MMP-9 activity ND Not determined N.D. N.D. [70] N.D. N.D. [69] 80 lg/disc (48 h) Inhibition of angiogenesis N.D. N.D. [47] 0.1% (2 days) 0.3 g/day (5 days) Inhibition of angiogenesis N.D. N.D. [68] Dose (Duration) Effects Signalings Transcription factors References

(A)

(B)

(C)

Type A

Name Ganoderic acid AM1 Ganoderic acid B Ganoderic acid C Ganoderic acid C2 Ganoderic acid D Ganoderic acid E Ganoderic acid F Ganoderic acid H Ganoderenic acid D Ganoderenic acid K Lucidenic acid A Lucidenic acid B Lucidenic acid C Lucidenic acid D2 Lucidenic acid E2 Lucidenic acid F Lucidenic acid N Lucidenic acid P Ganolucidic acid C

R1 -OH -OH =O -OH =O =O =O -OH =O -OH =O =O -OH =O -OH =O -OH -OH

R2 =O -OH -OH -OH -OH =O =O =O -OH -OH -OH -OH -OH =O =O =O -OH -OH

R3 =O =O =O -OH =O =O =O =O =O =O =O =O =O =O =O =O =O =O

R4 H H H H H H -OAc -OAc H -OAc H -OH -OH -OAc -OAc H H -OAc

Double bond 20,22 20,22

Fig. 2 Structures of the triterpenoids isolated from Ganoderma lucidum

such as lucidenic acid N and methyl lucidenate F, which were isolated from the dried fruiting bodies of G. lucidum [24, 50]. The common structures of triterpenoids isolated from G. lucidum are shown in Fig. 2. The triterpenoid fraction of the fruiting body of G. lucidum was shown to inhibit liver metastasis and secondary metastatic tumor growth in livers from mice that were intrasplenically injected with LLC. Ganoderic acid F was further identied as the active component of the triterpenoid fraction, as it was responsible for the anti-metastasis activity via inhibition of tumor-induced angiogenesis [56]. Triterpenoid components including ganoderic acids-R, -T, -U, -V, -W, -X, -Y, -Z, lucidimol-A, -B, ganodermanondiol, ganoderiol F and ganodermanontriol have been demonstrated to exert cytotoxic effects on cancer cells. Some of these compounds also possess anti-angiogenic activity [28, 45]. A pure lanostane triterpene, ganoderic acid Me (GA-Me), was puried from G. lucidum in the recent year [71], and was shown to increase immune function by enhancing the expression of T-helper type 1 (Th1) cytokines (IL-2 and IFN-c), leading to the inhibition of tumor growth and lung metastasis [72]. As the highly metastatic human lung tumor

123

366

Clin Exp Metastasis (2010) 27:361369

95-D cells were treated with 10 and 20 lg/ml GA-Me for 18 h, the adhesion ratio decreased 56.8 3.2% and 20.5 6.9%, respectively, with respect to the control. GAMe also effectively inhibited migration of 95-D cells in a dose- and time-dependent manner via induction of actin polymerization. In addition, GA-Me could suppress invasion of 95-D cells by inhibiting MMP2 and MMP9 gene expression [73]. According to the data mentioned above, ganoderic acid was thought to be a potent bioactive compound in G. lucidum. Thyagarajan et al. [38] suggested that ganoderic acids A, F and H in G. lucidum might contribute to the inhibition of oxidative stress-induced invasion of MCF-7 breast cancer cells via the suppression of IL-8 secretion. In addition, invasive behavior such as adhesion, migration and invasion of MDA-MB-231 breast cells were also inhibited through down-regulation of cyclin-dependent kinase (CDK) 4 and suppression of uPA secretion following treatment with ganoderic acids A and H [74]. Furthermore, ganoderic acid T (GA-T) possesses an anti-invasive effect in vitro and in vivo was also veried recently [75]. GA-T can promote homotypic aggregation, inhibit the adhesion and migration, and down-regulated expression of MMP-9, inducible nitric oxide synthase (iNOS) and uPA of HCT-116 cells (a human colon carcinoma cell line). It also suppressed the migration of 95-D cells in a dose- and time-dependent manner. In LLC bearing C57BL/6 mice model experiments demonstrated that GA-T suppresses LLC metastasis and down-regulates MMP-2 and -9 mRNA expressions in vivo. A recent study by our group has described the isolation of four triterpenoid components from the ethanol extract of G. lucidum, and identied them as lucidenic acids A, B, C, and N [76]. Our results demonstrate that these four lucidenic acids are the bioactive components in G. lucidum possessing anti-invasive properties on hepatoma (HepG2) cells through the reduction of phorbol-12-myristate-13-acetate (PMA)induced MMP-9 activity [76]. The inhibition of invasion and/or metastasis in vivo or in vitro following treatment with triterpenoid components in G. lucidum is summarized in Table 3.

concerning molecular mechanisms responsible for the antiinvasive or anti-metastatic effects of the mushroom were, therefore, focused on the G. lucidum extract and its triterpenoids component. The spores and fruiting body of G. lucidum inhibit invasive behavior of breast cancer MDA-MB-231 cells through suppressing cell adhesion to bronectin, down-regulating the expression of uPA and uPAR, as well as secretion of uPA to inhibit cell motility. The suppressions of uPA and uPAR in MDA-MB-231 cells by G. lucidum may be due to the inhibition of PI 3-kinase to reduce the levels of nuclear factor-kappa B (NF-jB) and activator protein-1 (AP-1) [15, 28, 34, 36]. Similar antiinvasive mechanisms were also found in PC-3 cells by treatment with G. lucidum extract [28, 34]. Jiang et al. [77] also demonstrated that G. lucidum could induce apoptosis, inhibit cell proliferation, and suppress cell migration of PC3 cells. The inhibitory activities of G. lucidum on PC-3 cells were further suggested to result from modulation of the phosphorylation of extracellular signal regulated kinase (ERK1/2) and protein kinase B (Akt kinase), subsequently inhibiting the activity of AP-1 and NF-jB, and downregulating the secretion of vascular endothelial growth factor (VEGF) and transforming growth factor-b1 (TGFb1) [14]. G. lucidum inhibits oxidative stress-induced phosphorylation of ERK1/2, leading to the inhibition of the transcriptional activity of AP-1 and NF-jB, suppressed of secretion of IL-8, and inhibition of migration of breast cancer MCF-7 cells [38]. Ganoderic acids A and H inhibited the invasive behavior of MDA-MB-231 breast cancer cells through the suppression of AP-1 and NF-jB, resulting in the down-regulation of CDK 4 and uPA secretion [74]. GA-T can inhibit NF-jB and the degradation of inhibitor of jB-a (IjBa) to reduce the expressions of MMP-9, iNOS and uPA, which leads to inhibit the invasion of HCT-116 cells [75]. Weng et al. [78] also demonstrated that lucidenic acid B could effectively inhibit PMA-induced invasion of hepatoma HepG2 cells by inhibiting the phosphorylation of ERK1/2 to reduce AP-1 and NF-jB DNA binding activities, leading to the down-regulation of MMP-9 expression. The mechanisms by which G. lucidum and its triterpenoids component inhibit invasion and metastasis are summarized in Tables 1 and 3.

Mechanisms of anti-invasion and anti-metastasis by G. lucidum and its bioactive compounds Conclusion and future perspectives G. lucidum extract and its bioactive compounds, polysaccharides and triterpenoids, have been shown to have antiinvasive or anti-metastatic activities by affecting various invasive behaviors in various cell lines or animal models. However, the signaling pathway or transcription factor involved in the effects of anti-angiogenesis, anti-adhesion and anti-migration of G. lucidum polysaccharides have not been identied (Table 2). The following discussion It is obvious that G. lucidum exhibits inhibitory activity against invasive and metastatic behaviors (e.g., adhesion, migration, and angiogenesis) in various cancer cells in vitro or implanted in BALB/c and C57BL/6 mice. Polysaccharides and triterpenoids may be the bioactive compounds in G. lucidum that are responsible for the observed antiinvasion and anti-metastasis action of GLE on tumors.

123

Clin Exp Metastasis (2010) 27:361369 Table 3 Inhibition of invasion and/or metastasis in vitro or in vivo following treatment with the triterpenoid components of G. lucidum Cell or animal model Dose (Duration) Effects Signalings Transcription factors

367

References

Triterpenoid fraction (including Ganoderic acid F) LLC implanted C57BL/6 mice Ganoderic acid Me LLC implanted C57BL/6 mice 28 mg/kg (10 days) Enhancement of 1. IL-2 and IFN-c 2. NK cells Inhibition of lung metastasis 95-D lung tumor cells 1020 lg/ml (1824 h) Inhibition of 1. adhesion 2. migration 3. MMP2/9 genes expressions Ganoderic acids A and H MDA-MB-231 breast cancer cells Inhibition of 1. expression of CDK4 2. secretion of uPA 3. adhesion, migration and invasion Ganoderic acid T HCT-116 human colon carcinoma cells 8.216.3 lM (124 h) Inhibition of 1. cell aggregation 2. adhesion 3. migration 4. MMP-9, iNOS and uPA 95-D lung tumor cells LLC implanted C57BL/6 mice 16.332.6 lM (24 h) 28 mg/kg (10 days) Inhibition of migration Inhibition of 1. lung metastasis 2. MMP-2/9 mRNA expressions Lucidenic acids A, B, C, and N HepG2 human hepatoma cells 50100 lM (24 h) Inhibition of 1. PMA-induced MMP-9 activity 2. invasion ND Not determined ERK1/2 AP-1 NF-jB [76, 78] N.D. N.D. N.D. NF-jB [75] N.D. AP-1 NF-jB [74] N.D. N.D. [26] N.D. N.D. [72] 100 and 200 mg/kg Inhibition of 1. angiogenesis 2. liver metastasis N.D. N.D. [56]

However, the mechanisms are not completely understood. G. lucidum and its bioactive compounds modulate the phosphorylation of ERK1/2, PI 3-kinase or Akt kinase, which in turn inhibit the activity or expression of AP-1 and NF-jB, resulting in the down-regulation of uPA, uPAR, MMP-9, VEGF, TGF-b1, IL-8, inducible NO and b1integrin in different cells or animal models. Data from a recent study in our laboratory suggested a possible mechanism by which G. lucidum inhibited the invasion of human hepatoma cells, as well as tumorigenesis and metastasis in nude mice [79]. The results revealed that the potential anti-growth and anti-invasive effects of

G. lucidum on hepatoma cells may be derived from lucidenic acid via the repression of MMP-9 transcriptional activity in addition to the down-regulation of AP-1 and NF-jB expression. Based on the scientic evidence available, G. lucidum in the form of a dietary supplement containing the major bioactive components could be benecial for the prevention of cancer metastasis. G. lucidum may be used as an effective anti-metastatic edible mushroom for the chemoprevention of cancer. In order to explore more bioactive components in it and the antimetastatic mechanisms underlying these compounds in detail, further research is needed to carry out.

123

368

Clin Exp Metastasis (2010) 27:361369 22. Zhang M, Cui SW, Cheung PCK et al (2007) Antitumor polysaccharides from mushrooms: a review of their isolation process, structural characteristics and antitumor activity. Trends Food Sci Tech 18:419 23. Hyun JW, Choi EC, Kim BK (1990) Studies on constituents of higher fungi of Korea IXII. Antitumor components of the basidiocarp of Ganoderma lucidum. Korean J Mycol 18:5869 24. Wu TS, Shi LS, Kuo SC (2001) Cytotoxicity of Ganoderma lucidum triterpenes. J Nat Prod 64:11211122 25. Akihisa T, Nakamura Y, Tagata M et al (2007) Ani-inammatory and anti-tumor-promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum. Chem Biodivers 4:224231 26. Chen Y, Yan Y, Xie MY et al (2008) Development of a chromatographic ngerprint for the chloroform extracts of Ganoderma lucidum by HPLC and LCMS. J Pharm Biomed Anal 47:469477 27. Shiao MS (2003) Natural products of the medicinal fungus Ganoderma lucidum: occurrence, biological activities, and pharmacological functions. Chem Rec 3:172180 28. Sliva D (2003) Ganoderma lucidum (Reishi) in cancer treatment. Integr Cancer Ther 2:358364 29. Sliva D (2006) Ganoderma lucidum in cancer research. Leuk Res 30:767768 30. Russell R, Paterson M (2006) GanodermaA therapeutic fungal biofactory. Phytochemistry 67:19852001 31. Lee SS, Wei YH, Chen CF et al (1995) Antitumor effects of Ganoderma lucidum. J Chin Med 6:112 32. Nonaka Y, Ishibashi H, Nakai M et al (2008) Effects of the antlered form of Ganoderma lucidum on tumor growth and metastasis in cyclophosphamide-treated mice. Biosci Biotechnol Biochem 72:13991408 33. Allison AC (2000) Immunosuppressive drugs: the rst 50 years and a glance forward. Immunopharmacology 47:6383 34. Sliva D, Labarrere C, Slivova V et al (2002) Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells. Biochem Biophys Res Commun 298:603612 35. Sliva D, Sedlak M, Slivova V et al (2003) Biological activity of spores and dried powder from Ganoderma lucidum for the inhibition of highly invasive human breast and prostate cancer cells. J Altern Complement Med 9:491497 36. Slivova V, Valachovicova T, Jiang J et al (2004) Ganoderma lucidum inhibits invasiveness of breast cancer cells. J Cancer Integr Med 2:2530 37. Thyagarajan A, Zhu J, Sliva D (2007) Combined effect of green tea and Ganoderma lucidum on invasive behavior of breast cancer cells. Int J Oncol 30:963969 38. Thyagarajan A, Jiang J, Hopf A et al (2006) Inhibition of oxidative stress-induced invasiveness of cancer cells by Ganoderma lucidum is mediated through the suppression of interleukin-8 secretion. Intl J Mol Med 18:657664 39. Song YS, Kim SH, Sa JH et al (2004) Anti-angiogenesis and inhibitory activity on inducible nitric oxide production of the mushroom Ganoderma lucidum. J Ethnopharmacol 90:1720 40. Zjawiony JK (2004) Biologically active compounds from Aphyllophorales (Polypore) fungi. J Nat Prod 67:300310 41. Jong SC, Birmingham JM (1992) Medicinal benets of the mushroom Ganoderma. Adv Appl Microbiol 37:101134 42. Shiao MS, Lee KR, Lin LJ et al. (1994) Natural products and biological activities of the chinese medicinal fungus Ganoderma lucidum. In: Ho CT, Osawa T, Huang MT, Rosen RT (eds) Food phytochemicals for cancer prevention II: teas, spices and herbs. Oxford University Press, Washington, DC 43. Chen Y, Xie MY, Gong XFJ (2007) Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum. J Food Eng 81:162170

References
1. Weiss L (1990) Metastatic inefciency. Adv Cancer Res 54: 159211 2. Liotta LA, Steeg PS, Stetter-Stevenson WG (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64:327336 3. Condeelis J, Segall JE (2003) Intravital imaging of cell movement in tumours. Nat Rev Cancer 3:921930 4. Sliva D (2008) Suppression of cancer invasiveness by dietary compounds. Mini Rev Med Chem 8:677688 5. Aarisawa M, Fujita A, Saga M et al (1986) Three new lanostanoids from Ganoderma lucidum. J Nat Prod 49:621625 6. Gao YH, Zhou SF (2004) Chemopreventive and tumoricidal properties of Ling Zhi mushroom Ganoderma lucidum (W. Curt.: Fr.) Lloyd (Aphyllophoromycetideae). II. Mechanism considerations. Int J Med Mushrooms 6:219230 7. Gao JJ, Nakamura N, Min BS et al (2004) Quantitative determination of bitter principles in specimens of Ganoderma lucidum using highperformance liquid chromatography and its application to the evaluation of Ganoderma products. Chem Pharm Bull 52:688695 8. Lin ZB, Zhang HN (2004) Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol Sin 25:13871395 9. Johnston N (2005) Medicinal mushroom cuts off prostate cancer cells blood supply. Drug Discov Today 10:2324 10. Liu GQ, Zhang KC (2005) Mechanisms of the anticancer action of Ganoderma lucidum (Leyss. Ex Fr.) Karst.: A new understanding. J Integr Plant Biol 47:129135 11. Yuen JWM, Gohel MDI (2005) Anticancer effects of Ganoderma lucidum: a review of scientic evidence. Nutr Cancer 53:1117 12. Paterson RR (2006) Ganodermaa therapeutic fungal biofactory. Phytochemistry 67:19852001 13. Xie JT, Wang CZ, Wicks S et al (2006) Ganoderma lucidum extract inhibits proliferation of SW 480 human colorectal cancer cells. Exp Oncol 28:2529 14. Stanley G, Harvey K, Slivova V et al (2005) Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-b1 from prostate cancer cells. Biochem Biophys Res Commun 330:4652 15. Sliva D, Rizzo D (2002) Phosphatidylinositol 3-kinase and NFjB regulate motility of invasive MDA-MB-231 human breast cancer cells by the secretion of urokinase-type plasminogen activator. J Biol Chem 277:31503157 16. Teow SS (1997) The effective application of Ganoderma nutriceuticals. In: Kim BK, Moon CK, Kim TS (eds) Recent progress in Ganoderma lucidum research. The Pharmaceutical Society of Korea, Seoul 17. Wang YY, Khoo KH, Chen ST et al (2002) Studies on the immuno-modulating and anti-tumor activities of Ganoderma lucidum (Reishi). Polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorgan Med Chem 10:10571062 18. Yang HL, Zhang KC (2002) The relationship between molecular structure and activity of Ganoderma acids. J Wuxi Univ Light Ind 21:249253 (in Chinese with an English abstract) 19. Ooi VE, Liu F (2000) Immunomodulation and anti-cancer activity of polysaccharideprotein complexes. Curr Med Chem 7:715729 20. Wasser SP (2002) Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol 60: 258274 21. Sullivan R, Smith JE, Rowan NJ (2006) Medicinal mushrooms and cancer therapy: translating a traditional practice into western medicine. Perspect Biol Med 49:159170

123

Clin Exp Metastasis (2010) 27:361369 44. Mizuno T, Wang G, Zhang J et al (1995) Reishi, Ganoderma lucidum and Ganoderma tsugae: bioactive substances and medicinal effects. Food Rev Int 11:151166 45. Min BS, Gao JJ, Nakamura N et al (2000) Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against meth-A and LLC tumor cells. Chem Pharm Bull 48:10261033 46. Adam JK, Odhav B, Bhoola KD (2003) Immune responses in cancer. Food Rev Int 99:113132 47. Cao QZ, Lin ZB (2004) Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide. Acta Pharmacol Sin 25:833838 48. Gao YH, Chan E, Zhou SF (2004) Immunomodulating activities of Ganoderma, a mushroom with medicinal properties. Food Rev Int 20:123161 49. Gao YH, Tang WB, Gao H et al (2004) Chemopreventive and tumoricidal properties of Lingzhi mushroom Ganoderma lucidum (W. Curt.:Fr.) Lloyd (Aphyllophorophoromycetideae). I. Preclinical and clinical studies (review). Int J Med Mushroom 6: 95106 50. Gao JJ, Min BS, Ahn E et al (2002) New triterpene aldehydes, lucialdehydes AC, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells. Chem Pharm Bull 50:837840 51. Lin ZB (2005) Cellular and molecular mechanisms of immunomodulation by Ganoderma lucidum. J Pharmacol Sci 99:144153 52. Liu X, Yuan JP, Chung CK et al (2002) Antitumor activity of the sporoderm-broken germinating spores of Ganoderma lucidum. Cancer Lett 182:155161 53. Lin LJ, Shiao MS (1989) 13C-NMR correlation of stereochemistry in lanostanoids triterpenes. J Nat Prod 52:595605 54. Lin CN, Tome WP, Won SJ (1990) A lanostanoid of formosan Ganoderma lucidum. Phytochemistry 29:673675 55. Lin CN, Tome WP (1991) Novel cytotoxic principles of formosan Ganoderma lucidum. J Nat Prod 54:9981002 56. Kimura Y, Taniguchi M, Baba K (2002) Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum: mechanism of action and isolation of an active substance. Anticancer Res 22:33093318 57. Kleiner DE, Stetler-Stevenson WG (1999) Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 43: S41S51 58. Westermarck J, Kahari VM (1999) Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13:781792 59. Rao JS (2003) Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 3:489501 60. Basset P, Okada A, Chenard MP et al (1997) Matrix metalloproteinases as stromal effectors of human carcinoma progression: therapeutic implications. Matrix Biol 15:535541 61. Nelson AR, Fingleton B, Rothenberg ML et al (2000) Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 18:11351149 62. Chung TW, Moon SK, Lee YC et al (2002) Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells. Arch Biochem Biophys 408:147154

369 63. Mazar AP (2001) The urokinase plasminogen activator receptor (uPAR) as a target for the diagnosis and therapy of cancer. Anticancer Drugs 12:387400 64. Ikekawa T, Nakanishi M, Uehara N et al (1969) Antitumor activity of aqueous extracts of edible mushrooms. Cancer Res 29:734735 65. Folkman J (1989) What is the evidence that tumor are angiogenesis dependent? J Natl Cancer Inst 82:46 66. Kim KJ, Li B, Winer J et al (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362:841844 67. Millauer B, Shawver LK, Plate KH et al (1994) Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 367:576579 68. Miura T, Yuan L, Sun B et al (2002) Isoavone aglycon produced by culture of soybean extracts with basidiomycetes and its antiangiogenic activity. Biosci Biotechnol Biochem 66:26262631 69. Wu QP, Xie YZ, Li SZ et al (2006) Tumor cell adhesion and integrin expression affected by Ganoderma lucidum. Enzyme Microb Tech 40:3241 70. Cao QZ, Lin SQ, Wang SZ (2007) Effect of Ganoderma lucidum polysaccharides peptide on invasion of human lung carcinoma cells in vitro. Beijing Da Xue Xue Bao 39:653656 71. Tang W, Gu TY, Zhong JJ (2006) Separation of targeted ganoderic acids from Ganoderma lucidum by reversed phase liquid chromatography with ultraviolet and mass spectrometry detections. Biochem Eng J 32:205210 72. Wang G, Zhao J, Liu JW et al (2007) Enhancement of IL-2 and IFN-gamma expression and NK cells activity involved in the antitumor effect of ganoderic acid Me in vivo. Int Immunopharmacol 7:864870 73. Chen NH, Liu JW, Zhong JJ (2008) Ganoderic acid Me inhibits tumor invasion through down-regulating matrix metalloproteinases 2/9 gene expression. J Pharmacol Sci 108:212216 74. Jiang J, Grieb B, Thyagarajan A et al (2008) Ganoderic acid suppress growth and invasive behavior of breast cancer cells by modulating AP-1 and NF-kappaB signaling. Int J Mol Med 21:577584 75. Chen NH, Liu JW, Zhong JJ (2010) Ganoderic acid T inhibits tumor invasion in vitro and in vivo through inhibition of MMP expression. Pharmacol Rep 62:150163 76. Weng CJ, Chau CF, Chen KD et al (2007) The anti-invasive effects of lucidenic acids isolated from a new Ganoderma lucidum strain. Mol Nutr Food Res 51:14721477 77. Jiang J, Slivova V, Valachovicova T et al (2004) Ganoderma lucidum inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3. Int J Oncol 24:10931100 78. Weng CJ, Chau CF, Hsieh YS et al (2008) Lucidenic acid inhibit PMA-induced invasion of human hepatoma cells through inactivating MAPK/ERK signal transduction pathway and reducing binding activities of NF-kB and AP-1. Carcinogenesis 29:147156 79. Weng CJ, Chau CF, Yen GC et al (2009) Inhibitory effects of Ganoderma lucidum on tumorigenesis and metastasis of human hepatoma cells in cells and animal models. J Agric Food Chem 57:50495057

123