Anticancer Effects of Taiwanofungus camphoratus Extracts, Isolated Compounds and its Combinational use

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REVIEW ARTICLE

Anticancer Effects of Taiwanofungus camphoratus Extracts, Isolated Compounds

and its Combinational use

Ying-Chen Chen

1

, Hsio-O Ho

1

, Chin-Hua Su

2

, Ming-Thau Sheu

1,3,* 1_{School of Pharmacy, College of Pharmacy, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan} 2_{School of Medicine, College of Medicine, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan} 3_{Clinical Research Center and Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan}

a r t i c l e i n f o

Article history: Received: Jun 23, 2010 Revised: Aug 1, 2010 Accepted: Aug 31, 2010 Available online 20 October 2010 KEY WORDS: anticancer; combination; crude extracts; pure compounds; Taiwanofungus camphoratus

Taiwanofungus camphoratus is an indigenous mushroom in Taiwan, which has been used as a traditional medicine to treat many health-related problems. Several biological activities have been reported on T. camphoratus ranging from anti-inflammatory antihypertension to anticancer and so on. Cancer is a major cause of death in Taiwan, and unfortunately, there is no satisfied treatment presently. Thus, a review article about the anticancer effect of T. camphoratus would be a great importance. This article reviews anticancer activities being performed with crude extracts and isolated compounds from T. camphoratus and their synergistic effects. The source of T. camphoratus might be from its fruiting bodies, mycelia, and fermented culture broth and be extracted from water, methanol, ethanol, ethyl acetate, or chloroform, which showed versatile anticancer activities. In addition, various compounds have been further purified from these extracts, such as terpenoids, maleic and succinic acids derivatives, polysaccharides, and other compounds, and they also showed potent cytotoxicity. Besides, T. camphoratus not only has cytotoxic effect but also produces synergistic anticancer effect with trichostatin A, lova-statin, and taxol. It is concluded that T. camphoratus could be considered as a potential anticancer agent to make cancer no longer a frightening nightmare. However, clinical trials of T. camphoratus on human subjects are absent, and the involved mechanism remains unclear. Hence, further investigations would be required.

1. Introduction

Taiwanofungus camphoratus (T. camphoratus), an orange to brown-red colobrown-red fungus, parasitizes only in the inner cavity of an endemic and endangered host, Cinnamomum kanehirai Hay (Lauraceae) (Bull camphor tree), which distributes at an altitude of 200e2000 m in the mountains of Taoyuan, Miaoli, Nantou, Kaohsiung, Taitung, and Hualien in Taiwan. T. camphoratus is a very scarce and an expensive mushroom because it grows extremely slow only from June to October and is difﬁcult to be cultivated in the greenhouse.1,2

T. camphoratus belongs to the following taxonomy: Kingdom: Mycoteae; Division: Amastigomycota; Subdivision: Basidiomytina; Class: Hymenomycetes; Order: Aphyllophorales; Family: Poly-poraceae; Genus: Taiwanofungus; Species: camphorautus.3

This unique Formosan mushroom was initially reported as Gano-derma camphoratum by Zang and Su4in 1990 because of a careless misidentiﬁcation from the contaminated specimen of Ganoderma

spores. However, according to the morphology of the fruiting body, the name Antrodia camphorata was proposed. In 2004, a phylogenetic analysis indicated that the ribosomal RNA of A. camphorata. is far related to other Antrodia species, and the fungus was designated the new name, T. camphoratus.3In Taiwan, it has been well known as “niu-chang-chih,” “chang-chih,” “niu-chang-ku,” or “chang-ku.”

Being an indigenous species, T. camphoratus was traditionally used by Taiwan aborigines to treat food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and improve the immune system and liver function.2This medicinal mushroom has been believed to improve health and increase longevity, and long been regarded as health foods. Several biological activities have been reported for T. camphoratus, such as anticancer,5e7 antihepatotoxic,8,9 antihypertensive,10,11 anti-in_ﬂammatory,12e15 antioxidant,16,17and neuroprotective18activities.

Cancer has been the leading health killer in Taiwan since 1982. In 2008, 38,913 people died of malignant tumor, which accounts for 27.3% death, and it means every 13.5 minutes, there was one case of death because of cancer. It did bring about lots of tragedies, and moreover, the incidence of cancer continues to increase so far. Therefore, many researchers devote themselves to the study of mechanism, treatment, and prevention in oncology. Unfortunately,

* Corresponding author. School of Pharmacy, College of Pharmacy, No. 250, Wu-Hsing Street, Taipei 110, Taiwan.

E-mail:mingsheu@tmu.edu.tw(M.-T. Sheu).

Contents lists available atScienceDirect

Journal of Experimental and Clinical Medicine

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various cytotoxic chemotherapeutic agents, such as paclitaxel, doxorubicin, and other widely used anticancer drugs currently, lack the speciﬁcity to only kill tumor cells without simultaneously damaging the healthy tissues and thus causing severe side effects. In order to solve the problems of dose-limiting toxicity associated with chemotherapeutic agents and drug resistance, development of cancer chemopreventive agents and improvement of cancer treat-ment are very important and urgent. The aim of this article was to review the literatures mentioning the method of extraction or isolation and anticancer effect of various extracts, isolated compounds, and their combinational use of T. camphoratus. 2. Anticancer Activities of Extracts

T. camphoratus has been used for a long time, and its pharmaco-logical effects have been investigated by many scientiﬁc researches. This part of review emphasizes the anticancer effects of crude extracts of T. camphoratus, and they are tabulated inTable 1. 2.1. Crude extract of T. camphoratus

Peng et al19 shook air-dried T. camphoratus powder with phos-phate-buffered saline at the ratio of 1:25 (w/v) at 25C for 10 hours, then centrifuged the extracts, and followed byﬁltering through a 0.2-

m

m pore sizeﬁlter. The growth of human urinary bladder cancer T24 cells was inhibited by 50

m

g/mL T. camphoratus crude extract in G2/M phase by suppressing the active form of matrix

metalloproteinase 9.14Furthermore, they also found that 100

m

g/mL T. camphoratus crude extract displayed antiproliferation effect in transitional cell carcinomas cell lines RT4, TSGH-8301, and T24. RT4 cells may proceed the p53-independent overexpression of p21 with simultaneous down alteration of pRb, and which was speculative of proceeding through a mechanism of replicative senescence. In the contrast, growth inhibitions of TSGH-8301 and T24 as affected by simultaneous downregulations of Cdc2 and cyclin B1 were attrib-uted to the insufﬁcient and destabilized Cdc2-cyclin B1 complex formation. In another study,20 prostate cancer cell lines LNCaP (androgen responsive) showed a G1/S phase arrest through the

procedure of Akt/ p53 / p21 / CDK4/cyclin D1 / G1/S-phase

arrest_{/ apoptosis, which involved inhibiting cyclin D1 activity} and preventing pRb phosphorylation with 150

m

g/mL T. camphor-atus crude extract. On the contrary, PC-3 cells (androgen inde-pendent) exhibited a G2/M-phase arrest mediated through

pathway p21/ cyclin B1/Cdc2 / G2/M-phase arrest with limited degree of apoptosis. Chen et al concluded that T. camphoratus might be a good adjuvant in anticancer therapy for prostate cancers regardless its androgen-responsive behaviors.

2.2. Ethanol extract from the fruiting bodies

The fruiting bodies of T. camphoratus show different plate-like, bell-like, or hoof-like shapes. Lu et al21reﬂuxed the dried fruiting bodies powder with ethanol at 75C in a 1:10 (w/v) ratio for 2 hours. The extracts were cooled and then precipitated overnight at 4C. The supernatant of extracts was furtherﬁltered and centrifuged, and the extracts were lyophilized. They found that ethanol extract could induce apoptosis of human leukemia (HL-60) cells through histone hypoacetylation, upregulation of histone deacetyltransferase 1, and downregulation of histone acetyltransferase activities containing GCN 5, CBP, and PCAF in a dose-dependent manner.

2.3. Ethyl acetate extract from the fruiting bodies

The fruiting bodies could be extracted with several solvents, and Hsu et al22soaked them in methanol for 3 days. The sample was

filtered with filter paper while the residue was further extracted under the same condition twice. Thefiltrates collected from three separate extractions were combined and evaporated to dryness under vacuum. Concentrated methanol extract was partitioned between water and ethyl acetate (1:3, v/v) three times to give an ethyl acetate extract, which was then evaporated to dryness under vacuum. The ethyl acetate extract decreased the cell growth of human hepatoma cancer cell line (Hep G2) and PLC/PRF/5 cells in a dose-dependent manner. In Fas/APO-1 positive-Hep G2 cells, the extract ascended the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand, and soluble Fas ligand with a p53-independent manner. Ethyl acetate extract also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep G2 and PLC/PRF/5 cells as well. Moreover, it suppressed the cell survival signaling by strengthening the amount of I

k

B

a

in cytoplasm and decreasing the level and activity of nuclear factor kB (NF-

k

B) in the nucleus and then attenuated the expression of Bcl-XL in Hep G2 and PLC/PRF/5 cells. The ethyl acetate extract also inhibited cell viability of hepa-toma Hep 3B cells by inducing apoptotic cell death with the increased level of Ca2þ in the cytoplasm and triggering the subsequent activation of calpain and caspase-12. It also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9. Furthermore, the mitochondrial apoptotic pathway ampli_{ﬁed the calpain pathway by Bid and Bax interaction and Ca}2þ translocation.23

2.4. CHCl3extract from the fruiting bodies

The powdered fruiting bodies were extracted with CHCl3 at

room temperature. Afterﬁltration, the solvent was evaporated under reduced pressure to obtain CHCl3 extracts. It showed

cytotoxic activity against Jurkat (human lymphocytic cancer cell line), Hep G2, Colon 205 (human colon cancer cell line), and MCF-7 (human breast cancer cell line) because of the inhibition of macrophage-mediated inﬂammatory mediators, such as nitric oxide (NO), tumor necrosis factor

a

(TNF-

a

), and interleukin 12 (IL-12), and cell cycle arrest in G0/G1 phase

in lipopolysaccharide/interferon (LPS/IFN)-

g

-activated murine peritoneal macrophages.24

2.5. Methanol extract from the fruiting bodies

In addition to chloroform extracts, Rao et al24also extracted the fruiting bodies with methanol at room temperature. Afterﬁltration, the solvent was evaporated under reduced pressure to obtain methanol extracts. It is cytotoxic to Jurkat cells (IC50¼ 40

m

g/mL)

through the growth inhibition of NO, TNF-

a

, and IL-12 and tumor cells proliferation.

2.6. Methanol/CHCl3extract from the fruiting bodies

Moreover, a CHCl3/MeOH extract exhibited signiﬁcant cytotoxicity

against P-388 murine leukemia cells (IC50¼ 4.1

m

g/mL); however,

the involving mechanism remains unclear.25 2.7. Aqueous extract from mycelia

The fruiting bodies have been demonstrated to possess anticancer activity. On the other hand, mycelia are also potential anticancer agents. Air-dried T. camphoratus mycelia powder was mixed with water, ﬁltered, and then air-dried. Mycelia powder of 100e200

m

g/mL induced apoptosis in human osteosarcoma MG63

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cells, exerted multiple effects on [Ca2þ]ivia inhibition of

extra-cellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) phosphorylation.26As to prostate cancer PC3 cells, it also worked on viability and [Ca2þ]i, caused apoptosis via

pathways unrelated to [Ca2þ]isignal, and phosphorylation of ERK,

c-Jun N-terminal kinases (JNK), and p38 MAPKs.27

Hseu et al16alsoﬁltered mycelia with water before being air-dried. Oxidative hemolysis and lipid/protein peroxidation of erythrocytes induced by the aqueous peroxyl radical and the endothelial cell damage induced by the aqueous peroxyl radical [2,2V-Azobis(2-amidinopropane) dihydrochloride] were sup-pressed by aqueous T. camphoratus extract from mycelia, and it also

Table 1 Anticancer activities of crude extracts from Taiwanofungus camphoratus

Extracts IC50(mg/mL) Mechanism Experimental model Reference

Fruiting body

Crude extract 50 Suppress the active form of MMP-9 Human urinary bladder cancer

T24 cells

49

Crude extract 100a _{Cyclin B1 destabilize Cdc2-cyclin B1 complex}

formation and downregulate Cdc2

Transitional cell carcinomas TSGH-8301 and T24

19

Crude extract 100 Overexpress p21 with simultaneous down

alteration of pRb

Transitional cell carcinomas RT4 cells

19

Crude extract 150a _Akt_{/ p53 / p21 / CDK4/cyclinD1 / G1/}

S-phase arrest/ apoptosis, inhibit cyclin D1 activity, and prevent pRb phosphorylation

Prostate cancer LNCaP cells (androgen responsive)

20

Crude extract 150a _p21_{/ cyclin B1/Cdc2 / G2/M-phase} _{Prostate cancer PC-3 cells}

(androgen independent)

20

Ethanol 104.82 Induce apoptosis through histone

hypoacetylation, upregulation of histone deacetyltransferase 1, and downregulation of histone acetyltransferase activities

Human leukemia HL 60 cells 21

Ethyl acetate 42.57 to Hep G2 cells

Ascend the expression level of Fas/APO-1, initiate mitochondrial apoptotic pathway, suppress the cell survival signaling, and then attenuate the expression of Bcl-XL

Human hepatoma Hep G2 cells 22

47.1 to PLC/PRF/5 cells

PLC/PRF/5 cells Ethyl acetate 78.3 Induce apoptotic death mediated through

calcium and calpain-dependent pathway

Human hepatoma Hep 3B cells 23

CHCl3extract 22 to Jurkat cells Inhibit macrophage-mediated inﬂammatory mediators

Human lymphocytic cancer Jurkat cells

24

150 to Hep G2 cells 65 to Colon 205 cells

Human hepatoma Hep G2 cells

95 to MCF-7 cells Human colon cancer Colon

205 cells

Human breast MCF-7 cells

Methanol 40 Inhibit macrophage-mediated inﬂammatory

mediators

Human lymphocytic cancer Jurkat cells

24

CHCl3/methanol 4.1 Unknown Murine leukemia P-388 cells 25

Mycelia

Mycelia powder 100e200a _{Effects on apoptosis, [Ca}2þ_{]i, and MAPKs} phosphorylation

Human osteosarcoma MG63 cells

26

Mycelia powder 100e200a _{Effects on apoptosis and [Ca}2þ_]i _{Prostate cancer PC3 cells} ₂₇

Aqueous extract Not shown Protection of oxidative damage Erythrocytes 16

Methanol 49.5 to Hep G2 cells Induce apoptosis through activation of caspase-3 and -8 cascades

Human hepatoma Hep G2 cells 28

62.7 to Hep 3B cells Human hepatoma Hep 3B cells

Methanol 0e200b _{Regulation of Fas pathway} _{Human heptoma Hep G2 cells} ₂₉

Ethanol extracts 36.9 to Hep G2 cells Unknown Human hepatoma Hep G2 cells 30

3.1 to Hep 3B cells Human hepatoma Hep 3B cells

Ethanol extracts 1e300b _{Protein kinase A-dependent pathway and}

suppress the activity of JNK and p38

Pheochromocytoma PC-12 cells 31

Extracts from solid-state fermentation

Ethanol extracts Not shown Downregulate human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitora, human calcium-dependent protease small subunit, and human annexin V

Human non-small cell lung carcinoma A549 cells

32

Fermented culture broth Fermented culture

broth

0e240b _{Reduce cyclin D1, cyclin E, CDK4, cyclin A, and}

proliferating cell nuclear antigen, and increase CDK inhibitor p27/KIP and p21/WAF1

Human breast cancer MDA-MB-231 cells

33

Fermented culture broth

55 and 110 mg/kgb _{Inhibit proliferation (cyclin D1 and PCNA) and}

induce apoptosis (Bcl-2 and TUNEL)

Nude mice inoculated with MDA-MB-231 cells

33

Fermented culture broth

316 to MF-7 cells Loss of chromatin condensation,

internucleosomal DNA fragmentation, release of cytochrome c, activation of caspase 3, and speciﬁc proteolytic cleavage of PARP, and inhibition of COX-2

Human breast cancer MCF-7 34,35

136 to MDA-MB-231 cells

MDA-MB-231 cells

MMP¼ matrix metalloproteinase; Jurkat ¼ human lymphocytic cancer cell line; Colon 205 ¼ human colon cancer cell line; MCF-7, MDA-MB-231 ¼ human breast cancer cell line.

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prevented the depletion of cytosolic antioxidant glutathione and adenosine triphosphate in erythrocytes.

2.8. Methanol extracts of mycelia

Like the fruiting bodies, mycelia could be extracted with different solvents. The methanol extracts of mycelia were obtained by ﬁltering and then concentrating to dryness. Cell cycle analysis revealed that methanol extract of mycelia induced apoptosis on Hep G2 cells in G0/G1cell cycle through the activation of caspase-3

and -8 cascades and regulation of the cell cycle progression to inhibit hepatoma cells proliferation.28 Furthermore, the author indicated that the mechanism could be Fas/Fas ligand death-receptor pathway through upregulation of Fas and downregulation of Bcl-2, DR3, DR4, tumor necrosis factor receptor (TNFR)I, and TNFRII in Hep G2 cells.29

2.9. Ethanol extracts of mycelia

The mycelia were extracted with 95% ethanol at 30C for 24 hours, and the ﬁltrates were dried under vacuum. Ethanol extracts of mycelia have antiproliferation against Hep G2 and Hep 3B cells.30 Lu et al31found that it also prevented serum deprivation-induced PC12 cell apoptosis via a protein kinase A-dependent pathway and by suppressing the activities of JNK and p38.

2.10. Extracts from solid-state fermentation

Air-dried T. camphoratus mycelia grown by solid-state fermentation were ground in liquid nitrogen and shaken with 100% ethanol at a ratio of 1:5 w/v overnight at room temperature. Then, the suspension was centrifuged, and the supernatant was ﬁltered through a 0.2-

m

m-pore size filter. The filtrate was evaporated to one-fifth of the original volume to collect the extract. Extract from solid-state fermentation can effectively interrupt the proliferation of human non-small cell lung carcinoma A549 cells by triggering the apoptosis through the induction of endoplasmic reticulum stress and downregulation of human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitor

a

, human calcium-dependent protease small subunit, and human annexin V.32

2.11. Fermented culture broth of T. camphoratus

Not only do the fruiting bodies and mycelia have anticancer effects but also the fermented culture broth inhibits the growth of cancer cells. The viability of MDA-MB-231 cells (human breast cancer cell line) was impeded by fermented culture broth in the G1 phase associated with the reduction in cyclin D1, cyclin E, CDK4, cyclin A, proliferating cell nuclear antigen, and the increase of cyclin-dependent kinases (CDK) inhibitor p27/KIP and p21/WAF1. More-over, it was also effective in the breast cancer nude mice inoculated with MDA-MB-231 cells to delay tumor incidence and reduce the tumor size. When the tumor tissue sections were examined histologically and immunohistochemically, the inhibition of the proliferation (cyclin D1 and PCNA) and induction of apoptosis (Bcl-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)) were observed.33

In another study, treatment of fermented culture broth of T. camphoratus with MCF-7 (25e150

m

g/mL)34and MDA-MB-231 (40e240

m

g/mL)35 resulted in a dose- and time-dependent sequences by apoptosis, as represented by loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation, and sub-G1 phase accumulation, the release of cytochrome c, activation of caspase-3 and speciﬁc proteolytic cleavage of poly

(adenosine diphosphate (ADP)-ribose) polymerase (PARP), and dysregulation of Bcl-2 and Bax. Furthermore, fermented culture broth of T. camphoratus inhibited cyclooxygenase (COX)-2 protein expression and prostaglandin E2production in MDA-MB-231 cells.

3. Anticancer Activities of Isolated Compounds FromT. camphoratus

So far, there have been 78 compounds isolated from T. camphoratus, which describes its complexity and difﬁculty in research. Terpe-noids predominate the fruiting body (39 compounds) and has been the focus of many pharmacological studies.18,36e39 Nevertheless, several other components were also identiﬁed comprising benze-noids,15,40 _{maleic/succinic acid derivatives,}6 _{polysaccharides,}41,42

sterols,25and sesquiterpene lactone37as well. The details of anti-cancer compounds will be discussed below and tabulated in

Table 2. 3.1. Terpenoids

The bitter taste with camphor aroma of T. camphoratus is because of terpenoids that have been considered as potential anticancer agents. Ethanol extract from the fruiting body was further extracted with solvents of increasing polarity (n-hexane, ethyl acetate, and ethanol). Ethyl acetate fraction was subsequently chromatographed using Sephadex LH-20 with CHCl3-MeOH (1:3) to get three

frac-tions, while Fraction 2 was separated by thin layer chromatography with CHCl3-MeOH (25:1) to obtain eight fractions, and zhankuic

acid A was then puriﬁed by octadecylsilica (ODS) high-performance liquid chromatography column. Zhankuic acid A exhibited signi ﬁ-cant cytotoxicity to HL 60 cells (IC50 ¼ 5.45

m

g/mL) in causing

antiproliferation, death, and apoptotic induction, and Lu et al21 suggested zhankuic acid A was the major component of ethanol extract from the fruiting body.

Zhankuic acid A could be isolated from other methods. Dried T. camphoratus was ground to powder and extracted with ethanol. The concentrated extracts were partitioned between ethyl acetate and water. The ethyl acetate fraction was chromatographed by Sephadex LH-20 column with ethanol as an eluent to yield four fractions. The third fraction was further isolated by a Si gel column and eluted with CHCl3and increasing concentrations of methanol

to provide 14 fractions. Fraction 2 was puriﬁed by Si gel to give zhankuic acid A, and zhankuic acids B and C were obtained from Fractions 4 and 7, respectively. While zhankuic acids A and C exhibited cytotoxicity against P-388 murine lymphocytic leukemia cells with IC50values of 1.8 and 5.4

m

g/mL, respectively.25

Yeh et al43extracted air-dried powder of T. camphoratus with CHCl3after solvent evaporation, and the residue was separated by

silica gel column chromatography with increasing polarity using mixtures of n-hexane/ethyl acetate to elute. Following the thin layer chromatography analysis, antcinate B, zhankuic acid A, and zhankuic acid C were isolated, and they displayed the tumor-speciﬁc cytotoxicity with an IC50range from 22.3 to 75.0

m

M against

the colon, breast, liver, and lung cancer cell lines in sub-G1 cell cycle through apoptosis induction by the cleavage of the downstream poly(ADP-ribose) polymerase, procaspase-3, and Bcl-2, while methyl antcinate B was slightly potent. Furthermore, these three compounds demonstrated synergistic cytotoxic effect (4

m

M each) in colon cancer HT-29 cells.

3.2. Maleic and succinic acids derivatives

Dried mycelia was extracted with methanol at room temperature; the crude methanol syrup was evaporated under reduced pressure and then partitioned with n-hexane:H2O (1:1) three times. The

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n-hexane layer extract was chromatographed by a silica gel column with a stepwise gradient of n-hexane and ethyl acetate to give 15 fractions. Fraction 5 was further puriﬁed by column chromatog-raphy on silica gel eluting with n-hexane and CH2Cl2to yield six

fractions, and antrodins B and C were isolated. These two mal-eimide derivatives inhibit the viability of murine macrophage cell line RAW264.7 to more than 30%.6

3.3. Polysaccharides

To obtain polysaccharide, mycelia of T. camphoratus were air-dried, disintegrated, and extracted with boiling water for 8e12 hours. The insoluble matter was removed, and water-soluble polysaccharide-enriched fraction was isolated by ethanol precipitation. The crude polysaccharides were then filtered through a Sephadex G50 gel filtration column and were further purified by an anion exchange column of diethylaminoethyl cellulose. The polysaccharide component alone did not show any direct cytotoxic effect to human leukemic U937 cells; however, it could inhibit 55.3% growth rate of U937 cells via activation of mononuclear cells. Intraperitoneal and

oral administration of polysaccharide (100 and 200 mg/kg, respectively) signiﬁcantly suppressed the tumor growth to 69.1% and 58.8%, respectively, in sarcoma 180-bearing mice by regulating some immunoparameters, such as the spontaneous proliferation of spleen cells, the cytolytic activity of spleen cells, and serum inter-leukin-12 levels. Liu et al7suggested that polysaccharides from T. camphoratus reveal its anticancer effect by promoting a Th1-dominant state and killer activities.

Cheng et al44extracted lyophilized mycelia with 80C water in a 1:100 (w/w) ratio for 6 hours. The extracts were cooled, and quadruple volumes of 95% ethanol were added and then stored overnight at 4C. The brownish precipitated polysaccharides were collected by centrifugation and lyophilized. The T. camphoratus polysaccharides inhibit cyclin D1 expression in endothelial cells through inhibition of vascular endothelial growth factor receptor signaling, leading to the suppression of angiogenesis.

In addition to extraction with water, lyophilized mycelia were also extracted with 0.1M sodium acetate (pH 5.5) buffer containing 5 mM cysteine, 100 mg papain as a digestion agent, and 5 mM EDTA at 60C for 24 hours. Supernatants were collected after

Table 2 Anticancer activities of isolated compounds of Taiwanofungus camphoratus

Compound name IC50(mg/mL) Mechanism Experimental model Reference

Zhankuic acid A 5.4521 _{Induce apoptosis,}21_suppress

the expression of apoptosis-associated proteins43

Human leukemia HL 60 cells21 _21,25,43

1.825 _{Murine lymphocytic leukemia}

P-388 cells25

22.3e75.0mMa,43 _{Human colon (HT-29, HCT116,}

SW480), breast (MCF-7, MDA-MB-231), liver (Huh 7, Hep G2, Hep 3B), and lung (A549, CL1-0) cancer cell lines43

Zhankuic acid C 5.425 _{Suppress the expression of}

apoptosis-associated proteins43

P-388 murine lymphocytic leukemia cells25

25,43

22.3e75.0mMa,43 _{Human colon (HT-29, HCT116,}

SW480), breast (MCF-7, MDA-MB-231), liver (Huh 7, Hep G2, Hep 3B), and lung (A549, CL1-0) cancer cell lines43

Methyl antcinate B 22.3e75.0mMa _{Suppress the expression of}

apoptosis-associated proteins

Human colon (HT-29, HCT116, SW480), breast (MCF-7, MDA-MB-231), liver (Huh 7, Hep G2, Hep 3B), and lung (A549, CL1-0) cancer cell lines43

43 Combined use of zhankuic acid A, zhankuic acid C, and methyl antcinate B

4.0mM (each) Suppress the expression of apoptosis-associated proteins

Colon cancer HT-29 cells 43

Antrodins B 3.6b _Unknown _{Murine macrophage cell line}

RAW264.7

5

Antrodins C 7.5b _Unknown _{Murine macrophage cell line}

RAW264.7

6

Polysaccharide 100 Activation of mononuclear cells Human leukemic U937 cells 7

Polysaccharide 100 mg/kg (intraperitoneal administration) and 200 mg/kg (oral administration)c Regulate some immunoparameters

Sarcoma 180-bearing mice 7

Polysaccharides 35.2 Inhibit cyclin D1 expression Endothelial cells 44

Sulfated polysaccharides

Not shown Antiangiogenic and

neuroprotective effects

Matrigel tube formation serum deprivation-induced apoptosis in neuronal-like PC12 cell 45 4,7-dimethoxy- 5-methyl-1,3-benzodioxole 1.721 to MCF-7, 0.992 to MDA-MB-231, 0.016 to Hep 3B, 2.462 to Hep G2, 4.46 to LNCap, and 2.21 to DU-145 cell lines

Unknown Human breast cancer MCF-7

and MDA-MB-231 cells, human hepatoma Hep 3B and Hep G2, human prostate cancer cells LNCaP, and human prostate cancer DU-145 cells

46

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centrifugation, and the same buffer was added to the precipitate for another 24 hours at 60C. The supernatants of the two extractions were collected, and a 3.75-fold volume of 95% ethanol was added, precipitated at 4C overnight, then spun, and the pellets were collected. The pellets were dried and resuspended in distilled water, dialyzed (molecular weight: 12,000e14,000 Da) against distilled water overnight at 4C, and centrifuged to collect the supernatant as sulfated polysaccharides. It is theﬁrst time to report that the sulfated polysaccharides inhibit in vitro Matrigel tube formation in an angiogenesis model. Furthermore, using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a stress model, the sulfated polysaccharides were effective in pre-venting serum-deprived apoptosis.45

3.4. Miscellaneous compounds with anticancer activities

US patent46 disclosed that 4,7-dimethoxy-5-methyl-1,3-benzo-dioxole inhibited the cell number of MCF-7, MDA-MB-231, Hep 3B, Hep G2, LNCaP human prostate cancer cells, and DU-145 human prostate cancer cells with IC50of 1.721, 0.992, 0.016, 2.462, 4.46, and

2.21

m

g/mL, respectively. The above IC50values are lower than the

IC50value of whole T. camphoratus extract (11.461, 26.812, 6.112,

18.931, 45.47, and 30.15

m

g/mL, respectively). Therefore, it is conﬁrmed that 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from T. camphoratus extract is capable of inhibiting cell prolifera-tion of breast cancer, liver cancer, and prostate cancer.

4. Synergistic Anticancer Activities ofT. camphoratus

Interestingly, T. camphoratus has more potent cytotoxicity when it is combined with other agents, such as trichostatin A (TSA), lova-statin, and taxole. The synergistic effects are discussed in the following mention andTable 3.

Solid-state cultured T. camphoratus mycelia were extracted in the ratio of 1:10 (v/v) with 95% ethanol at room temperature for 24 hours and then centrifuged. Supernatant was removed and ﬁltered. In C3A and PLC/PRF/5 hepatoma cell model, T. camphor-atus extract (1

m

g/mL) showed prominent adjuvant inhibitive effects on proliferation when it was combined with cisplatin (100

m

M) or mitomycin (100

m

M). Furthermore, in xenografted cancer animal model, the combined treatment of T. camphoratus extract

(200 mg/kg/d) with cisplatin (1 mg/kg/wk) for 21 consecutive days signiﬁcantly extended the median survival days of PLC/PRF/5 cells-implanted imprinting control region (ICR) nude mice. T. cam-phoratus extract showed its adjuvant effects through the inhibition of multidrug resistance (MDR) gene expressions and pathway of COX-2-dependent inhibition of AKT phosphorylation (p-AKT), which ultimately resulted in the induction of apoptosis in hepa-toma cells.47

US patent46illustrated that synergistic effects of taxol (0.0017

m

g/mL) and 4,7-dimethoxy-5-methyl-1,3-benzodioxole inhibited the viability of MCF-7, MDA-MB-231, Hep G2, LNCaP, and DU-145 cells signiﬁcantly. In addition, lovastatin (0.0043

m

g/mL) and 4,7-dimethoxy-5-methyl-I,3-benzodioxole (0.0007

m

g/mL) caused the death of half of Hep 3B human liver cancer cells. Therefore, Liu et al suggested that 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from T. camphoratus extract shows more inhibitory effects with lovastatin and taxol.

US patent48disclosed that a method of treating cancer with an effective amount of a mevalonate pathway inhibitor and T. cam-phoratus extract. Pulverized T. camcam-phoratus was suspended in 95% ethanol, and the suspension was stirred at room temperature for 48 hours and then ﬁltered to remove insoluble residue. The ﬁltrate was collected and concentrated by a rotary evaporator at 40e45C. The concentrate was evaporated to dryness under nitrogen at room temperature. Details indicated that mevalonate pathway inhibitor, for example, lovastatin, and T. camphoratus extract exhibited profound synergistic effect in inhibiting cell growth of Hep G2, DBTRG 05MG (human glioma cell line), CL1-0 (human lung adenocarcinoma cell lines), and PC12 (rat pheo-chromocytoma cell line). For example, lovastatin (2

m

M) and T. camphoratus extract (20

m

g/mL) inhibited growth of Hep G2 cells only by 11.5% and 2.8%, respectively, whereas their combination inhibited the growth by 99.3%. In addition, the cells surviving from the combinational treatment showed markedly shrunk morphology.

The combined use of 100

m

g/mL ethanol extract from the fruiting body and 100 nM of TSA (a potent reversible inhibitor of histone acetylase) caused synergistic inhibition of cell growth compared with single TSA or T. camphoratus treatment to HL 60 cells. T. camphoratus could induce apoptosis through the increase of cytotoxic sensitivity of TSA, upregulation of DR5, and NF

k

B activation.21

Table 3 Synergistic anticancer activities of Taiwanofungus camphoratus

Extract of T. camphoratus Adjuvant agents Mechanism Experimental model Reference

Solid-state cultured T. camphoratus mycelia (1mg/mL) Cisplatin (100mM), mitomycin (100mM)

Inhibit MDR gene expressions and the pathway of COX-2-dependent inhibition of AKT phosphorylation

Human hepatoma C3A and PLC/PRF/5 cells 47 Solid-state cultured T. camphoratus mycelia (200 mg/kg/d) Cisplatin (1 mg/kg/wk)

Inhibit MDR gene expressions and the pathway of COX-2-dependent inhibition of AKT phosphorylation PLC/PRF/5 cells-implanted nude mice 47 4,7-dimethoxy-5-methyl-1,3-benzodioxole (0.0007mg/mL) Lovastatin (0.0043mg/mL)

Unknown Human hepatoma Hep 3B cells 46

4,7-dimethoxy-5-methyl-1,3-benzodioxole (0.0007, 0.0009, 0.0129, 1.16, and 0.71mg/mL) Taxol (0.0017mg/mL)

Unknown Human breast cancer MCF-7

and MDA-MB-231 cells, human hepatoma Hep G2, human prostate cancer LNCaP cells, and human prostate cancer DU-145 cells

46

Ethanol extract from the fruiting body (20mg/mL)

Lovastatin (2mM) Unknown Human hepatoma Hep G2,

human glioma DBTRG 05MG, human lung adenocarcinoma CL1-0, and rat

pheochromocytoma PC12 cells

48

Ethanol extract from the fruiting body (100mg/mL)

Trichostatin A (100 nM)

Increase cytotoxic sensitivity of trichostatin, upregulation of DR5, and NFkB activation

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5. Summary and Perspective

This review summarized important anticancer activities being performed with T. camphoratus with focus on crude extracts, iso-lated compounds, and their synergistic effects. The source of T. camphoratus might be from its fruiting bodies, mycelia and fer-mented culture broth, and be extracted from water, methanol, ethanol, ethyl acetate, or chloroform, which showed versatile anticancer activities. In addition, various compounds have been further puriﬁed, such as, terpenoids, maleic and succinic acids, polysaccharides, and other compounds, and they also showed potent cytotoxicity. It is of signiﬁcant interest to isolate and identify such exact compounds that are responsible for the anticancer activity and make medicines from this fungus. For example, taxanes from Taxus brevifolia, and vincristine and vinblastine from Cathar-anthus roseus. Besides, T. camphoratus not only has cytotoxicity, but also it produces synergistic anticancer effect with TSA, lovastatin, and taxol.

The anticancer studies so far have mostly been conducted in vitro and in vivo, thus clinical studies are needed to confirm the anticancer effect. Regardless, the previous reports could be considered as pioneers for more innovative researches. In addition, the biological activities of the crude fungus and pure compound were not identical, and it suggested there are synergistic effects present in the mixture of T. camphorata. The biological activities need to be thoroughly assessed, but this complexity can also bring signi_{ficant advantages. For example, certain components in the} natural products can reduce the cytotoxicity of the whole product, and vice versa. Also, the interaction between different biologically active components can play a major role for their effects in vivo. Different compounds can modulate unrelated signaling and therefore, can possess synergistic effects. However, the molecular mechanism has not been fully elucidated and further studies are needed to explore the benefits and safety to cancer patients.

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