Anticancer Effects of Taiwanofungus Camphoratus Extracts, Isolated Compounds and Its Combinational Use

J Exp Clin Med 2010;2(6):274e281

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Journal of Experimental and Clinical Medicine

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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 article info Taiwanofungus camphoratus is an indigenous mushroom in Taiwan, which has been used as a traditional Article history: medicine to treat many health-related problems. Several biological activities have been reported on Received: Jun 23, 2010 T. camphoratus ranging from anti-inflammatory antihypertension to anticancer and so on. Cancer is Revised: Aug 1, 2010 a major cause of death in Taiwan, and unfortunately, there is no satisfied treatment presently. Thus, Accepted: Aug 31, 2010 a review article about the anticancer effect of T. camphoratus would be a great importance. This article Available online 20 October 2010 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 KEY WORDS: bodies, mycelia, and fermented culture broth and be extracted from water, methanol, ethanol, ethyl anticancer; acetate, or chloroform, which showed versatile anticancer activities. In addition, various compounds combination; have been further purified from these extracts, such as terpenoids, maleic and succinic acids derivatives, crude extracts; polysaccharides, and other compounds, and they also showed potent cytotoxicity. Besides, T. camphoratus pure compounds; Taiwanofungus camphoratus not only has cytotoxic effect but also produces synergistic anticancer effect with trichostatin A, lovastatin, 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. Copyright Ó 2010, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved.

1. Introduction spores. However, according to the morphology of the fruiting body, the name Antrodia camphorata was proposed. In 2004, a phylogenetic Taiwanofungus camphoratus (T. camphoratus), an orange to brown- analysis indicated that the ribosomal RNA of A. camphorata.isfar red colored fungus, parasitizes only in the inner cavity of an endemic related to other Antrodia species, and the fungus was designated the and endangered host, Cinnamomum kanehirai Hay (Lauraceae) (Bull new name, T. camphoratus.3 InTaiwan, it has beenwell known as “niu- camphor tree), which distributes at an altitude of 200e2000 m in chang-chih,”“chang-chih,”“niu-chang-ku,” or “chang-ku.” the mountains of Taoyuan, Miaoli, Nantou, Kaohsiung, Taitung, and Being an indigenous species, T. camphoratus was traditionally Hualien in Taiwan. T. camphoratus is a very scarce and an expensive used by Taiwan aborigines to treat food and drug intoxication, mushroom because it grows extremely slow only from June to diarrhea, abdominal pain, hypertension, skin itching, and improve October and is difficult to be cultivated in the greenhouse.1,2 the immune system and liver function.2 This medicinal mushroom T. camphoratus belongs to the following taxonomy: Kingdom: has been believed to improve health and increase longevity, and Mycoteae; Division: Amastigomycota; Subdivision: Basidiomytina; long been regarded as health foods. Several biological activities e Class: Hymenomycetes; Order: Aphyllophorales; Family: Poly- have been reported for T. camphoratus, such as anticancer,5 7 e poraceae; Genus: Taiwanofungus; Species: camphorautus.3 antihepatotoxic,8,9 antihypertensive,10,11 anti-inflammatory,12 15 This unique Formosan mushroom was initially reported as Gano- antioxidant,16,17 and neuroprotective18 activities. derma camphoratum by Zang and Su4 in 1990 because of a careless Cancer has been the leading health killer in Taiwan since 1982. misidentification from the contaminated specimen of Ganoderma 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. * Corresponding author. School of Pharmacy, College of Pharmacy, No. 250, Therefore, many researchers devote themselves to the study of Wu-Hsing Street, Taipei 110, Taiwan. E-mail: [email protected] (M.-T. Sheu). mechanism, treatment, and prevention in oncology. Unfortunately,

1878-3317/$ e see front matter Copyright Ó 2010, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.jecm.2010.08.003 Anticancer effects of Taiwanofungus camphoratus 275 various cytotoxic chemotherapeutic agents, such as paclitaxel, filtered with filter paper while the residue was further extracted doxorubicin, and other widely used anticancer drugs currently, lack under the same condition twice. The filtrates collected from three the specificity to only kill tumor cells without simultaneously separate extractions were combined and evaporated to dryness damaging the healthy tissues and thus causing severe side effects. under vacuum. Concentrated methanol extract was partitioned In order to solve the problems of dose-limiting toxicity associated between water and ethyl acetate (1:3, v/v) three times to give an with chemotherapeutic agents and drug resistance, development of ethyl acetate extract, which was then evaporated to dryness under cancer chemopreventive agents and improvement of cancer treat- vacuum. The ethyl acetate extract decreased the cell growth of ment are very important and urgent. The aim of this article was to human hepatoma cancer cell line (Hep G2) and PLC/PRF/5 cells in review the literatures mentioning the method of extraction or a dose-dependent manner. In Fas/APO-1 positive-Hep G2 cells, the isolation and anticancer effect of various extracts, isolated extract ascended the expression level of Fas/APO-1 and its two compounds, and their combinational use of T. camphoratus. forms of ligands, membrane-bound Fas ligand, and soluble Fas ligand with a p53-independent manner. Ethyl acetate extract also 2. Anticancer Activities of Extracts initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and T. camphoratus has been used for a long time, and its pharmaco- activation of caspase-9 in Hep G2 and PLC/PRF/5 cells as well. logical effects have been investigated by many scientific researches. Moreover, it suppressed the cell survival signaling by strengthening This part of review emphasizes the anticancer effects of crude the amount of IkBa in cytoplasm and decreasing the level and extracts of T. camphoratus, and they are tabulated in Table 1. activity of nuclear factor kB (NF-kB) in the nucleus and then attenuated the expression of Bcl-XL in Hep G2 and PLC/PRF/5 cells. 2.1. Crude extract of T. camphoratus The ethyl acetate extract also inhibited cell viability of hepatoma Hep 3B cells by inducing apoptotic cell death with the þ Peng et al19 shook air-dried T. camphoratus powder with phos- increased level of Ca2 in the cytoplasm and triggering the phate-buffered saline at the ratio of 1:25 (w/v) at 25C for 10 hours, subsequent activation of calpain and caspase-12. It also initiated then centrifuged the extracts, and followed by filtering through the mitochondrial apoptotic pathway through regulation of Bcl-2 a 0.2-mm pore size filter. The growth of human urinary bladder family proteins expression, release of cytochrome c, and activation cancer T24 cells was inhibited by 50 mg/mL T. camphoratus crude of caspase-9. Furthermore, the mitochondrial apoptotic pathway 2þ extract in G2/M phase by suppressing the active form of matrix amplified the calpain pathway by Bid and Bax interaction and Ca metalloproteinase 9.14 Furthermore, they also found that 100 mg/mL translocation.23 T. camphoratus crude extract displayed antiproliferation effect in transitional cell carcinomas cell lines RT4, TSGH-8301, and T24. RT4 2.4. CHCl3 extract from the fruiting bodies cells may proceed the p53-independent overexpression of p21 with simultaneous down alteration of pRb, and which was speculative of The powdered fruiting bodies were extracted with CHCl3 at proceeding through a mechanism of replicative senescence. In the room temperature. After filtration, the solvent was evaporated contrast, growth inhibitions of TSGH-8301 and T24 as affected by under reduced pressure to obtain CHCl3 extracts. It showed simultaneous downregulations of Cdc2 and cyclin B1 were attrib- cytotoxic activity against Jurkat (human lymphocytic cancer uted to the insufficient and destabilized Cdc2-cyclin B1 complex cell line), Hep G2, Colon 205 (human colon cancer cell line), formation. In another study,20 prostate cancer cell lines LNCaP and MCF-7 (human breast cancer cell line) because of the (androgen responsive) showed a G1/S phase arrest through the inhibition of macrophage-mediated inflammatory mediators, procedure of Akt / p53 / p21 / CDK4/cyclin D1 / G1/S-phase such as nitric oxide (NO), tumor necrosis factor a (TNF-a), and arrest / apoptosis, which involved inhibiting cyclin D1 activity interleukin 12 (IL-12), and cell cycle arrest in G0/G1 phase and preventing pRb phosphorylation with 150 mg/mL T. camphor- in lipopolysaccharide/interferon (LPS/IFN)-g-activated murine atus crude extract. On the contrary, PC-3 cells (androgen inde- peritoneal macrophages.24 pendent) exhibited a G2/M-phase arrest mediated through pathway p21 / cyclin B1/Cdc2 / G2/M-phase arrest with limited 2.5. Methanol extract from the fruiting bodies degree of apoptosis. Chen et al concluded that T. camphoratus might be a good adjuvant in anticancer therapy for prostate cancers In addition to chloroform extracts, Rao et al24 also extracted the regardless its androgen-responsive behaviors. fruiting bodies with methanol at room temperature. After filtration, the solvent was evaporated under reduced pressure to obtain 2.2. Ethanol extract from the fruiting bodies methanol extracts. It is cytotoxic to Jurkat cells (IC50 ¼ 40 mg/mL) through the growth inhibition of NO, TNF-a, and IL-12 and tumor The fruiting bodies of T. camphoratus show different plate-like, bell- cells proliferation. like, or hoof-like shapes. Lu et al21 refluxed the dried fruiting bodies powder with ethanol at 75 C in a 1:10 (w/v) ratio for 2 hours. The 2.6. Methanol/CHCl3 extract from the fruiting bodies extracts were cooled and then precipitated overnight at 4C. The supernatant of extracts was further filtered and centrifuged, and Moreover, a CHCl3/MeOH extract exhibited significant cytotoxicity the extracts were lyophilized. They found that ethanol extract could against P-388 murine leukemia cells (IC50 ¼ 4.1 mg/mL); however, induce apoptosis of human leukemia (HL-60) cells through histone the involving mechanism remains unclear.25 hypoacetylation, upregulation of histone deacetyltransferase 1, and downregulation of histone acetyltransferase activities containing 2.7. Aqueous extract from mycelia GCN 5, CBP, and PCAF in a dose-dependent manner. The fruiting bodies have been demonstrated to possess anticancer 2.3. Ethyl acetate extract from the fruiting bodies activity. On the other hand, mycelia are also potential anticancer agents. Air-dried T. camphoratus mycelia powder was mixed The fruiting bodies could be extracted with several solvents, and with water, filtered, and then air-dried. Mycelia powder of Hsu et al22 soaked them in methanol for 3 days. The sample was 100e200 mg/mL induced apoptosis in human osteosarcoma MG63 276 Y.-C. Chen et al.

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 49 T24 cells Crude extract 100a Cyclin B1 destabilize Cdc2-cyclin B1 complex Transitional cell carcinomas 19 formation and downregulate Cdc2 TSGH-8301 and T24 Crude extract 100 Overexpress p21 with simultaneous down Transitional cell carcinomas 19 alteration of pRb RT4 cells a Crude extract 150 Akt / p53 / p21 / CDK4/cyclinD1 / G1/ Prostate cancer LNCaP cells 20 S-phase arrest / apoptosis, inhibit cyclin D1 (androgen responsive) activity, and prevent pRb phosphorylation Crude extract 150a p21 / cyclin B1/Cdc2 / G2/M-phase Prostate cancer PC-3 cells 20 (androgen independent) Ethanol 104.82 Induce apoptosis through histone Human leukemia HL 60 cells 21 hypoacetylation, upregulation of histone deacetyltransferase 1, and downregulation of histone acetyltransferase activities Ethyl acetate 42.57 to Hep Ascend the expression level of Fas/APO-1, Human hepatoma Hep G2 cells 22 G2 cells initiate mitochondrial apoptotic pathway, 47.1 to PLC/PRF/5 suppress the cell survival signaling, and then PLC/PRF/5 cells cells attenuate the expression of Bcl-XL Ethyl acetate 78.3 Induce apoptotic death mediated through Human hepatoma Hep 3B cells 23 calcium and calpain-dependent pathway

CHCl3 extract 22 to Jurkat cells Inhibit macrophage-mediated inﬂammatory Human lymphocytic cancer 24 150 to Hep G2 cells mediators Jurkat cells 65 to Colon 205 Human hepatoma Hep G2 cells cells 95 to MCF-7 cells Human colon cancer Colon 205 cells Human breast MCF-7 cells Methanol 40 Inhibit macrophage-mediated inﬂammatory Human lymphocytic cancer 24 mediators Jurkat cells

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

Mycelia a 2þ Mycelia powder 100e200 Effects on apoptosis, [Ca ]i, and MAPKs Human osteosarcoma 26 phosphorylation MG63 cells a 2þ Mycelia powder 100e200 Effects on apoptosis and [Ca ]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 Human hepatoma Hep G2 cells 28 62.7 to Hep 3B cells caspase-3 and -8 cascades 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 Pheochromocytoma PC-12 cells 31 suppress the activity of JNK and p38

Extracts from solid-state fermentation Ethanol extracts Not shown Downregulate human galectin-1, human Human non-small cell lung 32 eukaryotic translation initiation factor 5A, carcinoma A549 cells human Rho GDP dissociation inhibitor a, human calcium-dependent protease small subunit, and human annexin V

Fermented culture broth Fermented culture 0e240b Reduce cyclin D1, cyclin E, CDK4, cyclin A, and Human breast cancer 33 broth proliferating cell nuclear antigen, and increase MDA-MB-231 cells CDK inhibitor p27/KIP and p21/WAF1 Fermented culture 55 and 110 mg/kgb Inhibit proliferation (cyclin D1 and PCNA) and Nude mice inoculated with 33 broth induce apoptosis (Bcl-2 and TUNEL) MDA-MB-231 cells Fermented culture 316 to MF-7 cells Loss of chromatin condensation, Human breast cancer MCF-7 34,35 broth 136 to MDA- internucleosomal DNA fragmentation, release MDA-MB-231 cells MB-231 cells of cytochrome c, activation of caspase 3, and speciﬁc proteolytic cleavage of PARP, and inhibition of COX-2

MMP ¼ 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. a means effective concentrations (microgram/milliliter); b means tested dose (microgram/milliliter).

2þ 16 cells, exerted multiple effects on [Ca ]i via inhibition of extra- Hseu et al also ﬁltered mycelia with water before being air- cellular signalregulated kinase (ERK) mitogen-activated protein dried. Oxidative hemolysis and lipid/protein peroxidation of kinase (MAPK) phosphorylation.26 As to prostate cancer PC3 cells, erythrocytes induced by the aqueous peroxyl radical and the 2þ it also worked on viability and [Ca ]i, caused apoptosis via endothelial cell damage induced by the aqueous peroxyl radical 2þ pathways unrelated to [Ca ]i signal, and phosphorylation of ERK, [2,2V-Azobis(2-amidinopropane) dihydrochloride] were sup- c-Jun N-terminal kinases (JNK), and p38 MAPKs.27 pressed by aqueous T. camphoratus extract from mycelia, and it also Anticancer effects of Taiwanofungus camphoratus 277 prevented the depletion of cytosolic antioxidant glutathione and (adenosine diphosphate (ADP)-ribose) polymerase (PARP), and adenosine triphosphate in erythrocytes. dysregulation of Bcl-2 and Bax. Furthermore, fermented culture broth of T. camphoratus inhibited cyclooxygenase (COX)-2 protein 2.8. Methanol extracts of mycelia expression and prostaglandin E2 production in MDA-MB-231 cells.

Like the fruiting bodies, mycelia could be extracted with different 3. Anticancer Activities of Isolated Compounds From T. solvents. The methanol extracts of mycelia were obtained by camphoratus filtering and then concentrating to dryness. Cell cycle analysis revealed that methanol extract of mycelia induced apoptosis on So far, there have been 78 compounds isolated from T. camphoratus, Hep G2 cells in G0/G1 cell cycle through the activation of caspase-3 which describes its complexity and difficulty in research. Terpe- and -8 cascades and regulation of the cell cycle progression to noids predominate the fruiting body (39 compounds) and has been e inhibit hepatoma cells proliferation.28 Furthermore, the author the focus of many pharmacological studies.18,36 39 Nevertheless, indicated that the mechanism could be Fas/Fas ligand death- several other components were also identified comprising benze- receptor pathway through upregulation of Fas and downregulation noids,15,40 maleic/succinic acid derivatives,6 polysaccharides,41,42 of Bcl-2, DR3, DR4, tumor necrosis factor receptor (TNFR)I, and sterols,25 and sesquiterpene lactone37 as well. The details of anti- TNFRII in Hep G2 cells.29 cancer compounds will be discussed below and tabulated in Table 2. 2.9. Ethanol extracts of mycelia 3.1. Terpenoids The mycelia were extracted with 95% ethanol at 30C for 24 hours, and the filtrates were dried under vacuum. Ethanol extracts of The bitter taste with camphor aroma of T. camphoratus is because of mycelia have antiproliferation against Hep G2 and Hep 3B cells.30 terpenoids that have been considered as potential anticancer Lu et al31 found that it also prevented serum deprivation-induced agents. Ethanol extract from the fruiting body was further extracted PC12 cell apoptosis via a protein kinase A-dependent pathway and with solvents of increasing polarity (n-hexane, ethyl acetate, and by suppressing the activities of JNK and p38. ethanol). Ethyl acetate fraction was subsequently chromatographed using Sephadex LH-20 with CHCl3-MeOH (1:3) to get three frac- 2.10. Extracts from solid-state fermentation tions, while Fraction 2 was separated by thin layer chromatography with CHCl3-MeOH (25:1) to obtain eight fractions, and zhankuic Air-dried T. camphoratus mycelia grown by solid-state fermentation acid A was then purified by octadecylsilica (ODS) high-performance were ground in liquid nitrogen and shaken with 100% ethanol at liquid chromatography column. Zhankuic acid A exhibited signifi- a ratio of 1:5 w/v overnight at room temperature. Then, the cant cytotoxicity to HL 60 cells (IC50 ¼ 5.45 mg/mL) in causing suspension was centrifuged, and the supernatant was filtered antiproliferation, death, and apoptotic induction, and Lu et al21 through a 0.2-mm-pore size filter. The filtrate was evaporated to suggested zhankuic acid A was the major component of ethanol one-fifth of the original volume to collect the extract. Extract from extract from the fruiting body. solid-state fermentation can effectively interrupt the proliferation Zhankuic acid A could be isolated from other methods. Dried of human non-small cell lung carcinoma A549 cells by triggering T. camphoratus was ground to powder and extracted with ethanol. the apoptosis through the induction of endoplasmic reticulum The concentrated extracts were partitioned between ethyl acetate stress and downregulation of human galectin-1, human eukaryotic and water. The ethyl acetate fraction was chromatographed by translation initiation factor 5A, human Rho GDP dissociation Sephadex LH-20 column with ethanol as an eluent to yield four inhibitor a, human calcium-dependent protease small subunit, and fractions. The third fraction was further isolated by a Si gel column 32 human annexin V. and eluted with CHCl3 and increasing concentrations of methanol to provide 14 fractions. Fraction 2 was purified by Si gel to give 2.11. Fermented culture broth of T. camphoratus zhankuic acid A, and zhankuic acids B and C were obtained from Fractions 4 and 7, respectively. While zhankuic acids A and C Not only do the fruiting bodies and mycelia have anticancer effects exhibited cytotoxicity against P-388 murine lymphocytic leukemia 25 but also the fermented culture broth inhibits the growth of cancer cells with IC50 values of 1.8 and 5.4 mg/mL, respectively. cells. The viability of MDA-MB-231 cells (human breast cancer cell Yeh et al43 extracted air-dried powder of T. camphoratus with line) was impeded by fermented culture broth in the G1 phase CHCl3 after solvent evaporation, and the residue was separated by associated with the reduction in cyclin D1, cyclin E, CDK4, cyclin A, silica gel column chromatography with increasing polarity using proliferating cell nuclear antigen, and the increase of cyclin- mixtures of n-hexane/ethyl acetate to elute. Following the thin dependent kinases (CDK) inhibitor p27/KIP and p21/WAF1. More- layer chromatography analysis, antcinate B, zhankuic acid A, and over, it was also effective in the breast cancer nude mice inoculated zhankuic acid C were isolated, and they displayed the tumor- with MDA-MB-231 cells to delay tumor incidence and reduce the specific cytotoxicity with an IC50 range from 22.3 to 75.0 mM against tumor size. When the tumor tissue sections were examined the colon, breast, liver, and lung cancer cell lines in sub-G1 cell cycle histologically and immunohistochemically, the inhibition of the through apoptosis induction by the cleavage of the downstream proliferation (cyclin D1 and PCNA) and induction of apoptosis (Bcl- poly(ADP-ribose) polymerase, procaspase-3, and Bcl-2, while 2 and terminal deoxynucleotidyl transferase-mediated dUTP nick methyl antcinate B was slightly potent. Furthermore, these three end-labeling (TUNEL)) were observed.33 compounds demonstrated synergistic cytotoxic effect (4 mM each) In another study, treatment of fermented culture broth of in colon cancer HT-29 cells. T. camphoratus with MCF-7 (25e150 mg/mL)34 and MDA-MB-231 (40e240 mg/mL)35 resulted in a dose- and time-dependent 3.2. Maleic and succinic acids derivatives sequences by apoptosis, as represented by loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation, Dried mycelia was extracted with methanol at room temperature; and sub-G1 phase accumulation, the release of cytochrome c, the crude methanol syrup was evaporated under reduced pressure activation of caspase-3 and specific proteolytic cleavage of poly and then partitioned with n-hexane:H2O (1:1) three times. The n- 278 Y.-C. Chen et al.

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 Human leukemia HL 60 cells21 21,25,43 1.825 the expression of apoptosis- Murine lymphocytic leukemia associated proteins43 P-388 cells25 22.3e75.0 mMa,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 P-388 murine lymphocytic 25,43 apoptosis-associated proteins43 leukemia cells25 22.3e75.0 mMa,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 22.3e75.0 mMa Suppress the expression of Human colon (HT-29, HCT116, 43 apoptosis-associated proteins SW480), breast (MCF-7, MDA- MB-231), liver (Huh 7, Hep G2, Hep 3B), and lung (A549, CL1-0) cancer cell lines43 Combined use of 4.0 mM (each) Suppress the expression of Colon cancer HT-29 cells 43 zhankuic acid A, apoptosis-associated proteins zhankuic acid C, and methyl antcinate B Antrodins B 3.6b Unknown Murine macrophage cell line 5 RAW264.7 Antrodins C 7.5b Unknown Murine macrophage cell line 6 RAW264.7 Polysaccharide 100 Activation of mononuclear cells Human leukemic U937 cells 7 Polysaccharide 100 mg/kg Regulate some Sarcoma 180-bearing mice 7 (intraperitoneal immunoparameters administration) and 200 mg/kg (oral administration)c Polysaccharides 35.2 Inhibit cyclin D1 expression Endothelial cells 44 Sulfated Not shown Antiangiogenic and Matrigel tube formation serum 45 polysaccharides neuroprotective effects deprivation-induced apoptosis in neuronal-like PC12 cell 4,7-dimethoxy- 1.721 to MCF-7, Unknown Human breast cancer MCF-7 46 5-methyl-1,3- 0.992 to MDA-MB- and MDA-MB-231 cells, human benzodioxole 231, 0.016 to Hep hepatoma Hep 3B and Hep G2, 3B, 2.462 to Hep G2, human prostate cancer cells 4.46 to LNCap, and LNCaP, and human prostate 2.21 to DU-145 cell cancer DU-145 cells lines

a b c Means effective concentration; Means ED50 (EC50 represents the plasma concentration required for obtaining 50% of a maximum effect); Means tested dose.

hexane layer extract was chromatographed by a silica gel column oral administration of polysaccharide (100 and 200 mg/kg, with a stepwise gradient of n-hexane and ethyl acetate to give 15 respectively) significantly suppressed the tumor growth to 69.1% fractions. Fraction 5 was further purified by column chromatog- and 58.8%, respectively, in sarcoma 180-bearing mice by regulating raphy on silica gel eluting with n-hexane and CH2Cl2 to yield six some immunoparameters, such as the spontaneous proliferation of fractions, and antrodins B and C were isolated. These two mal- spleen cells, the cytolytic activity of spleen cells, and serum inter- eimide derivatives inhibit the viability of murine macrophage cell leukin-12 levels. Liu et al7 suggested that polysaccharides from T. line RAW264.7 to more than 30%.6 camphoratus reveal its anticancer effect by promoting a Th1- dominant state and killer activities. 3.3. Polysaccharides Cheng et al44 extracted lyophilized mycelia with 80C water in a 1:100 (w/w) ratio for 6 hours. The extracts were cooled, and To obtain polysaccharide, mycelia of T. camphoratus were air-dried, quadruple volumes of 95% ethanol were added and then stored disintegrated, and extracted with boiling water for 8e12 hours. The overnight at 4C. The brownish precipitated polysaccharides were insoluble matter was removed, and water-soluble polysaccharide- collected by centrifugation and lyophilized. The T. camphoratus enriched fraction was isolated by ethanol precipitation. The crude polysaccharides inhibit cyclin D1 expression in endothelial cells polysaccharides were then filtered through a Sephadex G50 gel through inhibition of vascular endothelial growth factor receptor filtration column and were further purified by an anion exchange signaling, leading to the suppression of angiogenesis. column of diethylaminoethyl cellulose. The polysaccharide In addition to extraction with water, lyophilized mycelia were component alone did not show any direct cytotoxic effect to human also extracted with 0.1M sodium acetate (pH 5.5) buffer containing leukemic U937 cells; however, it could inhibit 55.3% growth rate of 5 mM cysteine, 100 mg papain as a digestion agent, and 5 mM EDTA U937 cells via activation of mononuclear cells. Intraperitoneal and at 60C for 24 hours. Supernatants were collected after Anticancer effects of Taiwanofungus camphoratus 279

Table 3 Synergistic anticancer activities of Taiwanofungus camphoratus

Extract of T. camphoratus Adjuvant agents Mechanism Experimental model Reference Solid-state cultured Cisplatin (100 mM), Inhibit MDR gene expressions Human hepatoma C3A and 47 T. camphoratus mycelia mitomycin and the pathway of COX- PLC/PRF/5 cells (1 mg/mL) (100 mM) 2-dependent inhibition of AKT phosphorylation Solid-state cultured Cisplatin Inhibit MDR gene expressions PLC/PRF/5 cells-implanted 47 T. camphoratus mycelia (1 mg/kg/wk) and the pathway of COX- nude mice (200 mg/kg/d) 2-dependent inhibition of AKT phosphorylation 4,7-dimethoxy-5-methyl-1,3- Lovastatin Unknown Human hepatoma Hep 3B cells 46 benzodioxole (0.0007 mg/mL) (0.0043 mg/mL) 4,7-dimethoxy-5-methyl-1,3- Taxol Unknown Human breast cancer MCF-7 46 benzodioxole (0.0007, (0.0017 mg/mL) and MDA-MB-231 cells, human 0.0009, 0.0129, 1.16, hepatoma Hep G2, human and 0.71 mg/mL) prostate cancer LNCaP cells, and human prostate cancer DU-145 cells Ethanol extract from the Lovastatin (2 mM) Unknown Human hepatoma Hep G2, 48 fruiting body (20 mg/mL) human glioma DBTRG 05MG, human lung adenocarcinoma CL1-0, and rat pheochromocytoma PC12 cells Ethanol extract from the Trichostatin A Increase cytotoxic sensitivity Human leukemia HL 60 cells 21 fruiting body (100 mg/mL) (100 nM) of trichostatin, upregulation of DR5, and NFkB activation centrifugation, and the same buffer was added to the precipitate for (200 mg/kg/d) with cisplatin (1 mg/kg/wk) for 21 consecutive days another 24 hours at 60C. The supernatants of the two extractions significantly extended the median survival days of PLC/PRF/5 cells- were collected, and a 3.75-fold volume of 95% ethanol was added, implanted imprinting control region (ICR) nude mice. T. cam- precipitated at 4C overnight, then spun, and the pellets were phoratus extract showed its adjuvant effects through the inhibition collected. The pellets were dried and resuspended in distilled of multidrug resistance (MDR) gene expressions and pathway of water, dialyzed (molecular weight: 12,000e14,000 Da) against COX-2-dependent inhibition of AKT phosphorylation (p-AKT), distilled water overnight at 4C, and centrifuged to collect the which ultimately resulted in the induction of apoptosis in hepa- supernatant as sulfated polysaccharides. It is the first time to report toma cells.47 that the sulfated polysaccharides inhibit in vitro Matrigel tube US patent46 illustrated that synergistic effects of taxol (0.0017 formation in an angiogenesis model. Furthermore, using serum mg/mL) and 4,7-dimethoxy-5-methyl-1,3-benzodioxole inhibited deprivation-induced apoptosis in neuronal-like PC12 cells as the viability of MCF-7, MDA-MB-231, Hep G2, LNCaP, and DU-145 a stress model, the sulfated polysaccharides were effective in pre- cells significantly. In addition, lovastatin (0.0043 mg/mL) and 4,7- venting serum-deprived apoptosis.45 dimethoxy-5-methyl-I,3-benzodioxole (0.0007 mg/mL) caused the death of half of Hep 3B human liver cancer cells. Therefore, Liu et al 3.4. Miscellaneous compounds with anticancer activities suggested that 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from T. camphoratus extract shows more inhibitory effects with US patent46 disclosed that 4,7-dimethoxy-5-methyl-1,3-benzo- lovastatin and taxol. dioxole inhibited the cell number of MCF-7, MDA-MB-231, Hep 3B, US patent48 disclosed that a method of treating cancer with an Hep G2, LNCaP human prostate cancer cells, and DU-145 human effective amount of a mevalonate pathway inhibitor and T. cam- prostate cancer cells with IC50 of 1.721, 0.992, 0.016, 2.462, 4.46, and phoratus extract. Pulverized T. camphoratus was suspended in 95% 2.21 mg/mL, respectively. The above IC50 values are lower than the ethanol, and the suspension was stirred at room temperature for IC50 value of whole T. camphoratus extract (11.461, 26.812, 6.112, 48 hours and then filtered to remove insoluble residue. The 18.931, 45.47, and 30.15 mg/mL, respectively). Therefore, it is filtrate was collected and concentrated by a rotary evaporator at confirmed that 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated 40e45C. The concentrate was evaporated to dryness under from T. camphoratus extract is capable of inhibiting cell prolifera- nitrogen at room temperature. Details indicated that mevalonate tion of breast cancer, liver cancer, and prostate cancer. pathway inhibitor, for example, lovastatin, and T. camphoratus extract exhibited profound synergistic effect in inhibiting cell 4. Synergistic Anticancer Activities of T. camphoratus growth of Hep G2, DBTRG 05MG (human glioma cell line), CL1- 0 (human lung adenocarcinoma cell lines), and PC12 (rat pheo- Interestingly, T. camphoratus has more potent cytotoxicity when it chromocytoma cell line). For example, lovastatin (2 mM) and T. is combined with other agents, such as trichostatin A (TSA), lova- camphoratus extract (20 mg/mL) inhibited growth of Hep G2 cells statin, and taxole. The synergistic effects are discussed in the only by 11.5% and 2.8%, respectively, whereas their combination following mention and Table 3. inhibited the growth by 99.3%. In addition, the cells surviving Solid-state cultured T. camphoratus mycelia were extracted in from the combinational treatment showed markedly shrunk the ratio of 1:10 (v/v) with 95% ethanol at room temperature for morphology. 24 hours and then centrifuged. Supernatant was removed and The combined use of 100 mg/mL ethanol extract from the fruiting filtered. In C3A and PLC/PRF/5 hepatoma cell model, T. camphor- body and 100 nM of TSA (a potent reversible inhibitor of histone atus extract (1 mg/mL) showed prominent adjuvant inhibitive acetylase) caused synergistic inhibition of cell growth compared with effects on proliferation when it was combined with cisplatin (100 single TSA or T. camphoratus treatment to HL 60 cells. T. camphoratus mM) or mitomycin (100 mM). Furthermore, in xenografted cancer could induce apoptosis through the increase of cytotoxic sensitivity animal model, the combined treatment of T. camphoratus extract of TSA, upregulation of DR5, and NFkB activation.21 280 Y.-C. Chen et al.

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