樟芝為台灣特有寄生於牛樟樹的真菌，已有很多研究證實，它具有抗癌及保肝等多樣化的藥理作用，但抗肺、膀胱癌及酒精性傷肝機制的動物實驗，尚未有文獻加以探討，故本研究分別以三部分的動物實驗，來探討樟芝的這些抗癌與保肝的能力。亦即，先以菌絲體萃取液的抗肺癌及其減少肺轉移之功效，繼而採用野生子實體抗肺癌及膀胱癌與DNA疫苗合併抗癌能力，最後以野生子實體來研究減少酒精對肝臟受損的能力。

第一部分
樟芝抗肺癌之研究只有著力於細胞和其蛋白質體之研究，本實驗擬以液態培養之樟芝菌絲體甲醇萃取液(ACME)作為研究材料，探討它對小鼠肺腫瘤之抗癌性和其抑制轉移功效之研究。以HPLC圖譜發現ACME，在275 nm波長有數個主要成分。經實驗證實ACME對人類肺腺癌細胞(A549)和小鼠路易斯肺癌細胞(LLC)具有毒殺作用。以流式細胞儀發現，ACME可使癌細胞停留在細胞週期的次G1期。以細胞移行試驗轉移盤(Transwell)發現，ACME有抑制細胞轉移的作用(p＜0.05)，也有抑制金屬間質蛋白質-9 (MMP-9)分泌的作用(p＜0.001)。以小雞胚胎絨毛膜血管(CAM)和血管內皮細胞之指標蛋白CD31分析，證實ACME具有抑制血管新生的作用。最後以動物實驗證實，ACME經腹腔投予，可顯著的減少小鼠原位肺癌腫瘤的體積和延長肺癌小鼠的生命，並減少腫瘤細胞轉移到肺臟產生結節的數量。由此實驗證實ACME對小鼠荷LLC癌細胞，具有抑制及轉移能力。

第二部分
癌症的治療主要以化學療法的促進癌細胞凋亡和抑制血管新生的方法，但疫苗來治療癌症，目前為運用於癌症的新療法。本研究擬以樟芝與DNA疫苗合?來抗癌，並瞭解其抗癌機制。本實驗為野生樟芝(WAC)提取物為材料，經口腔投予負荷原位膀胱癌的小鼠，加以探討WAC單獨或合併HER-2/neu DNA的DNA疫苗的抗癌性和其免疫性質。實驗證實疫苗和合併WAC可延長膀胱癌小鼠的生命，其抗癌作用歸因於此合併組合加強了Th-1細胞的免疫反應作用。證據來自小鼠腹股溝淋巴結的CD4+ T細胞，此細胞進而誘使分泌IFN-? mRNA之表現增加。而促使HER-2/neu有特異性的CTLs細胞也增加，進而增加浸潤到腫瘤裏的CD4+ 和 CD8+ T細胞數量；如消減此等細胞的增加，即可去除疫苗-WAC組合的療效。小鼠經WAC處理後，實驗證實可增加樹突細胞的活性及使淋巴結內的Th1細胞誘導細胞激素如IL-12和IFN-?，此等細胞激素的增加，即足以促使T細胞因ErbB2之作用，而導致細胞增生和產生較高量的IFN-?。由此實驗證明WAC可作為免疫調解輔佐劑之用，可以加強HER-2/neu DNA小鼠荷膀胱癌之抑制作用。

第三部分
樟芝具有保護肝臟功能，已有多篇研究被證實，但飲用酒精會促進脂肪肝產生，尚未有研究樟芝是否可解除脂肪及解酒功能的研究，本研究擬以大鼠來探討此功能的機制。本研究為先讓飲用水中含有20%酒精，誘導大鼠產生酒精性脂肪肝。然後，在飲用期間每日經口腔投予，給予每大鼠公斤體重0.25 g的silymarin及0.025 g的WAC，來試著分析其酒精性傷害肝臟程度，並瞭解樟芝是否可保護肝臟傷害的能力。由實驗結果，發現WAC可降低肝臟中膽固醇及脂肪的含量(p＜0.05)，可改變下列酒精傷肝代謝有關基因的表現活性(p＜0.05)，如增加乙醇去氫酵素(ADH)和乙醛去氫酵素(ALDH)表現，以及不影響細胞色素酵素(CYP2E1)，核因子-κB (NF-κB)，α-平滑肌蛋白(α-SMA)表現，和降低類Kruppel-6因子(KLF6)等基因表現，降低MMP-9含量等蛋白，WAC也可增加DPPH及還原力的抗氧化作用，可加強肝中的麩胱甘?(GSH)，Trolox當量的總抗氧化能力(TEAC)，降低肝中丙二醛(MDA)含量，和降低血中轉胺酵素(AST)的活性。肝組織切片觀察發現受損程度大為減輕，本實驗證實樟芝可減輕酒精性傷肝的能力。

Antrodia camphorata (AC), a native tree fungus to Taiwan, has been published recently for multiple medical purposes as an effective treatment as folk medicine for cancer therapy and hepatoprotection. However, no reference was published for investigating the function utilizing animal model for lung cancer anti-metastasis, bladder cancer anti-tumor and proection alcohol-induced liver damage. There are three parts for investigating the pharmacolical function. An anticancer effect of cultivated AC mycelia was first investigated on lung cancer. To analyzed promoting effect of wild fruiting body of AC (WAC) co-treatment on the anti-tumor efficacy of DNA vaccine. Last, to evalute of WAC protection for livers against chronic damage due to alcohol consumption.

Part I
A methanol extract procedure for AC cultured mycelia (ACME) was developed and optimized for this study. The results of HPLC analysis showed that ACME is a mixture of many components around the main absorbance peak at 275 nm. The ACME is cytotoxic for Lewis lung carcinoma cells (LLC) and human lung adenocarcinoma epithelial cells (A549) in vitro. The cell cycle was arrested at the sub G1 phase by ACME using the flow cytometry analysis (p＜0.01). ACME inhibited the migrations of LLC and A549 cancer cells (p＜0.01) in a transwell assay system and the activity of matrix metalloproteinase-9 (MMP-9) of A549 cells is reduced in a migration assay (p＜0.001). The blood vessel formation was suppressed by ACME in chicken embryo chorioallantoic membrane study. Taken altogether, solid tumors growth in animals with LCC lung tumors was significantly inhibited and the number of metastasized lung nodule in LLC mice was reduced by ACME.

Part II
The goal of this study was to investigate whether wild fruiting body of A. caphorata (WAC) is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with WAC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine–WAC combination. Evidence for this came from 1) the marked increase in the IFN-? mRNA expression in CD4+ T cells in the draining inguinal lymph nodes, 2) an increase in the number of functional HER-2/neu-specific CTLs, and 3) the increased tumor infiltration of both CD4+ and CD8+ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine–WAC therapy. Our results further indicate that treatment of mice with WAC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-α) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-γ production in response to ErbB2. Overall, these results clearly demonstrate that WAC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.

Part III
An alcoholic fatty liver disease was induced in rats by feeding drinking water containing 20% (w/w) alcohol. The therapeutic groups were either orally administered with silymarin or WAC. The treatment of WAC not only showed a hypo-cholesterolemic effect (p＜0.05) but also reduced (p＜0.05) hepatic lipids in alcohol-fed rats. These beneficial effects could be elevated gene expressions (p＜0.05) include ADH (alcohol dehydrogenase), ALDH (aldehyde dehydrogenase) and CYP2E1 outputs, α-SMA (alpha-smooth muscle actin), NF-κB (nuclear factor-κB), KLF6 (Kruppel-like factor 6) and MMP-9 protein. Moreover, WAC treatment can promote 1-diphenyl-2-picrylhydrazyl (p＜0.05), antioxidation rate in vitro, liver glutathione content, Trolox equivalent antioxidant capacity, concomitantly decreased the liver malondialdehyde content and lowered (p＜0.05) serum aspartate aminotransferase levels. Finally, the results of histological examination of WAC-fed rats showed that liver damage was reduced significantly.
In summary, A. camphorata is a promising Taiwanese fungus for medicinal purposes with potential applications for anti-cancer and hepatoprotective, and deserived further attention for research and development for either biotech products or phamaceutics.

Abstract I
Abstract in Chinese III
Acknowledgements V
Contents VI
List of tables X
List of figures XI
Abbreviations XIV
Chapter 1 Introduction 1
1.1 Motivations 1
1.2 Study framework 2
1.3 Literature reviews 3
1.3.1 What is Antrodia camphorata 3
1.3.2 Classification and morphology 3
1.3.3 Chemical constituents 4
1.3.4 Pharmacological effects 5

Chapter 2 Anticancer activities of artificially cultivated Antrodia camphorata mycelia (ACM)--methanolic extracts on lung cancer 9
2.1 Introduction 9
2.2 Materials and methods 11
2.2.1 Reagents 11
2.2.2 Instruments 12
2.2.3 Preparation of ACM 12
2.2.4 Chemical profiles of ACM 13
2.2.5 Determination of total triterpenoids 13
2.2.6 Determination of total water-soluble polysaccharides 14
2.2.7 Cell line and animals 14
2.2.8 Cytotoxicity assay 15
2.2.9 Cell cycle analysis 15
2.2.10 Cancer cell migration assay 15
2.2.11 Gelatin zymography 16
2.2.12 Solid tumor growth and metastasis inhibitions in vivo 16
2.2.13 Statistical analysis 17
2.3 Results 17
2.3.1 Determination of extraction time and solvent 17
2.3.2 HPLC analysis and contents of triterpenoids, polysaccharides 17
2.3.3 Cytotoxicity in vitro through apoptosis 18
2.3.4 Cell migration inhibitions 20
2.3.5 Gelatin zymography 20
2.3.6 Inhibition of solid tumor growth and metastasis in vivo 25
2.4 Discussion 25

Chapter 3 Promoting effect of wild fruiting body of Antrodia camphorata (WAC) as an immunomodulating adjuvant on the anti-tumor efficacy of HER-2/neu DNA vaccine 32
3.1 Introduction 32
3.2 Materials and methods 34
3.2.1 Reagents 34
3.2.2 Instruments 35
3.2.3 Cell line and animals 36
3.2.4 Preparation of aqueous extracts of WAC 36
3.2.5 MTT assay 36
3.2.6 Annexin V staining 36
3.2.7 TUNEL assay 36
3.2.8 Preparation of the HER-2/neu DNA vaccine 37
3.2.9 Therapeutic efficacy in a mouse model of established MBT-2 tumors 37
3.2.10 In vitro cytotoxicity assay 37
3.2.11 In vivo cytotoxicity assay 38
3.2.12 Intracellular cytokine staining and flow cytometry 38
3.2.13 Quantitative RT-PCR 38
3.2.14 Histological analysis of tumor-infiltrating lymphocytes 39
3.2.15 Depletion of subset T cells in vivo 39
3.2.16 Isolation of CD11c+ DCs 39
3.2.17 Surface molecule expression on and cytokine production by CD11c+ DCs 40
3.2.18 In vitro DC function assays 40
3.2.19 Statistical analysis 41
3.3 Results 41
3.3.1 WAC-mediated cytotoxicity and apoptosis in MBT-2 cells in vitro but not in vivo 41
3.3.2 WAC augments the antitumor effect of the HER-2/neu DNA vaccine 42
3.3.3 WAC enhances p185neu-specific cellular immune responses induced by the HER-2/neu DNA vaccine 47
3.3.4 WAC augments Th1-type immune responses induced by the HER-2/neu DNA vaccine 51
3.3.5 HER-2/neu DNA vaccine-WAC enhances CD4+ and CD8+ T cell infiltration into tumors 51
3.3.6 The anti-tumor effect of the HER-2/neu DNA vaccine-WAC combination requires CD4+ and CD8+ T cells 60
3.3.7 HER-2/neu DNA vaccine-WAC enhanced draining inguinal LN DC activation, IL-12 and IFN-alpha production, and Th1-like cellular immune responses 60
3.4 Discussions 65

Chapter 4 Wild fruiting body of Antrodia camphorata (WAC) protects livers against chronic damage of alcohol consumption 78
4.1 Introduction 78
4.2 Materials and methods 81
4.2.1 Reagents 81
4.2.2 Instruments 83
4.2.3 Anatomical microscope of WAC 83
4.2.4 Identification of WAC DNA from internal transcribed spacer region 83
4.2.5 Chemical profiles of WAC 84
4.2.6 Fungal material 85
4.2.7 Polysaccharides content 85
4.2.8 Determination of total triterpenoids 86
4.2.9 Determination of polyphenols content 87
4.2.10 Determination of total flavonoids content 87
4.2.11 Determination of condensed tannins 87
4.2.12 Free radical scavenging activity (DPPH) determination 88
4.2.13 Test for reducing power 88
4.2.14 Animal and diets 88
4.2.15 Collection of serum, liver, visceral fat 89
4.2.16 Determination of serum cholesterol and triacylglycerol and liver damage
indices 89
4.2.17 Determination of hepatic cholesterol, triacylglycerol 90
4.2.18 Gene expressions of hepatic alcohol metabolism and profibrosis 90
4.2.19 Preparation of liver homogenate 91
4.2.20 Determination of hepatic malondialdehyde (MDA) and contents as well as trolox equivalent antioxidant capacity (TEAC) 91
4.2 21 Gelatin zymography 92
4.2.22 Statistical analysis 93
4.3 Results 93
4.3.1 Identification of WAC from fungal morphology, DNA and chemical profiles 93
4.3.2 Contents of total polysaccharides, total triterpenoids, phenolics and flavornoids in WAC 96
4.3.3 DPPH radical scavenging activity of WAC 96
4.3.4 Reducing power of WAC 98
4.3.5 Effects of WAC on growth performances, serum, liver 98
4.3.6 Effects of WAC on gene expressions of hepatic alcohol metabolism, profibrosis and its MMPs protein expression 99
4.3.7 Effects of WAC on liver antioxidant capacity and liver damage 99
4.4 Discussions 107

Conclusions 117
References 119
Appendix 139

List of tables
Table 1-1 Compounds isolated from AC 140
Table 2-1 Extraction efficiency of methanol at various times 17
Table 2-2 Extraction efficiency of various solvents 18
Table 2-3 Total triterpenoids and polysaccharide contents of ACME 20
Table 2-4 Cytotoxicity of ACME on differential cell lines—the IC50 values 20
Table 3-1 Tumor-infiltrating lymphocytes in cryosectioned samples 64
Table 3-2 Inhibiton ratio of p.o. administration AC to mice bearing lung tumor 77
Table 4-1 Total polysaccharide, triterpenoids, flavonoids, phenolics and condensed tannins content of WAC 96


List of figures
Fig. 2-1 Metastasis cascade of tumors 10
Fig. 2-2 Chromatographic spectrum of varios solvent extracts of Antrodia camphorata mycelial powder 19
Fig. 2-3 Cytotoxicity of ACME in vitro 21
Fig. 2-4 Cell cycles of ACME 22
Fig. 2-5 Invasion assay of ACME 23
Fig. 2-6 Gelatin zymography of ACME 24
Fig. 2-7 Inhibition of solid tumor growth were administrated p.o. ACME 26
Fig. 2-8 Inhibition of solid tumor growth were administrated i.p. ACME 27
Fig. 2-9 Percent survival of solid tumor growth were administrated i.p. ACME 28
Fig. 2-10 Anti-metastasis of LLC lung was administred i.p. ACME 29
Fig. 3-1 The ErbB signaling network 33
Fig. 3-2 WAC reduces growth in MBT-2 cancer cell lines 43
Fig. 3-3 WAC induces apoptosis in MBT-2 cancer cell lines 44
Fig. 3-4 WAC did not significantly alter survival in MBT-2 tumor-bearing C3H/HeN mice 45
Fig. 3-5 WAC induces apoptosis in MBT-2 cancer cell lines 46
Fig. 3-6 Design of WAC and HER-2/neu DNA vaccination in MBT-2 tumor-carrying C3H/HeN mice 48
Fig. 3-7 Tumor volume of MBT-2 tumor–carrying C3H/HeN mice were administrated WAC and HER-2/neu DNA vaccination 49
Fig. 3-8 Percent survival of MBT-2 tumor–carrying C3H/HeN mice were administrated WAC and HER-2/neu DNA vaccination 50
Fig. 3-9 WAC-mediated CTL activation in spleen from hN’-neu vaccinated C3H/HeN mice 52
Fig. 3-10 WAC-mediated CTL activation in draining LN cells from hN’-neu vaccinated C3H/HeN mice 53
Fig. 3-11 WAC-mediated increase of HER-2/neu specific functional CD8+ T cells in spleen cells from hN’-neu vaccinated C3H/HeN mice 54
Fig. 3-12 WAC-mediated increase of HER-2/neu specific functional CD8+ T cells in draining LN cells from hN’-neu vaccinated C3H/HeN mice 55
Fig. 3-13 WAC-mediated HER-2/neu specific functional CD8+ T cells in spleen cells from hN’-neu vaccinated C3H/HeN mice were quantified by flow cytometry 56
Fig. 3-14 WAC-mediated HER-2/neu specific functional CD8+ T cells in draining LN cells from hN’-neu vaccinated C3H/HeN mice were quantified by flow cytometry 57
Fig. 3-15 Recombinant human ErbB20-pulsed splenocytes were used as target cells to assess in vivo CTL activity 58
Fig. 3-16 Recombinant human ErbB20-pulsed draining inguinal LNs were used as target cells to assess in vivo CTL activity 59
Fig. 3-17 Flow cytometric analysis of HER-2/neu-specific CD4+ cells that secrete IFN-gamma and IL-4 in vaccinated mice 61
Fig. 3-18 Expression of IFN-gamma and IL-4 mRNAs in HER-2/neu-specific CD4+ T cells from vaccinated mice by quantitative real-time RT-PCR analysis 62
Fig. 3-19 Tumor infiltrations of CD4+ and CD8+ T cells 63
Fig. 3-20 The effect of CD4+ or CD8+ T cell depletion on the therapeutic effects of co-administration of WAC and HER-2/neu DNA vaccination in MBT-2 tumor-carrying C3H/HeN mice 66
Fig. 3-21 Phenotypic characterization of CD11c+ DCs purified from draining inguinal LNs, spleen, or the tumor site in the different groups of mice 67
Fig. 3-22 IL-12 p40 mRNA expression of CD11c+ DCs purified from draining inguinal LNs, spleen, or the tumor site in different groups of mice 68
Fig. 3-23 IFN-alpha mRNA expression of CD11c+ DCs purified from draining inguinal LNs, spleen, or the tumor site in different groups of mice 69
Fig. 3-24 Proliferative capacity from tumor infiltrating DCs in different groups of mice 70
Fig. 3-25 Proliferative capacity from splenic DCs in different groups of mice 71
Fig. 3-26 Proliferative capacity was analyzed from draining inguinal LNs DCs in different groups of mice 72
Fig. 3-27 Analysis of IFN-gamma cytokine production from tumor infiltrating DCs in Different groups of mice 73
Fig. 3-28 Analysis of IFN-gamma cytokine production from splenic DCs in different groups of mice 74
Fig. 3-29 Analysis of IFN-gamma cytokine production from draining LN DCs in different groups of mice 75
Fig. 4-1 Schematic diagram of the major pathways of alcoholic fatty liver and Lipotoxicity 80
Fig. 4-2 Identification of WAC by morpholgical observation and anatomical microscopy 94
Fig. 4-3 Identification of WAC was confirmed by ITS region of the ribosomal RNA gene (rDNA) 95
Fig. 4-4 Identification of WAC was confirmed by chemical profile 97
Fig. 4-5 Scavenging activities of WAC on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals 100
Fig. 4-6 Reducing power of WAC 101
Fig. 4-7 Change of rat body weight gain measured at different weeks 102
Fig. 4-8 Visceral fat in the experimental rats 103
Fig. 4-9 Serum lipids (TG) in the experimental rats 104
Fig. 4-10 Serum lipids (TC) in the experimental rats 105
Fig. 4-11 Hepatic lipids (TG and TC) in the experimental rats 106
Fig. 4-12 Serum ALT, AST and ALP in the experimental rats 108
Fig. 4-13 Liver NF-κB, α-SMA and KLF-6 mRNA expression in the experimental rats 109
Fig. 4-14 Liver MMP-2 and MMP-9 mRNA expression in the experimental rats 110
Fig. 4-15 Liver ADH, ALDH and CYP2E1 mRNA expression in the experimental rats 111
Fig. 4-16 Hepatic malondialdehyde (MDA) in the experimental rats 112
Fig. 4-17 Hepatic trolox equivalent antioxidant capacity (TEAC) in the experimental rats 113

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