臺灣博碩士論文加值系統

靈芝多醣為從靈芝(Ganoderma lucidum)純化之富含岩藻糖成份的多醣，具有抑制腫瘤增生和調節人類免疫系統之作用。靈芝多醣已經被多方報導具有抗腫瘤的效果與活性，但是所產生的機轉與作用仍尚未完全明瞭。在此，我們使用靈芝多醣的特定的含有岩藻糖的成份（FFLZ）並且發現FFLZ有效地減少在小鼠模式中乳癌腫瘤的大小。我們也發現FFLZ抑制肺癌細胞與乳腺癌細胞的增殖，減少在異種移植物的小鼠肺癌腫瘤與乳腺腫瘤的生長和轉移，並會增加患腫瘤的小鼠的存活率。此外，FFLZ也會抑制癌症細胞的上皮間質轉化（EMT），並使上皮細胞生物標記上升。而轉化生長因子-β受器對於癌症細胞的轉移也是扮演重要的角色。我們發現FFLZ促進經由泛素依賴性蛋白酶體途徑（UPP）在肺癌以及乳腺癌細胞中轉化生長因子β受體（TGFRs）降解，也研究TGFRs和與乳腺癌轉移的TGFβ介導的信號通路的穩定性。FFLZ會加速泛素依賴性蛋白酶體途徑（UPP）以及FFLZ加速觸發Smurf2-Smad7與TGFR的結合，進一步讓Smurf2介導TGFRs降解，讓其蛋白質穩定性下降。我們發現更進一步發現，FFLZ經由其相關的脂質筏（lipid rafts）調控會增強UPP機制之TGFRs內吞作用，使其靈芝多醣對於抑制癌症的功效與機制更加明朗。最後我們也組合FFLZ和曲妥單抗（赫賽汀，抗Her2單克隆抗體）的協同抑制作用發現合併使用FFLZ與曲妥單抗可以有加成性的抑制癌症效果。因此，我們希冀靈芝多醣能於具抗藥性藥物在乳癌的治療中給予病患更多抗癌成功的希望。

Ganoderma lucidum (Ling-Zhi, reishi) has shown antitumor activity; however, the effect of polysaccharide of G. lucidum on the lung cancer and breast cancers is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduces tumor size and suppresses lung metastasis in LLC-1 and 4T1-xenograft breast cancer mouse model in vivo. In addition, we found that FFLZ decreases proliferation and colony formation of cancer cells in vitro. Furthermore, we found that FFLZ inhibits lung and breast cancer cell migration and invasion. Specifically, the epithelial-to-mesenchymal transition (EMT) phenotype was transformed to cobble-stone epithelial morphology after FFLZ treatment, concomitantly, epithelial markers were up-regulated. Transforming growth factor-β receptors (TGFRs) pathways play key mediators to promote tumor cell invasion and metastasis in cancer cells. We found that FFLZ down-regulates TGFRs level and downstream signaling pathways, including phosphorylation of smad2/3, and smad4 expression. To investigate the mechanisms, we found that FFLZ enhances Smurf2-dependent ubiquitination of TGFRs through disruption the balance of lipid-raft fraction and promotes “re-localization” of the TGFRs to caveolae and facilitates TGFRs degradation. Moreover, FFLZ promotes Tollip to associate with ubiquitination of TGFR, leading TGFR degradation. Together, the effectiveness of FFLZ in down-regulation of cancer progression is multifaceted, which associated with inhibition of EMT and prevention of metastasis via promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Alternatively, we found our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for breast cancer patients, acting via the caveolin-1/Smad7/Smurf2-dependent ubiquitin degradation of TGFR.

CONTENTS
誌謝 I
中文摘要 II
ABSTRACT III
ABBREVIATIONS IV
CONTENTS V
LITERATURE REVIEW 1
1. A specific fucose-containing polysaccharide fraction of Ling-Zhi extract (FFLZ) 1
2. Lung cancer 1
3. Breast cancer 2
4. Transforming growth factor-β receptor (TGFR)-mediated signal transductions in metastasis 3
5. The role of ubiquitin-dependent proteasomal pathways (UPP) 4
6. Motivation and aims 5
MATERIALS AND METHODS 7
1. Cell lines 7
2. Reagents and antibodies 7
3. Preparation of the fucose-containing fraction of Ling-Zhi (FFLZ) 8
4. Lung cancer animal model 8
5. Breast cancer animal model 9
6. Cell extracts 9
7. Western Blotting 10
8. Immunoprecipitation of TGFR as part of the in vitro ubiquitination assay 10
9. Design and transfection of shRNAs 11
10. Cell viability assay 12
11. Cell viability assay (MTT assay) 12
12. Colony formation 12
13. Migration and invasion assays 12
14. Immunofluorescence analysis 13
15. Cell cycle analysis 14
16. Apoptosis analysis 14
17. Synergy analysis 14
18. Statistical Analysis 15
CHAPTER 1
Fucose-containing polysaccharides of Ling-Zhi suppresses lung tumorigenesis through inhibition of EGFR signaling and induction of TGFβ receptor degradation 17
1.1. Abstract 17
1.2. Introduction 18
1.3. Results 20
1.3.1. FFLZ inhibits the lung tumorigenesis and metastasis of lung in LLC1-bearing C57BL/6 mice 20
1.3.2. FFLZ inhibits the proliferation and colony formation as well as induces G1 arrest in lung cancer cells 21
1.3.4. FFLZ induces changes of EMT and inhibits lung cancer cell mobility 21
1.3.5. FFLZ decreases TGFR-mediated Smad pathway related proteins and the expression of the EMT-related transcriptional factors Snail, Slug and Twist in A549 cells 22
1.3.6. FFLZ reduces TGFβ receptor expression in A549 cells via accelerated ubiquitin-dependent proteasome-mediated degradation 23
1.3.7. Combination of FFLZ and cisplatin synergistically suppresses lung tumorigenesis and metastasis in LLC1-bearing mouse 24
1.4. Discussion 25
1.5. Figures and figure legends 28
Figure 1.5.1. FFLZ inhibits tumorigenesis and metastasis in LLC1-bearing mice as well as reducing the expression of EMT relevant markers in A549-bearing CB17-SCID mice 28
Figure 1.5.2. FFLZ inhibits proliferation and colony formation in human NSCLC A549 and murine LLC1 cells 30
Figure 1.5.3. FFLZ affects the expression of TGFβ1-mediated EMT markers and inhibits the migration and invasion of A549 cells 32
Figure 1.5.4. FFLZ decreases TGFR-induced Smad-dependent pathway related proteins and the expression of the EMT-related transcriptional factors Snail, Slug and Twist in A549 cells 34
Figure 1.5.5. FFLZ reduces expression of the TGFβ1 and TGFβ receptors (TGFRI and TGFRII) proteins; these changes are, potentially modulated by accelerating protein degradation in A549 cells 36
Figure 1.5.6. Combination of FFLZ and cisplatin synergistically suppresses lung tumorigenesis and metastasis in LLC1-bearing mouse. 38
CHAPTER 2
Fucose-containing fraction of Ling-Zhi enhances lipid rafts-dependent ubiquitination of TGFβ receptor degradation and 39
attenuates breast cancer tumorigenesis 39
2.1. Abstract 39
2.2. Introduction 40
2.3. Results 42
2.3.1. FFLZ inhibits the carcinogenesis of breast in vivo 42
2.3.2. FFLZ inhibits proliferation, colony formation and the transformation capability in vitro 42
2.3.3. FFLZ inhibits spontaneous pulmonary metastasis in the mouse lungs and reduces the invasion of breast cancer cells 43
2.3.4. FFLZ reduces the expression of epithelial mesenchymal transition (EMT) markers via decreasing TGFR-induced Smad-dependent pathway relevant proteins 44
2.3.5. FFLZ reduces expression of the TGFRs (TGFRI and TGFRII) in MDA-MB-231 cells via accelerated ubiquitin-dependent proteasome-mediated degradation 45
2.3.6. FFLZ modulation of TGFRI protein in MDA-MB-231 cells via accelerating lipid rafts/caveolae mediated endocytosis of TGFR 47
2.3.7. FFLZ promotes caveolin-dependent degradation via triggering Smurf2/Smad7/Tollip to conjugate on TGFRI. 48
2.3.8. Study of synergistic effect of combining FFLZ and trastuzumab (Herceptin®) in treatment of trastuzumab-resistant human breast cancer cells, BT474 and SKBR3 49
2.4. Discussion 50
2.5. Figures and figure legends 53
Figure 2.5.1. Fucose-containing fraction of Ling-Zhi (FFLZ) inhibits carcinogenesis in vivo. 53
Figure 2.5.2. Fucose-containing fraction of Ling-Zhi (FFLZ) inhibits the carcinogenesis in vivo. 55
Figure 2.5.3. Fucose-containing fraction of Ling-Zhi (FFLZ) inhibits the carcinogenesis in vitro. 56
Figure 2.5.4. FFLZ inhibits spontaneous pulmonary metastasis in mouse lungs and liver and changes the morphology of breast cancer cells. 58
Figure 2.5.5. FFLZ inhibits spontaneous pulmonary metastasis in the mouse lungs and reduces the invasion of breast cancer cells. 59
Figure 2.5.6. FFLZ modulates the morphology of breast cancer cells and the expression of EMT markers. 61
Figure 2.5.7. FFLZ reduces the expression of EMT markers FFLZ via decreasing TGFR-induced Smad-dependent pathway related proteins. 63
Figure 2.5.8. FFLZ is downregulated TGF-β Receptors via accelerated ubiquitin-dependent proteasome-mediated degradation. 66
Figure 2.5.9. FFLZ reduces expression of the TGFβ receptors (TGFRI and TGFRII) in MDA-MB-231 cells via accelerated ubiquitin-dependent proteasome-mediated degradation. 67
Figure 2.5.10. Examination of the role of Smurf2, Tollip, Cav-1, and Smad7 in FFLZ mediated degradation or endocytosis of TGFRI. 69
Figure 2.5.11. Synergistic anti-cancer activity of the combination of FFLZ and Trastuzumab in BT474 and SKBR1 human breast cancer cell. 71
Figure 2.5.12. Proposed scheme of FFLZ-enhanced TGFR degradation via lipid rafts-dependent ubiquitination. 73
CONCLUSION 74
REFERENCES 76

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