臺灣博碩士論文加值系統

膀胱癌是國人泌尿道最常見的癌症，然目前臨床上膀胱癌治療藥物的效果仍不甚理想。在癌症生長過程中 hypoxia/HIF-1α 訊息路徑活化對腫瘤血管新生、腫瘤生長、轉移及化療藥物抗性等扮演一關鍵角色。近年來中西醫藥物整合治療癌症的方向已漸被重視，由中藥材「厚朴」中所萃取出的活性成份厚朴酚(magnolol)，已證實具有多種藥理作用包括抗發炎及抗癌作用等，然其對膀胱癌的血管新生、腫瘤轉移及是否能改善癌症或化療藥物所誘發之惡病質等至今未有報告。
因此本論文即是要建立一個有效率及完整的癌症研究平台，研究厚朴酚對人類膀胱癌的血管新生、轉移及預防惡病質產生的效果評估及機轉探討:
首先，評估在缺氧狀態下magnolol對抑制膀胱癌及對其重要血管新生訊號傳遞路徑的影響。經由細胞及動物實驗結果呈現，magnolol經由抑制缺氧所誘發的ROS/HIF-1α/VEGF/ VEGFR2訊號傳遞路徑達到抑制腫瘤血管新生；接著，進一步評估在缺氧狀態下magnolol是否藉由調控HIF-1α及其相關的重要癌轉移訊號傳遞達到抑制癌轉移效用。經由細胞及動物實驗結果呈現，magnolol經由抑制HIF-1α/EMT訊號傳遞路徑達到抑制腫瘤癌轉移。最後，評估magnolol與臨床常用化療藥物如健澤(Gemzar)及順鉑(Cisplatin)併用後是否可減輕其副作用及提升膀胱癌之治癒效果，並研究其相關作用機轉。於原位注射動物癌症模式證實，合併給予magnolol可藉由降低FoxO3相關的降解路徑及增加IGF-1相關的生成路徑達到減少癌症及化療藥物所誘發之肌肉萎縮、發炎等副作用。由上述我們一系列研究成果顯示，magnolol對膀胱癌的生長、血管新生、轉移及癌症所誘發之惡病質等皆有明顯的抑制作用。希望從本論文中能提供未來治療膀胱癌一個新的方向。

Bladder cancer is the most common cancer of the urinary tract in Taiwan. However, the current therapeutic drugs used for bladder cancer are still unsatisfied. During the pathogenesis of cancer, the activation of hypoxia/HIF-1α signal pathway plays a key role in tumor angiogenesis, metastasis and development of drug resistance. Magnolol isolated from Magnolia officinalis, a traditional Chinese medicine, has been reported to have a variety of pharmacological activities. Although magnolol exerts an anti-tumor activity, there is little information about the effects of magnolol on angiogenesis, metastasis and cachexia in human bladder cancer.
Thus, the goals of this study are to investigate whether magnolol has a protective effect against angiogenesis, metastasis and cachexia in bladder cancer through a series of in vitro and in vivo studies. Moreover, the underlying mechanisms involved of magnolol are also investigated. Direction and results of research into the following three parts:
First of all, to investigate the effects of magnolol on hypoxia-induced angiogenesis signal pathways in T24 baldder cancer. Our results presented via in vitro and in vivo, magnolol suppresses tumor progression by inhibiting angiogenesis via ROS/HIF-1α/VEGF/VEGFR signaling pathways in human bladder cancer cells. Furthermore, to investigate the anti-metastatic activity of magnolol is associated with regulation of hypoxia-induced HIF-1α/EMT cascade. Our results presented via in vitro and in vivo, magnolol suppresses metastasis via elimination of HIF-1α/ TWIST/EMT cascade, migration and invasion in T24 cells. At last, to investigate the effect of magnolol supplement on chemotherapy-induced muscle atrophy in bladder cancer-bearing mice. Our results have demonstrated that supplementation of magnolol attenuates skeletal muscle atrophy in bladder cancer-bearing mice undergoing chemotherapy via suppression of FoxO3 Activation and induction of IGF-1.
Taken together, our results have demonstrated that magnolol has an inhibitory effect on tumor growth, angiogenesis and metastasis in bladder cancer. Furthermore, magnolol also has a beneficial effect in attenuation of tumor or chemotherapy drug-induced cachexia. Hopefully, from this whole study, we can provide more valuable information about the anti-tumor effects of magnolol in bladder cancer, which may be useful for future development of a potential anti-tumor drug.

List of figures
Abbreviation list
中文摘要
Abstract
Chapter I: Introduction
1.1. Bladder cancer: background and clinical treatment
1.2. Hallmarks of Cancer
1.3. Tumor angiogenesis
1.4. Regulation of HIF-1α
1.5. Tumor metastasis
1.6. Tumor and chemotherapy-induced cachexia
1.7. Role of muscle atrophy-related pathways in cachexia
1.8. Magnolol
1.9. The aim of this thesis
Chapter II: Materials and Methods
2.1. Reagents
2.2. Antibodies
2.3. Cell culture and hypoxic treatment
2.4. Quantitative real-time PCR assay
2.5. Western blotting
2.6. Immunofluorescence assay
2.7. Cell viability assay
2.8. H2O2 measurement
2.9. Chromatin immune precipitation (ChIP) assay
2.10. Enzyme-linked immunosorbent assay (ELISA)
2.11. Co-immunoprecipitation (Co-IP) assay
2.12. Gelatin Zymography Assay
2.13. Cell Migration and Invasion Assay
2.14. Capillary-like tube formation assay
2.15. Chicken chorioallantoic membrane (CAM) assay
2.16. In vivo Matrigel plug angiogenesis assay
2.17. Animal model
2.18. Histological examination and immunohistochemical staining
2.19. Reactive Oxygen Species (ROS) Assay
2.20. Intestinal Function
2.21. Proteasome Activity
2.22. Statistical analysis
Chapter III: Results
3. The aim of this study
3.1. Magnolol inhibits hypoxia-induced HIF-1α expression and transcriptional activity in T24 cells
3.2. Magnolol inhibits hypoxia-induced VEGF expression, VEGFR2 phosphorylation, interaction of VEGF and VEGFR2 in T24 cell
3.3. The PI3K/AKT/mTOR pathway involves hypoxia-induced HIF-1α protein accumulation and VEGF secretion
3.4. Magnolol promoted HIF-1α protein degradation and decreased its protein synthesis in hypoxic T24 cells
3.5. Magnolol inhibits angiogenesis in vitro and semi in vivo
3.6. Magnolol inhibited tumor growth, HIF-1α/VEGF protein expression and the down-stream signaling pathway in a xenograft mouse model
3.7. The proposed schematic diagram of signaling pathways for magnolol-mediated anti-angiogenesis
3.8. Effect of magnolol on EMT-related morphologic changes in hypoxic T24 cells
3.9. Effect of magnolol on MMP-2 and MMP-9 expression
3.10. Magnolol inhibited migration and invasion of hypoxic T24 cells
3.11. Magnolol inhibited tumor metastasis
3.12. The proposed schematic diagram of signaling pathways for magnolol-mediated anti-metastasis in hypoxic T24 cells
3.13. Effects of magnolol on body weight, daily food intake and tumor growth
3.14. Effects of magnolol on intestinal damage and digestive enzyme dysfunction
3.15. Magnolol reduces muscle atrophy and proteasome activity
3.16. Magnolol attenuates muscle atrophy-related gene expression and increases IGF-1-regulated signaling
3.17. Effects of magnolol on pro-inflammatory cytokine production and NF-κB activation
3.18. The proposed schematic diagram of signaling pathways for the anti-cachectic activity of magnolol
Chapter IV: Discussion
Chapter V: Conclusion
Chapter VI: References
Chapter VII: Table and figures
Chapter VIII: Publications

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