臺灣博碩士論文加值系統

過氧化小體增生因子活化受體伽碼 (peroxisome proliferator-activated receptor γ, PPARγ) 是一種受體活化細胞核接受器，具有調控細胞內脂質和葡萄糖代謝的功能，並在不同癌症中扮演抑制的角色。然而，PPARγ在臨床上肝癌的顯著性仍舊沒有明確的研究結果。厚朴酚(honokiol)是一種天然雙酚類化合物，近年來被發現在肝癌細胞中具有PPARγ促效劑的功能。但目前PPARγ對於厚朴酚的重要性以及PPARγ是否在厚朴酚抑制細胞生長的機轉中扮演角色仍不清楚。此篇研究主要目標為探討PPARγ在臨床肝癌診斷上的價值和其抑制腫瘤進展的角色，並且探討PPARγ是否參與在厚朴酚抑制細胞生長的機轉中。此篇研究中共有83個肝癌病人組織，利用免疫組織染色的方式檢測PPARγ的表現量，透過病理學的方式探討其表現量和臨床參數上的相關性。低表現量之PPARγ相較於高表現量之PPARγ的肝癌病患偏年輕者居多(p = 0.006)，並具有較多的腫瘤數量(p = 0.038)，較多的巨觀血管侵襲(p = 0.008)，和較為惡化的診斷後期(p = 0.013)。此外，分析結果中發現到低表現量之PPARγ可作為預測肝癌病患具有較多巨觀血管侵襲的獨立因子。而針對PPARγ的功能性探討則是利用病毒轉植技術分別於肝癌細胞Mahlavu中使其過度表現及於PLC/PRF/5細胞中降低其表現量。體外細胞實驗中的結果顯示PPARγ具有抑制肝癌細胞生長，移動，以及血管新生的功能。其中cyclin D1和STAT3蛋白可能參與在PPARγ抑制肝癌細胞生長的訊息傳遞路徑中。此外，厚朴酚透過調控細胞週期和KLF4, cyclin D1, p21, 以及cdc2這些細胞週期調控蛋白來抑制肝癌細胞生長。其中，SK-Hep1肝癌細胞株可以觀察到最明顯的效果。由於不同肝癌細胞株的特性，使得過量表現PPARγ於不同肝癌細胞對於增強厚朴酚抑制細胞生長的影響有所差異。此外，透過分析台灣健保資料庫百萬抽樣人檔，結果顯示第二型糖尿病患者使用糖尿病用藥噻唑烷二酮類(thiazolidinedione, TZDs)，PPARγ促效劑的一種，能降低後續發生肝癌的風險。總結本研究結果，PPARγ的功能在肝癌細胞中扮演一個抑制腫瘤的角色並且可作為治療肝癌的標的。

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor that regulates cellular lipid and glucose metabolism and also plays an inhibitory role in various cancers. However, the clinical significance of PPARγ in hepatocellular carcinoma (HCC) remains unclear. Honokiol, a natural biphenolic compound was found to function as a PPARγ agonist in HCC cells recently. However, the functional importance of PPARγ in honokiol-mediated growth inhibition is not known. This study aimed to investigate the prognostic value of PPARγ in HCC and its role in inhibiting tumor progression, and to investigate whether PPARγ is involved in honokiol-mediated cell growth inhibition of HCC cells. Immunohistochemical PPARγ staining was examined in 83 HCC specimens to investigate the clinicopathological correlations between PPARγ expression and various parameters. The functional role of PPARγ was determined via PPARγ overexpression and knockdown in HCC cell lines Mahlavu and PLC/PRF/5, respectively. Patients with low HCC tissue PPARγ expression were significantly younger (p = 0.006), and exhibited more tumor numbers (p = 0.038), more macroscopic vascular invasion (MVI) (p = 0.008), and more advanced TNM (tumors, nodes, and metastasis) stages at diagnosis (p = 0.013) than patients with high HCC tissue PPARγ expression. These results suggest that low PPARγ expression is an independent predictor of more MVI in HCC patients. In vitro experiments showed that PPARγ contributes to suppression of HCC cell growth, migration, and angiogenesis. Cyclin D1 and STAT3 may be involved in PPARγ-mediated signaling pathways that inhibit HCC cell growth. In addition, honokiol exhibits inhibitory role on cell growth through regulation of cell cycle and the expression of KLF4, cyclin D1, p21, and cdc2. This could be observed especially in SK-Hep1 cells. Particularly, PPARγ enhances honokiol on growth inhibition and results in differential response in different human HCC cell lines. Moreover, the analyses from Taiwan National Health and Insurance Research Database showed that anti-diabetic drugs thiazolidinediones (TZDs), PPARγ agonists, reduces HCC development in patients with type II DM. In conclusion, PPARγ functions as a tumor suppressor in HCC cells and may be therapeutic target in HCC.

Table of Contents 1
Chinese abstract 4
Abstract 6
Abbreviations 8
I. Introduction 11
1.1 Liver cancer 11
1.1.1 Epidemiology 11
1.1.2 Types of liver cancer 11
1.1.2.1 Benign liver tumors 11
1.1.2.2 Primary liver cancer 12
1.1.2.3 Secondary liver cancer 12
1.1.3 Risk factors for hepatocellular carcinoma 13
1.1.4 Diagnosis 13
1.1.5 Stage 14
1.1.5.1 AJCC TNM stage system 14
1.1.5.2 BCLC system 14
1.1.6 Therapeutic strategies 15
1.1.6.1 Surgery—Partial hepatectomy and liver transplantation 15
1.1.6.2 Tumor ablation 15
1.1.6.3 Embolization 16
1.1.6.4 Radiation therapy (RT) 17
1.1.6.5 Chemotherapy 18
1.1.6.6 Targeted therapy 19
1.1.6.7 Combination therapy 19
1.1.7 Ongoing clinical studies 20
1.2 Peroxisome proliferator-activated receptor γ (PPARγ) 20
1.2.1 Molecular structure of PPARγ 20
1.2.2 Expression of PPARγ in HCC 22
1.2.3 Functions of PPARγ in HCC 22
1.3 Functions of PPARγ ligands TZDs in HCC 23
1.4 Use of TZDs and the risk of HCC in patients with type II DM 25
1.5 Honokiol 26
II. Specific aims and rationale 28
III. Materials and Methods 30
3.1 Human tissue specimens and patient information 30
3.2 Immunohistochemical study 30
3.3 Cell culture and drug treatment 31
3.4 Overexpression and knockdown of PPARγ 32
3.5 Measurement of cell proliferation 32
3.6 Cell cycle analysis by flow cytometry 33
3.7 Quantitative real-time polymerase chain reaction (RT-qPCR) analysis 33
3.8 Western blot analysis 34
3.9 Wound healing assay 35
3.10 Matrigel tube formation assay 35
3.11 Big data source and design 36
3.12 Statistical analyses 37
IV. Results 38
4.1 PPARγ protein expression in human HCC tissues and associated clinicopathological characteristics 38
4.2 PPARγ suppresses HCC cell proliferation 39
4.3 PPARγ inhibits HCC cell migration 40
4.4 PPARγ decreases HCC cell angiogenesis 40
4.5 PPARγ enhances honokiol on growth inhibition in human HCC cells 41
4.6 Honokiol-induced cell cycle phase alteration is partially PPARγ-dependent 42
in human HCC cells 42
4.7 Honokiol up-regulates cell cycle arrest-related proteins and down-regulated 43
cell cycle promotion-related proteins 43
4.8 PPARγ agonists TZDs inhibits cell growth in HCC cells 44
4.9 TZDs decreases HCC development in patients with type II DM 45
V. Discussion 47
5.1 Clinical significance of PPARγ in HCC 47
5.2 PPARγ functions as a tumor suppressor in HCC development 48
5.3 Honokiol serves as a PPARγ agonist and has an inhibitory role in HCC 50
5.4. Anti-diabetic medications in HCC treatment 53
VI. Conclusion 54
VII. References 55
VIII. Tables 74
IX. Figures 80
X. Appendices 95
XI. Publications 97

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