黑色素細胞癌是所有皮膚癌中最惡性、最容易轉移，且致死率最高的皮膚癌類型。全球高度轉移性黑色素細胞癌的發生率越來越高，治療策略如那些聚集於高侵略性黑色素癌細胞下游β-catenin / MITF訊號傳遞路徑軸線的治療策略是迫切需要的。治療標的針對內質網壓力 (ER stress) 已知可以促進癌細胞死亡並抑制轉移性腫瘤的上皮細胞間質轉化（EMT）。本研究目的在於確認厚朴酚是否可以促進內質網壓力有關的細胞凋亡及調節轉移性黑色素細胞癌。使用高轉移性黑色素細胞癌異種移植小鼠之腹膜轉移模型及電腦斷層掃描影像來評估厚朴酚的治療效果。內質網壓力標記物Calpain-10，是一種新的蛋白水解切割酶。利用基因靜默(gene silencing) 技術抑制CHOP / GADD153所調節的細胞凋亡作用以確定β-catenin / MITF訊號傳遞路徑軸線在黑素癌細胞中所扮演的角色。研究結果發現厚朴酚經由活化內質網壓力及抑制上皮細胞間質轉化可有效地降低黑色素細胞癌腹膜腔轉移和器官轉移。利用基因減弱技術抑制Calpain-10基因或CHOP / GADD153基因的表現可抑制由厚朴酚誘發之訊號傳遞路徑軸線β-catenin / MITF蛋白分解並抑制ERSE (ER stress response element) 或TCF / LEF (T cell-specific transcription factor/Lymphoid enhancer-binding factor) 螢光素酶活性和β-catenin蛋白激酶活性之相關生物效應。這些研究發現顯示了厚朴酚可以經由調節Calpain-10和CHOP / GADD153而抑制β-catenin / MITF訊號傳遞路徑軸線，進而顯著地阻止高度轉移性黑色素細胞癌的進展。
乾癬是一種自體免疫慢性發炎的皮膚疾病，影響約2％-3% 的人口。對於患者造成重大的心理負擔以及影響其生活品質的程度，不亞於其他重大慢性疾病，如關節炎、糖尿病、高血壓等。不少患者從青壯年時就得開始面對乾癬，在無治癒的希望下，許多患者合併有憂鬱、焦慮、酒精濫用的現象，甚至有些患者有自殺念頭。最近研究發現aryl hydrocarbon receptor (AhR) 在乾癬的致病機轉中扮演很重要的角色，然而是否AhR agonists對乾癬有治療效果仍然是未知的，所以我們將已知的AhR agonists: Omeprazole (OMP)及Leflunomide (LEU)用於由Imiquimod (IMQ)所誘導類似乾癬皮膚發炎之小鼠模式來評估AhR agonists是否有療效，動物實驗結果顯示OMP 及LEU 可藉由減少表皮免疫細胞的浸潤及白细胞介素-17的表現，進而減少由IMQ所誘導類似乾癬之皮膚發炎。此外，OMP及LEU可以增強表皮細胞間的細胞緊密連接 (cell tight junction) 及細胞粘著（cell-cell adhesion），進而增強皮膚屏障(skin barrier) 之功能。此外，高光譜影像 (Hyperspectral imaging) 已經被廣泛的應用於各種民生與軍事的用途，包括農藥殘留檢驗、食品安全檢驗、翡翠寶石鑑定、藝術品鑑定、地表的監測、生態環境的變遷、地理資訊的蒐集等應用，最近已有硏究將高光譜影像應用於醫學領域，包括評估腫瘤的良性與惡性及評估缺血性組織的血液灌注狀況。所以我們將高光譜影像應用於評估乾癬的嚴重度，動物實驗結果顯示高光譜影像之水及膠原蛋白的訊號在IMQ所誘導類似乾癬發炎之老鼠皮膚會明顯減少，而在正常老鼠的皮膚則沒有變化。我們的硏究結果顯示高光譜影像是快速、準確且可信任的技術，可應用於臨床評估乾癬的嚴重度。目前治療乾癬傳統用藥包括acitretin、methotrexate及cyclosporine，然而這些藥物有肝毒性、腎毒性或骨髓抑制等副作用。目前已有生物製劑用於治療中、重度乾癬，雖然生物製劑的副作用比傳統乾癬用藥少，但是生物製劑價格比起傳統用藥昂貴許多，我們的硏究顯示OMP及LEU確實可明顯減少由IMQ所誘導類似乾癬之皮膚發炎，而OMP及LEU的價格低且無明顯副作用。我們深信該研究將打開更多的視野，暸解AhR agonists減緩由IMQ誘導類似乾癬皮膚發炎之機轉 ，進而可將Omeprazole及Leflunomide應用於臨床治療乾癬。There is increasing global incidence of highly metastatic melanoma and therapeutic strategies like those focusing on the downstream beta-catenin/MITF axis of invading melanoma cells are urgently needed. Targeting endoplasmic reticulum (ER) stress can promote cancer cell death and inhibit epithelial mesenchymal transition (EMT) in metastatic tumors. However, whether Honokiol could promote ER stress-dependent apoptosis and regulate metastatic melanoma is still unkown. We therefore used the highly metastatic melanoma xenograft mouse model and computed tomography imaging to assess the therapeutic efficacy of Honokiol for peritoneal metastasis. The ER stress marker, Calpain-10, delineated a novel proteolytic cleavage enzyme, while CHOP/GADD153-regulated apoptosis was used for gene silencing to determine the role of the β-catenin/MITF axis in melanoma cells. Our results showed that Honokiol effectively decreased peritoneal dissemination and organ metastasis via ER stress activation and EMT marker inhibition. Knockdown Calpain-10 or CHOP/GADD153 blocked all of the biological effects in Honokiol-induced β-catenin/MITF cleavage, ERSE or TCF/LEF luciferase activity, and β-catenin kinase activity. These experimental outcomes suggest that Honokiol can significantly thwart the progression of highly metastatic melanoma using the β-catenin/MITF axis via prompt Calpain-10 and CHOP/GADD153 regulated cascades.
We also explored the molecular mechanisms and therapeutic effects of potential agents in psoriasis. Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation and by and influx of inflammatory cell that is associated with multiple coexisting conditions. The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of immune cells by pharmacological effect of a receptor agonist has not been described. Here we first establish and present of short-wave infrared (SWIR, defined here as ∼1000 to 2000 nm) spectroscopy and imaging techniques for biological tissue optical property characterization. We conducted and identify a novel spectroscopic instrument (GAIA) application of AhR-mediated signaling in imiquimod (IMQ)-induced psoriatic inflammation, a mouse model that shares feature with the human disease. We found that GAIA precisely evaluated psoriatic inflammation and consistent with pathological histology, which specially survey the water and collagen signals of tissue constituents but not lipids. Simultaneously, we also show that deficiency of AhR expression by knockout (AhRKO) mice is induced and aggravated in spectroscopic image, immune cells and cytokine IL-17. In contrast, activation of AhR with an AhR agonists, Leflunomide (LEU) and Omeprazole (OMP) efficiently attenuates immune activity and IL-17 of production. In vitro study, primary goldfish keratocytes (PFK) demonstrated that AhR is essential for keratinocyte barrier function by trans epithelial electrical resistance (TEER) measurement. Our studies introduce AhR as another regulator of immune cell activity in vivo, regulation of inflammatory responses and skin barrier function. We open the possibility for novel therapeutic strategies in chronic inflammatory disorders and identify AhR agonist may be a potential therapeutic target for psoriasis.摘要 i
Abstract iii
Table of Contents v
List of Tables viii
Lists of Figures ix
List of Abbreviations xi
Chapter 1. Exploiting Honokiol-induced ER stress CHOP activation inhibits the growth and metastasis of melanoma by suppressing the MITF and β-catenin pathway 1
1.1 Introduction 3
1.2 Materials and Methods 6
1.2.1 Melanoma cells and Cell culture 6
1.2.2 Mouse xenograft model of animal peritoneal dissemination 6
1.2.3 Positron emission tomography/computed tomography (PET/CT) 7
1.2.4 Western blot analysis and immuno-precipitation 8
1.2.5 Calpain activity 8
1.2.6 TOP/FOP luciferase reporter assay 8
1.2.7 Immuno-fluorescence confocal laser scanning microscopy 9
1.2.8 In vitro kinase activity 9
1.2.9 Colony-forming assay 9
1.2.10 Cell migration assay 10
1.2.11 Web-based correlation 10
1.2.12 Statistical analyses 10
1.3 Results 12
1.3.1 Honokiol blocked highly metastatic melanoma in vivo 12
1.3.2 Honokiol prompted ER stress and Calpain-10 activity and delayed EMT markers 12
1.3.3 Honokiol enhanced Calpain10 and MITF-m interaction and caused MITF-m cleavage 13
1.3.4 Honokiol-induced CHOP/GADD153 regulated β-catenin activation 14
1.3.5 Honokiol induced ER stress and reduced MITF, β-catenin, and CDK2 expressions in animal melanoma tissues 14
1.3.6 High expressions of MITF, β-catenin, and CDK2 decreased the overall survival probability 15
1.4 Discussion 16
Chapter 2. Amelioration of psoriasis skin inflammation by aryl hydrocarbon receptor activation 23
Abstract 24
2.1 Introduction 25
2.2 Materials and Methods 30
2.2.1 Mice and treatments 30
2.2.2 Fish keratinocyte primary 30
2.2.3 Reagents 30
2.2.4 Hyperspectral imaging (HSI) system 31
2.2.5 Immunohistochemistry 31
2.2.6 Immunofluorescence 32
2.2.7 Hydroxyproline assay 32
2.2.8 ATP/ADP Measurements 32
2.2.9 Measurement of trans epithelial electrical resistance (TEER) 33
2.2.10 Statistical Analyses 33
2.3 Results 34
2.3.1 IMQ-induced psoriasis-like skin inflammation with decreased hyperspectral imaging signals of water, collagen, and water combined with collagen, but not lipid 34
2.3.2 Exacerbated psoriasis-like skin inflammation in AhR-deficient mice 35
2.3.3 Pathogenic cytokine IL-17 and immune cells are significantly increased in the IMQ-induced psoriasis-like skin of AhR deficient mice. 37
2.3.4 AhR activation by LEU and OMP obviously attenuate IMQ-induced psoriasis-like skin inflammation 38
2.3.5 AhR activation by LEU and OMP efficiently decrease the immune activity and production of IL-17. 41
2.3.6 AhR activation by LEU and OMP enhances cell tight junction. 42
2.3.7 AhR activation by LEU and OMP enhances cell-cell adhesion. 43
2.4 Discussion 45
2.5 Future direction 53
Tables 54
Figures 58
Reference 8
