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研究生:翁茂琦
研究生(外文):Mao-Chi Weng
論文名稱:癌瑞格在肝癌和肺癌細胞與動物模式的作用機制與療效評估
論文名稱(外文):Evaluation of mechanism and therapeutic efficacy of regorafenib in hepatoma and lung cancer in vitro and in vivo
指導教授:王信二
指導教授(外文):Hsin-Ell Wang
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:72
中文關鍵詞:癌瑞格核因子活化B細胞κ輕鏈增強子肝癌非小細胞肺癌分子影像
外文關鍵詞:RegorafenibNF-κBhepatocellular carcinomaNon-small cell lung cancermolecular imaging
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目的:文獻指出癌瑞格(Regorafenib)於SK-Hep1肝癌細胞內,可透過抑制胞外信號調節激酶(ERK)/核因子活化B細胞κ輕鏈增強子(NF-κB)的活化來引發細胞凋亡,然而此調控路徑在生物體內對肝癌的抑制效果尚未見報導。非小細胞肺癌(non-small cell lung cancer, NSCLC)患者通常治療預後不佳,亦有文獻指出NF-κB在肺癌進程中扮演重要的調控者,然而癌瑞格對非小細胞肺癌的療效亦無文獻報導。本研究的第一個目標,欲釐清癌瑞格於肝癌小鼠模式,如何經由ERK/NF-κB調節路徑來抑制癌症進程;第二個目標為探討癌瑞格在非小細胞肺癌細胞及荷NSCLC腫瘤裸小鼠的抗癌效果及相關機制。
材料與方法:已建立pGL4.50冷光素酶報導基因載體(luciferase reporter vector)轉染SK-Hep1肝癌細胞(SK-Hep1/luc2)及Hep3B 2.1-7肝癌細胞動物模式,經口餵食(gavage)生理食鹽水(vehicle)或癌瑞格(20 mg/kg/day)共14天,使用游標尺及生物冷光造影(BLI)、西方墨點法免疫組織化學染色(ex vivo IHC staining)進行分析、並追蹤體重及肝組織病理檢驗。另一方面,將CL-1-5-F4肺癌細胞分為三組,分別加入癌瑞格、NF-κB抑制劑(QNZ)及AKT抑制劑 (LY294002),培養24及48小時後觀察細胞存活率、測試NF-κB基因表現、進行細胞遷移實驗(transwell invasion assay)及細胞凋亡流式細胞儀分析,並觀察癌瑞格透過ERK/NF-κB調控細胞內基因表現的機制及抗癌效果。於CL-1-5-F4肺癌動物模式,經口餵食生理食鹽水或癌瑞格等28天後,藉由量測腫瘤大小、細胞核及細胞質染色(H&E stain)及免疫組織化學染色等評估療效,並探討癌瑞格的抑癌機制。
結果:本研究結果顯示,癌瑞格可抑制肝癌腫瘤細胞生長,也可抑制磷酸化ERK、NF-κB p65 (Ser536)、磷酸化AKT、腫瘤增長相關蛋白的表現;癌瑞格可誘發外因性(extrinsic)及內因性(intrinsic)細胞凋亡的調控路徑,但不會改變動物體重及肝細胞型態;另外,癌瑞格可調控外因性及內因性凋亡路徑,經AKT/NF-κB路徑抑制與腫瘤增長及侵犯相關的蛋白,最終導致非小細胞肺癌死亡。冷光造影(BLI)及斷層掃描(CT)均可用於評估癌瑞格對肝癌和非小細胞肺癌的抑制效果。
結論:本研究結果顯示,於SK-Hep1/luc2及Hep3B 2.1-7肝癌小鼠模式,癌瑞格可透過抑制ERK/NF-κB調控路徑抑制腫瘤生長;此外,癌瑞格也透過AKT/NF-κB調控路徑,於細胞及生物體內引發非小細胞肺癌的凋亡。
Purpose: Regorafenib has been demonstrated in previous study to trigger apoptosis through suppression of extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB, the oncogenic transcription factor) activation in hepatocellular carcinoma (HCC) SK-Hep1 cells in vitro. However, the effect of Regorafenib on NF-κB-modulated tumor progression in HCC in vivo has never been reported. Non-small cell lung cancer (NSCLC) is a malignant lung cancer type with poor prognosis. NF-κB has also been recognized as an important mediator in progression of NSCLC. However, whether Regorafenib were effective in retarding the progression of NSCLC is still unknown. The first aim of the present study is to investigate the effect of Regorafenib on NF-κB-modulated tumor progression in HCC-bearing mouse models. The second aim was to evaluate anticancer efficacy and underlying mechanism of regorafenib on NSCLC tumor progression in vitro and in vivo.
Materials and Methods: pGL4.50 luciferase reporter vector transfected SK-Hep1 (SK-Hep1/luc2) and Hep3B 2.1-7 tumor-bearing mice were established and used for the present study. Mice were treated with vehicle or regorafenib (20 mg/kg/day by gavage) for 14 days. Effects of regorafenib on tumor growth and protein expression together with toxicity of regorafenib were evaluated based on tumor growth inhibition and bioluminescence imaging (BLI), ex vivo Western blotting immunohistochemistry (IHC) staining, and measurement of body weight and pathological examination of liver tissue, respectively, in mice bearing either SK-Hep1/luc2 or Hep3B 2.1-7 tumors. CL-1-5-F4 NSCLC cells in 96-well plate were treated with Regorafenib, NF-κB (QNZ) or AKT (LY294002) inhibitor, respectively, for 24 or 48 h. Then, cell viability assay, NF-κB reporter gene assay, transwell invasion assay and apoptosis related flow cytometry assay on cellular level were conducted to elucidate the possible mechanism of anti-cancer effect for regorafenib. Mice bearing CL-1-5-F4 tumor were treated with vehicle or regorafenib for 28 days. The mechanism of regorafenib and its therapeutic efficacy were evaluated by tumor growth inhibition, whole body computed tomography (CT) scan, and examination of Hematoxylin and Eosin (H&E) stain and immunohistochemistry (IHC) stain of tissue and tumor samples.
Results: The results indicated regorafenib significantly reduced both SK-Hep1/luc2 tumor and Hep3B 2.1-7 tumor growth and expression of phosphorylated ERK, NF-κB p65 (Ser536), phosphorylated AKT, and tumor progression-associated proteins. In addition, we found regorafenib induced both extrinsic and intrinsic apoptotic pathways. Body weight and liver morphology were not affected after regorafenib treatment. The results also demonstrated that Regorafenib significantly inhibited CL-1-5-F4 NSCLC tumor growth and induced apoptosis through extrinsic/intrinsic pathways in vitro and in vivo. Furthermore, we also found the suppression of AKT/NF-κB signaling was essential in inhibition of progression-related and invasion-related protein expression by regorafenib. This study also successfully evaluated the tumor growth inhibition of Regorafenib on HCC and NSCLC tumors by using molecular imaging, BLI and CT.
Conclusions: This study displayed that the inhibition of tumor progression by regorafenib is via suppression of ERK/NF-κB signaling in SK-Hep1/luc2 and Hep3B 2.1-7 tumors. The induction of apoptosis and suppression of AKT/NF-κB signaling were associated with Regorafenib-inhibited progression of NSCLC.
Contents
1. Introduction 1
1.1 Human hepatocellular carcinoma 1
1.2 Non-small cell lung cancer 2

PART.I
2. Materials and methods 4
2.1 Reagents and antibodies 4
2.2 Cell culture 5
2.3 Plasmid transfection and stable clone selection 5
2.12 Animal study 6
2.4 In vivo bioluminescent imaging (BLI) 7
2.5 Immunohistochemistry (IHC) staining 8
2.6 Ex vivo Western blotting 8
2.7 Pathological examination 9
2.8 Statistical analysis 9
3. Results 10
3.1 Regorafenib reduces tumor growth in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice 10
3.2 Regorafenib inhibits expression of ERK/NF-κB-modulated downstream effector proteins and triggers expression of apoptotic proteins in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice 12
3.3 General toxicity analysis of regorafenib in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice 17
4. Discussion 19

PART.II
5. Materials and Methods 22
5.1 Drug and antibodies 22
5.2 Non-small cell lung cancer cell culture 22
5.3 Cell viability 23
5.4 Plasmid transfection and stable clone selection 23
5.5 In vitro and in vivo NF-κB reporter gene assay 24
5.6 Trans-well invasion assay 25
5.7 Western blotting 25
5.8 Apoptosis analysis 25
5.9 Extrinsic apoptosis analysis 26
5.10 Intrinsic apoptosis analysis 26
5.11 DNA damage analysis 26
5.12 Animal experiment 27
5.13 In vivo whole-body computer tomography 27
5.14. Immunohistochemistry 27
5.15 Statistical analysis 28
6. Results 29
6.1 Regorafenib markedly reduced cell viability and AKT/NF-κB activation on CL1-5-F4 NSCLC cells 29
6.2 Regorafenib treatment inhibited invasion ability of CL1-5-F4 NSCLC cells 31
6.3 Regorafenib treatment triggered apoptosis and DNA damage ability of CL1-5-F4 NSCLC cells 33
6.4 Regorafenib treatment induced death receptor dependent apoptosis signaling of CL1-5-F4 NSCLC cells 36
6.5 Regorafenib treatment suppressed tumor growth of CL1-5-F4 bearing animal model 38
6.6 Regorafenib treatment suppressed NF-κB mediated proliferation, migration and anti-apoptosis protein expression in vivo 40
7. Discussion 44
8. References 48
9. Publications
A. Regorafenib inhibits tumor progression through suppression of ERK/NF-κB activation in hepatocellular carcinoma bearing mice
B. Apoptosis induction and AKT/NF-κB inactivation are associated with regroafenib-inhibited tumor progression in non-small cell lung cancer in vitro and in vivo

List of Figures
Fig. 2.1 Schematic depiction of experimental protocol 7
Fig. 3.1 Effect of regorafenib on tumor growth in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice 11
Fig. 3.2 Effect of regorafenib on expression of P-ERK, AKT (Ser473), NF-κB p65 (Ser473), and NF-κB-modulated downstream effector proteins in SK-Hep1/luc2 tumor and Hep3B 2.1-7 bearing mice 16
Fig. 3.3 Toxicity investigation of regorafenib in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice 18
Fig. 6.1 Cytotoxicity and AKT/NF-κB inhibition effect of regorafenib treatment on CL1-5-F4 cells. 30
Fig. 6.2 Invasion inhibition effect of regorafenib treatment on CL1-5-F4 cells. 32
Fig. 6.3 Apoptosis signaling was activated by regorafenib treatment on CL1-5-F4 cells. 35
Fig. 6.4 Death receptor mediated apoptosis was triggered by regorafenib treatment on CL1-5-F4 cells. 37
Fig. 6.5 Tumor growth was suppressed by regorafenib treatment on CL1-5-F4 bearing animal model. 40
Fig. 6.6 NF-κB and it downstream proteins were decreased after regorafenib treatment. 42
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