目的: 研究Withaferin A(WA)和游離輻射在非小細胞肺癌細胞株合併使用的生物效應。
材料及方法:
我們選用的是A549和NCI-H1299二株非小細胞肺癌細胞株；此二種細胞株具有不同的p53型式。游離輻射採用Varian 21EX的直線加速器，以六百萬電子伏特的電子射束給予。我們使用Trypan Blue Exclusion Assay來評估細胞存活率，並根據細胞存活率實驗的結果計算出WA的百分之二十抑制濃度(IC20)及百分之五十抑制濃度(IC50)，並應用於之後的實驗；腫瘤生長抑制實驗將不同的WA濃度與游離輻射併用，使用群落生成法(Clonogenic Assay)。WA和游離輻射的增效作用(Synergistic Effect)則藉由群落生成法的結果經中位效應方程式(median effect equation)和組合指數(Combination Index)計算而得；彗星拖尾試驗(comet assay)和TUNEL試驗(TUNEL assay)則分別用來評估DNA損傷以及自體凋亡(apoptosis)的程度；半胱胺酸蛋白酶(Caspase)的活性是使用Caspase Colorimetric Activity Assay來評估；最後，以西方點墨法(Western Blots)及螢光染色(Fluorescence Stain)來進行蛋白質的定性及定量。
結果:
WA和游離輻射單獨使用時，均能抑制肺癌細胞株在培養皿中的生長，當劑量愈高，抑制的情形就愈明顯。而當WA和游離輻射並用時，根據計算出的組合指數 (Combination Index)，可判斷出二者之間具有增效作用。WA和游離輻射並用也會使自體凋亡和DNA損傷的程度增加，半胱胺酸蛋白酶-3和半胱胺酸蛋白酶-9的活性也會顯著上升。在NCI-H1299細胞中，當4 Gy的游離輻射和WA 併用時，Bax蛋白的量會增加，而Bcl-2蛋白的量會減少。西方點墨法也確認WA和IR併用時會抑制ATM及Cdc25c的表現量。此外，在A549細胞中放射線處理後造成的 HIF-1α蛋白上升可被WA所抑制，WA也會降低β-catenin的表現量。
結論:
當WA和游離輻射併用時，具有細胞毒殺的增效作用、能抑制DNA損傷的修復機制、造成G2/M細胞阻滯(cell cycle arrest)，並且抑制HIF-1α和β-catenin蛋白的表現。

Purpose: To investigate the combined biological effects of withaferin A (WA) and ionizing radiation (IR) in Non-small-cell-lung cancer (NSCLC) cell lines
Methods &; Materials:
A549 and NCI-H1299 cells, two NSCLC cell lines with different p53 status, were chosen. IR was administered with 6MeV electron beam using Varian 21EX linear accelerator. Cell viability was evaluated with trypan blue exclusion assay. IC20 and IC50 from cell viability assay were used in caspase colorimetric assay, comet assay, TUNEL assay, and Western blots. Clonogenic assay was used to evaluate tumor growth in vitro with different combination of WA and IR. Synergistic effects of WA and IR were calculated using median effect equation and combination index according to the results of clonogenic survival assays. Comet assay and TUNEL assay was used to evaluate the extent of DNA damage and apoptosis, respectively. Caspase colorimetric activity assay was used to evaluate the activities of different caspases. Protein levels were evaluated with western blots and fluorescence stain.
Results:
Both WA and IR alone could inhibit tumor growth in vitro in a dose-dependent manner. When WA was combined with IR, the inhibition of tumor growth in vitro was synergistic according to combination index. The extent of DNA damage and apoptosis both increased when WA was added to IR. When WA was combined with IR, activity of caspase-3 and 9 significantly increased compared to WA or IR alone. When 4 Gy IR was combined with WA, Bax increased and Bcl-2 decreased significantly after adding WA in NCI-H1299 cells. Western blots in both cell lines demonstrated that adding WA to IR could inhibit the activation of ATM and Cdc25c. Besides, the activation of HIF-1α after IR could be inhibited with WA in A549 cells. The level of β-catenin was also attenuated significantly after adding WA to IR in A549 cells.
Conclusion:
When combined with IR, WA had synergistic cytotoxic effects, could inhibit DNA repair mechanisms, cause G2/M cell cycle arrest, and inhibit the expression of HIF-1α and β-catenin in NSCLC cell lines

Table of Contents
1 English Abstract 4
2 Chinese Abstract 5
3 List of Abbreviations 6
4 Introduction: 7
4.1 Non-Small Cell Lung Cancer (NSCLC)– Clinical Aspects 7
4.1.1 Histological Subtype of NSCLC 7
4.1.2 Clinical stages and prognosis of NSCLC 8
4.1.3 Radiation Therapy (RT) in NSCLC 8
4.1.4 Concomitant chemoradiotherapy (CCRT) for NSCLC 8
4.1.5 Chemotherapeutic drugs in CCRT for NSCLC 8
4.2 Connectivity Map 9
4.3 Withaferin A (WA) 10
4.3.1 General Introduction of WA 10
4.3.2 The in vivo evidences of WA in inhibiting tumor growth &; chemoprevention 10
4.3.3 The potential role of WA as a therapeutic agent for cancer 10
4.4 WA as a radiation sensitizer 14
4.4.1 Rationale 14
4.4.2 Existing Evidences 15
4.5 Radiation damage and IR induced signaling pathways 15
4.5.1 IR induced DNA damage 16
4.5.2 Radiation-induced signaling pathways 16
4.6 Vimentin 17
4.7 HIF-1α 17
4.8 Wnt/β-catenin 18
5 Material and Methods 19
5.1 Experimental Instruments 19
5.2 Reagents 19
5.3 General principles 20
5.4 Protocol of IR administration 20
5.5 Cell Lines 21
5.6 Cell Culture Protocol 21
5.7 Clonogenic Survival Assay 21
5.7.1 Cell Culture Protocol 21
5.8 Cell viability analysis 22
5.8.1 Principles of trypan blue exclusion assay 22
5.8.2 Methods 22
5.9 Comet assay 23
5.9.1 The principle of comet assay 23
5.9.2 Methods 23
5.10 Terminal deoxynucleotidyltransferase (TdT) dUTP Nick End Labeling (TUNEL)Assay 24
5.11 Caspase colorimetric assay 25
5.12 Immunofluorescence stain 25
5.13 Western Blots 26
5.13.1 Protein extraction 26
5.13.2 SDS-PAGE 26
5.13.3 Transferring the protein from the gel to the membrane 26
5.13.4 Antibody incubation 27
6 Result 27
6.1 The Effect of WA on A549/NCI-H1299 Cells 27
6.1.1 WA inhibits A549/NCI-H1299 cell tumor growth in vitro 27
6.1.3 WA reduced the cell viability 28
6.1.4 WA increases the extent of DNA damages in A549/NCI-H1299 cells 28
6.1.5 WA increases apoptosis of A549/NCI-H1299 cells 28
6.1.6 WA increases the mitochondrial pathway of apoptosis in A549/NCI-H1299 cells 29
6.2 The effects of IR on A549/NCI-H1299 cells 29
6.2.1 Radiation inhibits A549/NCI-H1299 cell tumor growth in vitro 29
6.3 The combination effect of WA and IR on A549 and NCI-H1299 cell lines 29
6.3.1 Combined WA and IR increased the effect on tumor growth inhibition 29
6.3.2 WA and IR had synergistic effect according to combination index (CI) 30
6.3.3 WA increased the extent of DNA damages when combined with IR 31
6.3.4 WA increased apoptosis when combined with IR 31
6.4 Western blot &; Fluorescence Stain 32
6.4.1 A549 Cells 32
6.4.2 NCI-H1299 Cells 34
7 Discussion: 35
7.1 Clonogenic survival with or without IR 35
7.2 Cell viability and cell morphology 35
7.3 Combination of WA and IR on cell clonogenic survival, increased DNA damage, and apoptosis 36
7.4 Increased intrinsic pathway activity of apoptosis 37
7.5 Western blots 38
7.5.1 Proteins related to apoptosis 38
7.5.2 Proteins related to cell cycle control and/or DNA repair 39
7.5.3 HIF-1α, HIF-2α, β-catenin, and vimentin 39
8 Conclusions: 41
9 Perspectives 42
10 References: 43
11 Figures and Tables 51
12 Appendix 69
13 Publications 69

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