指導教授：阮雪芬臺灣大學：分子與細胞生物學研究所李明樺Li, Ming-HuaMing-HuaLi2014-11-252018-07-062014-11-252018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261104Broad-band infrared (IR) light is used in scientific, military, medical and industrial applications, but only a few about narrow band IR radiation is applied in biomedical uses. Breast cancer is one of the cancer-related leading causes of death worldwide. Breast cancer-related mortality is associated with the development of metastatic potential to lymph nodes and distant organs. Therefore, we aimed to investigate the effects of narrow band MIR on metastatic breast cancer cells. Middle infrared (MIR) in the wavelength range of 3 to 5 μm region was used to irradiate MDA-MB-231 breast cancer cells. In our results, we found MIR exposure inhibited breast cancer cell proliferation and induced morphological changes but did not affect normal breast cells. To figure out the MIR-triggered molecular mechanism in breast cancer cells, we performed isobaric tags for relative and absolute quantification (iTRAQ)-coupled LC-MS/MS system and identified 81 up-regulated and 86 down-regulated proteins. We identified these differentially expressed proteins were contributing to cell cycle progression and focal adhesion by bioinformatics functional enrichment analysis. Furthermore, we used western blotting to validate the differentially expressed proteins and the results showed the consistent with iTRAQ data. We also found that MIR increased the accumulation of G2/M cell population during cell cycle progression and the microtubule arrangement of MIR-exposed cells showed formed microtubule-organizing center (MTOC) without condensed chromatin, which is the feature of late interphase. These evidences suggested that MIR reduced cell proliferation by inhibiting mitosis process and migration activity and invasion ability by altering the localization of the focal adhesion molecules. In summary, MIR could inhibit MDA-MB-231 cell proliferation, migration activity and invasion ability by changing the protein expression level. Our finding suggest that MIR could be a potential implementation of breast cancer therapy.致謝............ I 中文摘要.... II ABSTRACT III CONTENTS V LIST OF FIGURES IX LIST OF TABLES XI Chapter 1 Introduction 1 1.1 Treatment of breast cancer 1 1.1.1 Surgery 1 1.1.2 Radiation therapy 1 1.1.3 Chemotherapy 2 1.1.4 Hormone therapy 2 1.1.5 Targeted therapy 3 1.1.6 Treatment of triple-negative breast cancer 4 1.2 Infrared 5 1.3 Quantitative proteomics analysis 5 1.3.1 Proteomics 5 1.3.2 Quantitative proteomics analysis using iTRAQ labeling 6 1.4 Experimental design 7 Chapter 2 Material & Methods 9 2.1 Cell culture 9 2.2 Middle infrared radiation (MIR) at 3-5 μm 9 2.3 Cell viability 10 2.4 MTT assay 10 2.5 Colony formation assay 11 2.6 Protein extraction 11 2.7 iTRAQ labeling 11 2.8 Strong cation exchang (SCX) chromatography 13 2.9 ZipTip desalting 14 2.10 LC-MS/MS analysis 14 2.11 Peptides search 16 2.12 Selection of differentially expressed proteins 16 2.13 Functional enrichment analysis and network construction 17 2.14 Western blot 18 2.15 Cell cycle analysis by flow cytometry 18 2.16 Immunocytochemical staining 19 2.17 Cell migration and invasion assay 20 Chapter 3 Results 21 3.1 MIR altered the morphology and inhibited the cell growth of breast cancer cells but did not affect normal breast cell 21 3.2 MIR exposure caused irreversible effects on long-term growth inhibition. 21 3.3 iTRAQ proteomics profiling of MIR irradiated MDA-MB-231 cells 22 3.4 The results of western blotting were consist with iTRAQ labeling 23 3.5 Enriched network analysis of MIR-induced differentially expressed proteins 23 3.6 MIR exposure resulted in cell cycle arrest at G2/M phase 24 3.7 MIR exposure induced DNA damage in MDA-MB-231 cells 25 3.8 MIR exposure induced microtubule rearrangement 26 3.9 MIR exposure altered arrangements of cell adhesion molecules 26 3.10 MIR affected cells migration activity and invasion ability 27 Chapter 4 Discussion 28 REFERENCE 33 FIGURES.... 38 TABLES....... 56 APPENDIX 906888892 bytesapplication/pdf論文公開時間：2014/08/25論文使用權限：同意有償授權(權利金給回饋學校)中波段紅外光乳癌蛋白質體學相對和絕對定量的等量異位標籤[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261104/1/ntu-103-R01B43018-1.pdf