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Studies on endometrial cancer : cell deaths and cross talks with endometrial stromal cells

Studies on endometrial cancer : cell deaths and cross talks with endometrial stromal cells,

조한태 (Hantae Jo, The Graduate School of Ajou University)

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TABLE OF CONTENTS
ABSTRACT (English) i
CHAPTER 1. Cell death induction of endometrial cancer cells by nitric oxide 1
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TABLE OF CONTENTS
ABSTRACT (English) i
CHAPTER 1. Cell death induction of endometrial cancer cells by nitric oxide 1
1.1. INTRODUCTION 2
1.2. MATERIALS&METHODS 4
1.2.1 Cell culture and Reagent 4
1.2.2 Nitric oxide generation and quantification 4
1.2.3 Nitric oxide Griess assay 4
1.2.4 Cell viability assay 5
1.2.5 Immunoblot assay 5
1.2.6 Annexin V and PI staining assay 6
1.2.7 Measurement of intracellular reactive oxygen species level 6
1.2.8 Statistical analysis 6
1.3. RESULTS 7
1.3.1 The plasma device generates nitric oxide gas whose concentrations depend on the O2 gas level. 7
1.3.2 Concentrations of nitric oxide dissolved in the cell culture media linearly depends on NO gas applied to the media 7
1.3.3. Nitric oxide reduces endometrial cancer cell viability but not endometrial stromal cells. 7
1.3.4 Nitric oxide induces apoptosis of HEC-1A cells. 8
1.3.5. Nitric oxide increases cell death signaling favoring apoptosis. 9
1.3.6. Nitric oxide induces ROS-mediated apoptosis in HEC-1A cells. 9
1.4. DISCUSSION 11
1.5. FIGURES LEGENDS 14
1.6. REFERENCES 25
CHAPTER 2. Phytoncides enhance anti-cancer activity of natural killer cells 29
2.1. INTRODUCTION 30
2.2. MATERIALS&METHODS 32
2.2.1 Cell culture and reagent 32
2.2.2 Mice tumor xenografts and phytoncide treatment regimens 32
2.2.3 Tumor volume estimation 33
2.2.4 Mice natural killer cell isolation and measurement of cytotoxicity 33
2.2.5 Component analysis of phytoncide scents by gas chromatography mass spectrometry 34
2.2.6 Natural Killer cell NK-92mi activation 34
2.2.7 Reverse transcription and real-time PCR 35
2.2.8 Natural killer cell cytotoxicity assay 35
2.2.9 Immunoblot assay 36
2.2.10 Statistical analysis 36
2.3. RESULTS 38
2.3.1. Phytoncide inhibited the growth of CT-26 colon cancer cell xenografts in balb/c mice. 38
2.3.2. Phytoncide enhances mouse spleen Natural Killer activity. 38
2.3.3. Ingredient analysis of phytoncide scents. 39
2.3.4. Phytoncides elevate the expression of cytotoxic effector proteins in NK-92mi cells 39
2.3.5. α-pinene increase NK-92mi cell cytotoxicity. 39
2.3.6. α -pinene induce Natural killer cytotoxicity activation through Erk, Akt pathways 40
2.4. DISCUSSION 41
2.5. FIGURES LEGENDS 43
2.6. TABLES LEGENDS 46
2.7. REFERENCES 59
CHAPTER 3. Endometrial cancer cell and endometrial stromal cell cross-talk induces the epithelial to mesenchymal transition via cytokines 65
3.1. INTRODUCTION 66
3.2. MATERIALS&METHODS 68
3.2.1 Cell culture and reagent 68
3.2.2 Wound healing assay 68
3.2.3 Reverse transcription PCR and real-time PCR 69
3.2.4 Human Cytokine assay 70
3.2.5 Overexpression of growth differentiation factor-15 (GDF15) and chemokine (C-C motif) ligand 2 (CCL2) in HEC-1A cells 70
3.2.6 Statistical analysis 71
3.3. RESULTS 72
3.3.1 Co-cultured endometrial cancer cells cross-talk with endometrial stromal cells via cytokines. 72
3.3.2 Co-culture of endometrial cancer cells increases its motility 72
3.3.3 Cytokine expression profiles in conditioned media 73
3.3.4 The overexpression GDF15 and CCL2 induces epithelial-mesenchymal transition (EMT) in HEC-1A cells 73
3.3.5 The estrogen-TNFα-CCL2 axis plays a role in cancer invasion/metastasis by amplifying its EMT 74
3.4. DISCUSSION 75
3.5. FIGURES LEGENDS 79
3.6. TABLES LEGENDS 82
3.7. REFERENCES 94
ABSTRACT (Korean) 101