Dynamic interplay between macrophages and gastric cancer in chemotherapy
- Alternative Title
- 항암화학요법에서 대식세포와 위암 세포 사이의 역동적 상호작용에 대한 연구
- Abstract
- Inflammation is related to a large number of diseases like diabetes, cardiovascular disorders, Alzheimer's disease, autoimmune and pulmonary diseases, arthritis, and cancer. Immune cells, especially macrophages, play a major part by the production of inflammatory mediators such as cytokines through the activation of various pathways. Cancer is a major health burden worldwide. The incidence rate of gastric cancer is especially high in Eastern Asia. The control or regulation of cell cycle by induction of cell death through activation cell cycle arrest or induction of apoptosis are the major aim of cancer experiments. In the past decades, various bioactive compounds from plants have been investigated for the purpose of discovering a treatment for human diseases. Aster incisus is a common plant mostly found in Asia and has traditionally been used for medicinal purposes in South Korea. In this thesis, we evaluated the potential anti-inflammatory and anticancer effects of a methanol extract from Aster incisus in murine macrophages (Raw 264.7 cells), human gastric adenocarcinoma (AGS cells).
Results presented in this thesis show that Aster incisus successfully inhibited the production of nitric oxide (NO) and the expression of inflammatory cytokines (TNFα, IL-1β, IL-6, iNOS, Cox-2) through the regulation of NFkB, MAPK and Akt pathways in LPS-stimulated Raw 264.7 macrophages treated with Aster incisus. Additionally, Aster incisus scavenged DPPH therefore we were able to determine that Aster incisus has antioxidant effect. In gastric adenocarcinoma, Aster incisus inhibited the proliferation of AGS cells. Further analysis demonstrated that Aster incisus inhibited the proliferation of AGS cells by the induction of cell cycle arrest in G1 phase and by apoptosis. G1 phase cyclins (cyclin D1/3, cyclin E2) and CDK (CDK4/6, CDK2) were down-regulated while inhibitors (p16, p18, p21, p27) were over-expressed. Pro-apoptotic proteins (Bid, Bad, Bak, cytochrome c, AIF, cleaved caspase-3, -8, -9 and cleaved PARP) were also highly expressed in AGS cells treated with Aster incisus.
Inflammation has been proven to play a crucial role in tumor microenvironment. Macrophages (Tumor-Associated-Macrophages) are the most abundant cells in the tumor microenvironment and play a major role in the resistance of cancer cells to chemotherapy. In this thesis, we tested the effect of 5-FU treatment on AGS cells before and after coculture with tumor-associated macrophages. Tumor-associated macrophages induced the survival of AGS gastric cancer cells through PI3K/Akt pathway, inhibited H2O2-induced oxidation through Nrf2-HO1. Additionally, tumor-associated macrophages increased migration and invasion of 5-FU-treated-AGS cells cocultured with tumor-associated macrophages by the regulation of integrin β3, FAK, paxillin, E-cadherin proteins.
염증은 당뇨병, 심혈관계 질환, 알츠하이머, 자가 면역 질환, 폐 질환, 관절염 및 암과 같은 많은 질병에 관련되어 있다. 면역 세포, 특히 대식세포는 다양한 경로를 통하여 활성화되고, cytokine과 같은 염증매개체의 생성에 중요한 역할을 한다. 암은 전세계적으로 큰 건강 문제로 여겨지고 있으며, 특히 위암의 경우 동아시아에서 높은 발병률을 보인다. 세포 주기를 정지시키거나, 세포자살을 유도하여 세포 주기를 조절하는 것을 이용하여 세포의 사멸을 유도하는 것은 암과 관련된 실험에서의 주요 목표이다. 지난 수십 년 동안 인간의 질병에 대한 치료법을 발견하기 위해 식물이 가지고 있는 다양한 생리활성 물질들이 사용되었다. Aster incisus는 아시아에서 흔히 볼 수 있는 식물로서 한국에서는 전통적으로 약초로 사용되어왔다. 본 논문에서는 쥐의 대식세포(Raw 264.7 세포주)와 인간 위암(AGS 세포주)에서 Aster incisus의 메탄올 추출물의 항염증 효과와 항암 효과를 확인하였다.
이 논문의 결과에서는 Aster incisus를 처리하였을 때, NFkB, MAPK 및 Akt 신호전달 경로의 조절을 통하여 LPS에 의해 자극된 Raw 264.7 대식세포에서 산화 질소 (NO)와 염증 관련 cytokine들 (TNFα, IL-1β, IL-6, iNOS, Cox-2) 의 생성이 성공적으로 억제되는 것을 확인하였다. 또한, Aster incisus는 DPPH의 radical을 제거하므로 항산화 효과가 있는 것 역시 확인하였다. 위선암에 대한 효과로는 Aster incisus를 처리하였을 때, AGS 세포의 증식이 억제되었다. 추가적인 분석을 진행하였을 때, Aster incisus가 AGS 세포의 성장을 억제하는 것은 G1 기에서 세포 주기를 정지시키는 것과 세포자살을 유도하는 것에 의해 일어나는 것을 확인하였다. G1 기에서 발현되는 cyclin들(cyclin D1 / 3, cyclin E2)과 CDK (CDK4 / 6, CDK2)의 발현은 억제되었고, 이들에 대한 억제 단백질들 (p16, p18, p21, p27)은 발현이 증가하였다. 세포자살 유도 단백질들(Bid, Bad, Bak, cytochrome c, AIF, cleaved caspase-3, -8, -9 및 cleaved PARP) 역시 Aster incisus를 처리한 AGS 세포에서 높게 발현되었다.
염증은 종양 미세 환경에서 중요한 역할을 하는 것으로 알려져 있다. 대식세포 (종양 관련 대식세포)는 종양 미세 환경에서 가장 많이 존재하며, 화학 요법에 대한 암세포의 저항성에 깊게 관련되어있다. 이 논문에서 우리는 종양 관련 대식세포와 공동 배양하기 전후에서의 AGS 세포에 대한 5-FU의 효과를 확인하였다. 종양 관련 대식세포는 PI3K / Akt 경로를 통해 AGS 위암 세포의 생존을 유도하고, Nrf2-HO1을 통해 H2O2에 의해 유도되는 산화반응을 억제하였다. 또한 종양 관련 대식세포는 integrin β3, FAK, paxillin, E-cadherin 단백질의 조절을 통하여 종양 관련 대식세포와 공동 배양되고 있는 5-FU가 처리된 AGS 세포의 전이와 침습을 증가시켰다.
- Issued Date
- 2019
- Awarded Date
- 2019. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 미생물학과
- Advisor
- 김군도
- Table Of Contents
- Contents i
List of Figures v
List of Abbreviations viii
Abstract x
CHAPTER ONE: INTRODUCTION 1
1.1. Inflammation 3
1.1.1. Molecular mediators of Inflammation 4
1.1.2. Cellular mediators of Inflammation 5
1.1.3. Inflammation in diseases 8
1.2. Gastric cancer 9
1.2.1. Anatomy and Physiology of Normal stomach 9
1.2.2. Epidemiology and Risk Factors of gastric cancer 10
1.2.3. Diagnosis and Staging 12
1.2.4. Treatment of gastric cancer 18
1.3. Macrophages in Tumor Microenvironment 21
1.3.1. Tumor microenvironment 21
1.3.2. Tumor-associated macrophages 22
1.3.2.1. Macrophage Phenotype and activation 23
1.3.2.2. Tumor associated macrophages 24
1.3.2.3. Macrophages and chemoresistance 26
1.3.3. Targeting Macrophages in Cancer Therapy 26
1.4. Aim of this thesis 28
1.5. References 29
CHAPTER TWO: Antioxidant and anti-inflammatory effects of Aster incisus in RAW 264.7 cells 37
2.1. Abstract 38
2.2. Introduction 39
2.3. Materials and Methods
2.3.1. Cell lines and reagents 40
2.3.2. Gas Chromatography-Mass Spectroscopy (GS-MS) 41
2.3.3. DPPH assay 41
2.3.4. Cell culture 42
2.3.5. Cell viability assay 42
2.3.6. NO assay 42
2.3.7. Western blot analysis 43
2.3.8. RT-PCR 43
2.3.9. Immunofluorescence 44
2.3.10. Statistical analysis 44
2.4. Results
2.4.1. GC-MS results 45
2.4.2. Anti-oxidant capacities of AIE 48
2.4.3. AIE effects on cells viability 50
2.4.4. Inhibition of nitric oxide production by AIE 52
2.4.5. Inhibition of inflammatory mediators by AIE 54
2.4.6. Inhibition of NFkB phosphorylation and translocation in LPS-stimulated Raw 264.7 cells by AIE 57
2.4.7. AIE inhibited the activation of MAPK pathway 59
2.4.8. AIE inhibited the activation of PI3K/Akt pathway 61
2.5. Discussion 63
2.6. References 67
CHAPTER THREE: Anticancer effects of Aster incisus in AGS gastric adenocarcinoma cells
3.1. Abstract 74
3.2. Introduction 75
3.3. Materials and Methods
3.3.1. Cell culture and viability assay 77
3.3.2. DAPI staining 78
3.3.3. Western blot 78
3.3.4. Immunofluorescence 79
3.3.5. FACS analysis 79
3.3.6. Wound healing assay 80
3.3.7. Cell invasion 80
3.3.8. Statistical analysis 80
3.4. Results
3.4.1. AIE inhibited the proliferation of human cancer cells 81
3.4.2. AIE induced apoptotic changes in treated cancer cell lines 83
3.4.3. AIE induced apoptosis in AGS gastric cell lines 85
3.4.4. AIE induced G1/S phase cell cycle arrest in AGS gastric cancer cells 88
3.4.5. AIE inhibited cell migration and invasion in AGS cells 91
3.5. Discussion 93
3.6. References 99
CHAPTER FOUR: Macrophages mediate the resistance to fluorouracil in AGS gastric adenocarcinoma 105
4.1. Abstract 106
4.2. Introduction 107
4.3. Materials and Methods
4.3.1. Cell culture 109
4.3.2. Cell viability assay 109
4.3.3. Polarization of macrophages 109
4.3.4. Coculture experiments 110
4.3.5. NO assay 110
4.3.6. Western Blot analysis 111
4.3.7. Cell invasion assay 111
4.3.8. Immunofluorescence 112
4.3.9. Statistical analysis 113
4.4. Results
4.4.1. LPS and IL-4 successfully polarized Raw 264.7 cells into M1 and M2 macrophages respectively 113
4.4.2. M2 macrophages induced the inhibition of oxidative stress through Nrf2/HO-1 and HIF-1α 117
4.4.3. M2 macrophages induced cell survival in AGS cells through PI3K/Akt pathway 120
4.4.4. M2 macrophages induced cell migration and invasion in AGS cells treated with 5FU 124
4.5. Discussion 129
4.6. References 134
국문요약 140
Acknowledgements 142
- Degree
- Doctor
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