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Anticancer activities of the bioactive compounds from Citrus unshiu leaf: molecular targets and mechanisms

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Abstract
ABSTRACT Citrus leaves are reputedly general tonics for gastrointestinal disorders and fevers. The leaf extract is consumed as an herbal tea and the leaf hydrosol from numerous Citrus species is used for some traditional dishes in Tunisian cuisine. The appropriate tree canopy for Citrus unshiu should be maintained by skirting and pruning. Therefore, Citrus unshiu leaves are readily available agricultural byproducts. Although there are reports on the biological effects and chemical compositions of certain species of citrus leaves, very limited information about the anticancer properties of Citrus unshiu leaves has been reported. Therefore, Citrus unshiu leaves usages as anticancer reagents in food and pharmaceutical industries has been neglected. In CHAPTER II, we assessed antiproliferative activities of the methanolic extract of Citrus unshiu leaves (MECL) using several cancer cell lines and marked cytotoxicity was observed in AGS human gastric cancer cells. Treatment with MECL induced non-apoptotic cell death and increased formation of the acidic vesicular organelles and LC3 puncta. MECL-induced cell death was significantly reduced by pretreatment with an autophagy inhibitor, 3-methyladeinine. Gas chromatographymass spectrometry identified the major compound of MECL to be phytol (47.35%). In CHAPTER III, we investigated cytotoxic effects of phytol, the major component of MECL, on cancer cells. Phytol induces apoptosis in AGS cells, as evidenced by an increase of the sub-G1 population, activation of caspases, and depolarization of the mitochondrial membrane. Co-treatment with chloroquine (CQ), a lysosomal inhibitor, strongly enhanced phytol-induced apoptosis in AGS cells, suggesting that phytol could induce protective autophagy. Furthermore, N-acetyl-Lcysteine, a reactive oxygen species (ROS) scavenger, increased the phytol-induced cytotoxicity, but decreased the levels of p62 and the formation of the acidic vesicular organelles, suggesting that the Nrf2 pathway was induced by phytol. In CHAPTER IV, we fractionated methanolic extract of Citrus unshiu leaves by using n-hexane, chloroform, ethyl acetate, n-butanol and water; we found 2,4-ditert-butylphenol (DTBP) as the most potent candidate for anticancer agent in Citrus unshiu leaves. DTBP increased acetylation of α-tubulin, p21 and Rb expression, but reduced β-catenin expression and cell proliferation. Increase of α-tubulin acetylation by DTBP resulted in tubulin polymerization, consequently, inducing aberrant mitosis.
Author(s)
송연우
Issued Date
2017
Awarded Date
2017. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007962
Alternative Author(s)
Yeon Woo Song
Department
대학원 응용생명공학과
Advisor
김소미
Table Of Contents
ABSTRACT i
LIST OF TABLES . vii
LIST OF FIGURES viii
CHAPTER I. Introduction 1
1. Anticancer properties of phytochemicals 2
1-1. Nuclear factor erythroid-2-related factor 2 (Nrf2) . 5
1-2. Histone modifications 7
1-3. Histone deacetylases (HDACs) . 9
2. Cell death mechanisms . 11
2-1. Apoptosis 11
2-2. Autophagy 12
2-3. Necroptosis . 13
2-4. Caspase-independent cell death . 15
2-5. Senescence . 16
3. Research rationale 17
CHAPTER II. Citrus unshiu leaf extract induces autophagic cell death in human gastric adenocarcinoma AGS cells 19
1. ABSTRACT 20
2. INTRODUCTION 21
3. MATERIALS AND METHODS . 23
3-1. Plant samples 23
3-2. Cell culture . 23
3-3. Reagents . 23
3-4. Cell viability . 24
3-5. Cell morphology analysis . 24
3-6. Flow cytometry 24
3-7. LC3-GFP transfection 25
3-8. GC-MS analysis . 25
3-9. Statistical analysis 26
4. RESULTS . 27
4-1. Citrus unshiu leaves extract reduced viability of AGS cells 27
4-2. Analysis of cell cycle and the effects of 3-MA on MECL-induced cell death 29
4-3. MECL induced autophagy in AGS cells 31
4-4. Compositional analysis by GC-MS 33
5. DISCUSSION 36
CHAPTER III. Phytol, a major compound of methanolic extract of Citrus unshiu leaf, induces apoptosis and ROS-mediated autophagy in AGS cells 38
1. ABSTRACT . 39
2. INTRODUCTION 40
3. MATERIALS AND METHODS . 45
3-1. Cell culture . 44
3-2. Reagents . 44
3-3. Cell viability assay . 44
3-4. Cell morphology . 45
3-5. Flow cytometry 45
3-6. Western blotting analysis 45
3-7. Reverse transcription-polymerase chain reaction (RT-PCR) . 46
3-8. Statistical analysis 47
4. RESULTS . 49
4-1. Phytol induced apoptosis in AGS cells 48
4-2. Phytol induced autophagy in AGS cells . 51
4-3. Phytol induced protective autophagy in AGS cells 53
4-4. Phytol induced ROS-mediated Nrf2 cytoprotective pathways 55
5. DISCUSSION 58
CHAPTER IV. 2,4-Di-tert-butylphenol, a component of chloroform fraction of Citrus unshiu leaf, induces senescence and mitotic catastrophe in AGS cells 62
1. ABSTRACT . 63
2. INTRODUCTION 64
3. MATERIALS AND METHODS . 66
3-1. Cell culture . 66
3-2. Cell viability assay . 66
3-3. Colony formation assay 66
3-4. Flow cytometry 66
3-5. Western blotting analysis 67
3-6. Immunofluorescence staining 67
3-7. Tubulin polymerization assay 67
3-8. Senescence associated-β-galactosidase staining 68
3-9. In silico docking . 68
3-10. Statistical analysis 68
4. RESULTS . 69
4-1. DTBP as a potential therapeutic compound detected in Citrus unshiu leaves extract . 69
4-2. Growth inhibitory effect of DTBP . 72
4-3. Effects of DTBP on the cell cycle 74
4-4. DTBP induces senescence in AGS cells . 76
4-5. DTBP induces aberrant mitosis and multinucleated cells 78
4-6. DTBP inhibits HDAC6 activity . 80
5. DISCUSSION 87
REFRENCES 90
Degree
Doctor
Publisher
제주대학교 일반대학원
Citation
송연우. (2017). Anticancer activities of the bioactive compounds from Citrus unshiu leaf: molecular targets and mechanisms
Appears in Collections:
General Graduate School > Biomaterials Science and Technology
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