@phdthesis{oai:sucra.repo.nii.ac.jp:00018679,
 author = {ANCHALEE RAWANGKAN},
 month = {},
 note = {x, 89 p., Green tea catechin, （-）-epigallocatechin gallate （EGCG）, and green tea are well known effective non-toxic cancer preventives. To understand the mechanisms of action of EGCG that results in wide beneficial effects on cancer prevention, I studied whether EGCG enhances anti-tumor immunity by focusing on the immune checkpoint. Programmed cell death-ligand 1 （PD-L1） in tumor cells is an immune checkpoint molecule involved in immune evasion of tumor: It inhibits the immune response of T cells by binding to PD-1 on T cells. The PD-L1 expression is induced by cytokines and growth factors in the inflammatory tumor microenvironment. In this thesis, I studied the effects of EGCG on inducible PD-L1 expression in human lung cancer cells, and the stimulation of T-cell functions, examined by the model experiment using tumor specific cluster of differentiation 3 positive （CD3+） T cells co-cultured with F10-OVA cells.
First, I found that PD-L1 protein levels varied among human lung cancer cell lines: Lu99 cells showed the highest PD-L1 expression, A549 cells were moderate, and H1299 were very low. Treatment with interferon gamma （IFN-γ） increased cell-surface PD-L1 level in A549 and H1299 cells as determined by flow cytometry, but not in Lu99 cells. However, treatment with epidermal growth factor （EGF） increased PD-L1 expression in Lu99 cells. Pretreatment with EGCG and green tea extract （GTE） for 3 h dose-dependently reduced IFN-γ-induced PD-L1 mRNA and cell-surface PD-L1 level in A549 and H1299 cells by inhibiting phosphorylation of STAT1. EGCG also inhibited EGF-induced PD-L1 expression by inhibiting the AKT pathway in Lu99 cells.
Next, I examined the relationship between inhibition of PD-L1 expression and lung cancer prevention. The intraperitoneal injection of 4-（methylnitrosamino）-1-（3-pyridyl）-1-butanone （NNK） induced lung tumors in 100% A/J mice in 16 weeks and oral administration of 0.3% GTE reduced the number of tumors per mouse from 4.1 to 2.6. In addition, the percentage of positive PD-L1 cells in tumors in NNK + GTE group was reduced from 9.6 to 2.9 （70% reduction）.
Finally, I studied whether EGCG enhances anti-tumor immunity. I performed an ex vivo co-culture experiment using tumor specific CD3+ T cells with F10-OVA cells, to determine restoration of T cells activity: Pretreatment with EGCG reduced PD-L1 mRNA level in F10-OVA cells, and it increased IL-2 mRNA expression 1.7-fold. However, EGCG had no effect in non-co-cultured T cells. According to the reduction of PD-L1 by EGCG, apoptosis of F10-OVA cells was increased 3-fold by co-culture with tumor specific CD3+ T cells.
This is the first finding to show that EGCG and green tea extract act as alternative immune checkpoint inhibitors. This new function of EGCG in anti-tumor immune response plays a vital role in cancer prevention and enhancement of anti-cancer activities., ABSTRACT I
ACKNOWLEDGEMENTS III
TABLE OF CONTENTS V
LIST OF FIGURES VIII
Chapter 1: General introduction and objectives 1
1.1. Research background 1
1.1.1. Characteristics of green tea and green tea catechins 1
1.1.2. Cancer preventive activity of EGCG and green tea extract (GTE) in rodents 2
1.1.3 Cancer preventive activity of green tea in human 3
1.1.4. Synergistic enhancement of anti-cancer activity 5
1.1.5. Mechanisms of action of green tea catechins for anti-cancer activity 6
1.1.6. PD-L1/PD-1 immune checkpoint 9
1.1.7. Promotion of cancer immune escape by PD-L1 expression 11
1.1.8. Immune checkpoint inhibitors 12
1.2. Hypothesis and general objectives 14
Chapter 2: Green tea catechins inhibit PD-L1 expression and lung tumor growth as an alternative immune checkpoint inhibitor 15
2.1. Introduction 15
2.2. Materials and Methods 19
2.2.1. Cell lines and reagents 19
2.2.2. Treatment with green tea catechins and IFN-γ or EGF 22
2.2.3. RNA extraction and quantitative real-time RT-PCR (qRT-PCR) 22
2.2.4. Western blotting 24
2.2.5. Flow cytometry 25
2.2.6. NNK-induced lung carcinogenesis experiment in A/J mice 25
2.2.7. Immunohistochemical staining 26
2.2.8. Establishment of ovalbumin-expressing B16-F10 (F10-OVA) cells 27
2.2.9. Isolation of tumor specific CD3+ T cells 27
2.2.10. Cytotoxic assay with tumor specific CD3+ T cells 28
2.2.11. Analysis of apoptosis 29
2.2.12. Statistical analysis 29
2.3. Results 30
2.3.1. Green tea catechins inhibited PD-L1 expression and lung tumor development 30
2.3.1.1. Intrinsic PD-L1 expression levels varied among NSCLCs 30
2.3.1.2. IFN-γ and EGF inducted PD-L1 mRNA and protein in three NSCLC cell lines 30
2.3.1.3. Green tea catechins and GTE reduced IFN-γ-induced cellsurface PD-L1 in A549 cells 32
2.3.1.4. EGCG inhibited IFN-γ-induced PD-L1 mRNA and protein in A549 and H1299 cells 32
2.3.1.5. EGCG inhibited IFN-γ-induced phosphorylation of STAT1 compared with STAT1- and JAK2-inhibitors 33
2.3.1.6. EGCG inhibited EGF-induced PD-L1 mRNA and protein in Lu99 cells via AKT signal pathway 34
2.3.1.7. EGCG inhibited PD-L2 mRNA expression induced by IFN-γ or EGF 34
2.3.1.8. Oral administration of GTE inhibited lung tumor development induced by NNK and reduces PD-L1 positive cells in the tumors 35
2.3.2. EGCG stimulated the anti-tumor response of T cells by inhibition of PD-L1 expression 36
2.3.2.1. EGCG inhibited IFN-γ-induced PD-L1 mRNA and protein in B16-F10 mouse melanoma cells 36
2.3.2.2. EGCG inhibited PD-L1 expression in F10-OVA cells induced by co-cultured with tumor specific CD3+ T cells 37
2.3.2.3. EGCG restored IL-2 mRNA expression in tumor specific CD3+ T cells co-cultured with F10-OVA cells 38
2.3.2.4. EGCG stimulated apoptosis of F10-OVA cells by co-cultured with tumor specific CD3+ T cells 38
2.4. Discussion 40
2.5. Conclusion 44
ABBREVIATIONS 45
REFERENCES 46
FIGURES 63, 指導教員 : 菅沼雅美, text, application/pdf},
 school = {埼玉大学},
 title = {Mechanistic basis of lung cancer prevention with green tea catechins focused on anti-tumor immunity},
 year = {2018},
 yomi = {アンチェリー ラワンガーン}
}