지브라피쉬를 이용한 방사선보호물질 탐색과 이를 통해 도출된 후보 물질의 방사선 조사성 구내염에 대한 치료효과 분석과 보호 기전 연구
Screening of radioprotective agents using zebrafish and Therapeutic effects and protective mechanism of radioprotective candidates on radiation-induced oral mucositis
- 주제(키워드) Korean red ginseng (KRG); head and neck cancer; radiation-induced mucositis; apoptosis; radioprotection
- 발행기관 아주대학교
- 지도교수 김철호
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000019009
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
-Abstract- Screening of radioprotective agents using zebrafish and Therapeutic effects and protective mechanism of radioprotective candidates on radiation-induced oral mucositis Radiation-induced oral mucositis is a dose-limiting toxic side effect for patients with head and neck cancer. About 50–90% of cancer patients who undergo radiation therapy experience oral mucositis. Current oral mucositis therapies are mostly palliative. Some of these include antibiotics, analgesics, general oral hygiene, and the use of various mucosal barrier agents and radioprotective agents, such as amifostine. Despite the clinical significance of oral mucositis, there is no definite approved treatment In this study, we have used the in vivo zebrafish model to screen for radioprotective agents. The zebrafish, a small vertebrate species, has been widely used as an in vivo model system to study human disease because of its marked similarity to mammals in many of the key genes involved in developmental processes, cell cycle progression and proliferation, and differentiation. Unlike other vertebrate species, however, zebrafish are rapidly bred and easily maintained in the laboratory. Zebrafish embryos provide a versatile model system for assaying radioprotectors/radiomitigators in a vertebrate organism, both on a systemic and organ-specific basis. Through in vivo zebrafish screening, we found several candidates to decrease radiation induced damage. Korean red ginseng (KRG, steamed root of Panax ginseng CA Meyer) is one of the candidates. KRG has been an established traditional herbal medicine for millenia. The spectrum of medicinal effects of KRG include antibacterial, antiviral, antioxidative, antitumor, antimutagenic, and immune-modulatory activities. Many of these medicinal effects are attributed to the triterpene glycosides known as ginsenosides (saponins). Our aim was to investigate the effectiveness of KRG on radiation-induced damage in the human keratinocyte cell line HaCaT and in an in vivo zebrafish model. Radiation inhibited HaCaT cell proliferation and migration in a cell viability assay and wound healing assay, respectively. KRG protected against these effects. KRG attenuated the radiation-induced embryotoxicity in the zebrafish model. Irradiation of HaCaT cells caused apoptosis and changed in mitochondrial membrane potential (MMP). KRG inhibited the radiation-induced apoptosis and intracellular generation of reactive oxygen species (ROS), and stabilized the radiation-induced loss of MMP. Western blots revealed KRG-mediated reduced expression of ataxia telangiectasia mutated protein (ATM), p53, c-JunN-terminal kinase (JNK), p38 and cleaved caspase-3, compared with their significant increase after radiation treatment. The collective results suggest that zebrafish embryotoxicity model is a valuable model for radiation protection drug screening and KRG protects HaCaT cells by blocking ROS generation, inhibiting changes in MMP, and inhibiting the caspase, ATM, p38 and JNK pathways. We suggest that KRG can potentially be used as a protective agent against radiation-induced oral mucositis, which is a common complication of radiotherapy for head and neck cancers. Keywords: Korean red ginseng (KRG); head and neck cancer; radiation-induced mucositis; apoptosis; radioprotection
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v
ABBREVIATIONS vi
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 3
A. Preparation of Korean red ginseng extracts 3
B. Cell culture 3
C. Zebrafish screening model 3
D. Cell viability assay 4
E. Treatment effects on zebrafish morphology and survival 4
F. Colony-forming assay 5
G. Wound-healing assay 5
H. Annexin V-fluorescein isothiocynate/propidium iodide staining 6
I. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick
end labeling (TUNEL) assay 6
J. MMP assessment by JC-1 staining 7
K. Measurement of intracellular ROS generation 7
L. Western blot analysis 8
M. Statistical analyses 8
Ⅲ. RESULTS
A. Korean Red Ginseng increase HaCaT cell viability after irradiation 10
B. Korean red ginseng does not decrease the therapeutic effect of radiation
against cancer cells 13
C. Korean red ginseng increases clonogenic survival after radiation 15
D. Korean Red Ginseng restores the migration ability of HaCaT cells, which was decreased after irradiation 18
E. Korean red ginseng inhibits radiation-induced apoptosis in HaCaT cells 21
F. KRG inhibits intracellular ROS generation by radiation 24
G. Korean red ginseng protects mitochondria by stabilizing MMP 26
H. Korean red ginseng rescues HaCaT cells by inhibiting the caspase-3 pathway
And the activation of ATM and p53 28
IV. DISCUSSION 31
V. CONCLUSION 34
REFERENCES 35
국문요약 39
