폐암세포에서 Death Receptor의 과발현과 STAT3 활성 억제를 통한 Tectochrysin 의 항암효과

국문 초록 (Abstract)

사람 폐암 세포인 A549와 NCI-H460 에서 tectochrysin 이 Death receptor (DR) 들의 과발현과 STAT3 인산화의 억제를 통하여 암세포 성장을 억제시킨다는 기전을 연구하였다. Alpinia oxyphylla에서 추출한 tectochrysin은 농도 의존적으로 세포자살의 기전을 통하여 폐암세포인 A549와 NCI-H460 의 성장을 억제하였다. DR3와 FAS의 발현도 또한 두 암세포에서 수반하여 증가하였지만, 항 세포자살 단백질인 Bcl-2 의 발현은 감소하였다. Cleaved caspase-3, Cleaved caspase-9, Cleaved caspase-8 그리고 Bax를 포함한 DR의 하부 기전 단백질들의 발현도 또한 증가하였다. Tectochrysin 투여는 또한 A549와 NCI-H460 세포에서 STAT의 인산화를 억제시켰다. 게다가, siRNA에 의한 DR3와 FAS의 발현의 억제는 두 세포에서 모두 STAT3의 억제뿐 만 아니라 tectochrysin을 통한 세포 성장 억제 효과도 또한 반전시켰다. 그러므로 이번 실험의 결과로서 tectochrysin은 DR3와 FAS의 과발현과 STAT3 인산화의 억제를 통해 폐암세포에서 세포의 성장 억제를 일으키는 것을 확인하였다.

다국어 초록 (Multilingual Abstract)

Tectochrysin, a phenolic compound isolated from Alpinia oxyphylla Miquel, was investigated whether it inhibits cancer cell growth through suppression of STAT3 phosphorylation and enhancement of death receptor (DR)s in the human lung cancer cells; A549 and NCI-H460. Tectochrysin (0-80 M) suppressed the growth of A549 and NCI-H460 lung cancer cells by inducing of apoptotic cell death in a concentraion dependent manner. Expression of DR3 and Fas as well as DR downstream pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax were concomitantly increased, but the expression of anti-apoptotic proteins; Bcl-2 was decreased in both cancer cells. In addition, tectochrysin treatment also inhibited phosphorylation of STAT3 in A549 and NCI-H460 cells. However, deletion of DR3 and Fas by small interfering RNA significantly reversed tectochrysin-induced cell growth inhibitory effect as well as down regulation of STAT3 in A549 and NCI-H460 lung cancer cells. Moreover, combination of low concentration tectochrysin (20 M) and TWEAK (6 nM) or FasL (6 nM) synergistically inhibited both lung cancer cells growth accompanied with inhibition of STAT3. Docking model showed interaction of tectochrysin with STAT3. Therefore, our results indicate that tectochrysin leads to apoptotic cell death in lung cancer cells through activation of DR3 and Fas expression via inhibition of STAT3 phosphphorylation.

목차 (Table of Contents)

• Ⅰ. Introduction
• 1. Lung cancer 1
• 2. Death receptors 2
• 3. Role of STAT3 in lung cancer cell growth 5
• 4. Tectochrysin 6
• 5. Purpose of this study 9
• Ⅱ. Material and method
• 1. Chemicals 10
• 2. Docking experiment 11
• 3. Cell culture 12
• 4. MTT assay 12
• 5. Colony formation assay 13
• 6. Western blot analysis 13
• 7. Transfection 14
• 8. Detection of apoptosis 15
• 9. Gel electrophoretic mobility shift assay 16
• 10. Data analysis 17
• Ⅲ. Results
• 1. Inhibitory effect of tectochrysin on cell growth in lung cancer and lung cells 18
• 2. Apoptotic cell death by tectochrysin 21
• 3. Effect of tectochrysin on expression of apoptotic cell death regulatory proteins 23
• 4. Inhibition of STAT3 activity by tectochrysin 25
• 5. Reversed effect of DR3 and FAS siRNAs on tectochrysin-induced cell growth inhibition and STAT3 activity 27
• 6. Combination effect of DR3 and FAS ligand with tectochrysin in NSCLC cell growth 30
• 7. Structure of tectochrysin and Interaction between tectochrysin and STAT3 32
• Ⅳ. Disscussion 34
• Ⅴ. References 41
• Ⅵ. Summary in Korean 50