대장암 세포주에서 Curcumin에 의한 세포사멸 기전 = The molecular mechanism of cell death by curcumin in human colorectal HCT116 cells

다국어 초록 (Multilingual Abstract)

Curcumin, a diferuloylmenthane derived from the rhizome of curcuma longa, is active component of the spice tumeric. Several studies report that curcumin inhibits cancer cell proliferation and induces apoptosis in various cancer cells. To investigate whether curcumin could affect cancer cell viability and gene expression, we performed cell viability assay and oligo DNA microarray experiment. A dramatic decrease of cell viability was detected in 25 ?M curcumin-treated cells. In addition, we found that 6,855 genes were up-regulated more than 2-folds whereas 8,134 genes were down-regulated more than 2-folds by 25 ?M curcumin treatment. Among up-regulated genes, we selected 3 genes (activating transcription factor-3 [ATF-3], growth arrest and DNA-damage-inducible, alpha [GADD45A], and nuclear receptor subfamily 4, group A, member 1 [NR4A1]) to confirm microarray data. The results of real-time PCR was highly accorded with those of microarray data. Among the up-regulated genes by curcumin, we selected activating transcription factor 3 (ATF3) for further experiment, because ATF3 is a transcription factor involved in cell growth, apoptosis, and invasion. ATF3 expression was induced by 25 ?M treatment of several other phytochemicals (capsaicin, genistein, resveratrol, curcumin). Among treated phytochemicals, curcumin is the highest inducer of ATF3. The knock-down of ATF3 recovered cell death induced by curcumin. This result indicates that ATF3 overexpression is directly involved in cell death by curcumin. In addition, ectopic expression of ATF3 induced the expression of apopotosis-inducing genes. And, we also performed 2D-gel electrophoresis and MALDI-TOF to identify differentially expressed proteins by curcumin. Overall, these results may help to understand molecular mechanisms of cell death induced by curcumin in human colorectal HCT116 cells.

목차 (Table of Contents)

• 목차
• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법
• 1. 세포배양 및 재료 6
• 2. Cell Viability Assay (MTS assay) 6
• 3. Curcumin에 의한 세포의 형태적 변화 관찰 7
• 4. Total RNA 추출 7
• 5. Oligo DNA microarray 실험 8
• 6. Reverse transcription-polymerase chain reaction (RT-PCR) 9
• 7. Quantitative real-time PCR 10
• 8. Transient transfection 12
• 9. Flow cytometric analysis 13
• 10. 2D-PAGE 및 MALDI-TOF analysis 13
• Ⅲ. 결과 및 고찰
• 1. 파이토케미칼이 HCT116 세포주 성장에 미치는 영향 15
• 2. Curcumin처리에 의한 HCT116 세포주의 형태적 변화 17
• 3. Oligo DNA microarray 실험을 이용한 유전체 전체 수준에서의 유전자 발현 분석 18
• 4. Real-time PCR을 통한 DNA microarray 실험 검증 25
• 5. 파이토케미칼 처리 및 농도별 curcumin 처리에 의한 ATF3 유전자의 발현 27
• 6. ATF3-siRNA에 의한 ATF3 유전자 발현 억제 29
• 7. Curcumin에 의한 세포사멸이 ATF3의 발현억제에 의해 회복 31
• 8. ATF3 유전자의 과대발현에 의해 차별적으로 발현되는 유전자의 분석 33
• 9. Curcumin 처리에 의해 차별적으로 발현되는 단백질 분석 35
• Ⅳ. 요 약 38
• Ⅴ. 참고문헌 40
• Ⅵ. 영문초록 46
• 그림목차
• Fig. 1. Chemical structure of curcumin 5
• Fig. 2. Effects of phytochemicals on HCT116 cell viabilities 16
• Fig. 3. Morphological changes in curcumin-treated HCT116 cells 17
• Fig. 4. Confirmation of microarray analysis with real time PCR 26
• Fig. 5. ATF3 expression by curcumin and several phytochemicals 28
• Fig. 6. The knock-down of ATF3 expression by ATF3-siRNA... 30
• Fig. 7. Flow cytometric analysis in curcumin treated-HCT116 cells 32
• Fig. 8. ATF3 overexpression by pCG-ATF3 transfection 34
• Fig. 9. 2D-gel electrophoresis in curcumin treated-HCT116 cells 36
• 표목차
• Table 1. Oligonucleotide primers used for real-time... 11
• Table 2. Up-regulated genes more than 2-folds... 19
• Table 3. Down-regulated genes more than 2-folds... 24
• Table 4. Several up-regulated genes more than... 34
• Table 5. Lists of proteins identified with MALDI-TOF 37