초록 ▼

◆ 연구목표
<최종목표>
- 항암제 내성 인체 대장암 세포주를 이용한 유전체 및 단백질체 연구를 통하여 내성 관련 유전자 및 단백질들을 발굴하고 그 역할을 규명함으로써 새로운 내성 관련 기작의 이해를 확대
- 임상적으로 적용이 가능한 항암제 내성 예측인자나 항암보조제 개발의 토대를 마련

<당해연도목표>
- 항암제 내성에 큰 차이를 보이는 대장암 세포주들간의 단백질체 분석을 통한 내성 관련 단백질들의 동정
- 기존 연구를 통하여 얻어진 내성 관련 후보 단백질의 발현 조절과 활성 조절이 대장암 세

◆ 연구목표
<최종목표>
- 항암제 내성 인체 대장암 세포주를 이용한 유전체 및 단백질체 연구를 통하여 내성 관련 유전자 및 단백질들을 발굴하고 그 역할을 규명함으로써 새로운 내성 관련 기작의 이해를 확대
- 임상적으로 적용이 가능한 항암제 내성 예측인자나 항암보조제 개발의 토대를 마련

<당해연도목표>
- 항암제 내성에 큰 차이를 보이는 대장암 세포주들간의 단백질체 분석을 통한 내성 관련 단백질들의 동정
- 기존 연구를 통하여 얻어진 내성 관련 후보 단백질의 발현 조절과 활성 조절이 대장암 세포주의 항암제 내성에 미치는 효과 분석
- 내성 관련 단백질의 효과적인 임상 적용 고찰
- iCPL (Isotpe Coded Protein Labelling) 방법을 LC-MALDI system에 접목 2-DE gelindependent한 새로운 고감도 대용량 단백질체 분석 방법 토대 마련

◆ 연구내용 및 방법
- 인체 대장암 세포주, SNU-C4, SNU-C5, SNU-769A, SNU-769B 로부터 얻어진 5-FU 내성 세포주들의 비교 단백질체학(comparative proteomics)을 계속하여 수행함
① 세포주로부터 얻어진 단백질들의 분획(sub-fractionation: subcellular fractionation, phosphoprotein isolation, glycoprotein isolation etc)을 통한 proteome 분석의 효율을 증대시킴
② iCPL 과 LC-MALDI system 구축을 통한 2-DE independent proteomics 실현 및 high-throughput automatic MS/MS analysis를 통한 효율적 protein-protein interaction 분석

- 기존 연구를 통하여 얻어진 내성 관련 후보 단백질의 발현 조절과 활성 조절이 대장암
세포주의 항암제 내성에 미치는 효과 분석
① siRNA system을 이용한 효과적 유전자 발현 억제 방법 확립을 통해 내성관련 후보 단백질들이 내성 억제 및 증진에 미치는 영향을 효과적으로 분석
② 내성관련 후보 단백질의 항체를 이용한 immunoprecipitate 내의 단백질 분석을 통해 내성 후보 단백질과 interaction 하는 단백질을 동정하여 내성 단백질의 세포내 내성과 관련한 역할을 이해할 수 있는 자료를 획득함

◆ 연구성과
-정량적 성과
1) 총연구기간내 목표 연구성과로 기 제출한 값
-정성적 성과
·iCPL을 이용한 LC-MALDi MS/MS system 확립
·임상의 항암제 내성 예측을 위한 secretome (in vitro) data 구축

Abstract ▼

1. Decreased galectin-3 expression is significantly associated with elevated 5-FU resistance. Galectin-3 suppression results in diminished human colon cancer cell proliferation. However, galectin-3 suppression in the presence of 5-FU does not affect the proliferation rate, and slightly augments apop

1. Decreased galectin-3 expression is significantly associated with elevated 5-FU resistance. Galectin-3 suppression results in diminished human colon cancer cell proliferation. However, galectin-3 suppression in the presence of 5-FU does not affect the proliferation rate, and slightly augments apoptosis. Suppression of heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) interacting with galectin-3 has similar effects as galectin-3 on colon cancer cell proliferation, both in the absence and presence of 5-FU. Interestingly, while galectin-3 protein is not affected by the hnRNP Q level, its suppression is accompanied by a decrease in hnRNP Q expression. Furthermore, the hnRNP Q protein becomes unstable in the presence of 5-FU. The results demonstrate that galectin-3 may regulate hnRNP Q-mediated 5-FU resistance.

2. Galectin-3 interacted with the d-subunit of the F0 complex, but not the alpha-subunit of the F1 complex. Galectin-3 was co-purified with ATP synthase until mitochondrial inner membrane vesicle fraction. Interestingly, ATP synthase activity decreased in the presence of galectin-3. However, after suppression of galectin-3 activity by siRNA transfection, the pattern of total intracellular ATP content in human colon cancer cells remained similar to that of the control. Interestingly, galectin-3 suppression is accompanied by a decrease in ATP synthase d-subunit expression. In conclusion, galectin-3 co-localized with ATP synthase in mitochondria of human colon cancer cells possibly inhibits enzyme activity by interacting with the F0 complex, however, its interaction may stabilize ATP synthase protein itself.

3. Comparative assessment of secretome obtained from human colon cancer cell line SNU-C4 and its 5-FU resistant derivative cells has been performed using iCPL and LC-MALDI-MS/MS. From this assessment 235 secreted proteins have been identified. Among the proteins dysregulated in secretome of 5-FU resistant cells, most up-regulated proteins were the enzymes involved in glycolysis, and the level of pyruvate kinase - rate determining enzyme of glycolysis - was 1.7 fold higher compared to that in the secretome from parent cells. To investigate whether level of pyruvate kinase in serum from colorectal cancer patient can be the predictive marker for the response of 5-FU-based chemotherapy, 16 patients with previously untreated mCRC were enrolled. Serum samples have been obtained before chemotherapy, and the chemotherapy response of 12 patients was stable disease (SD), and 3 were partial response (PR). Only one patient's response was defined as progress disease (PD). Interestingly, level of pyruvate kinase was about 3 times higher in sera from chemotherapy non-responders (SD and PD) compared to that in sera from chemotherapy responders (PR). Our present results show a successful example that in vitro information of secretome of human colon cancer cell line with induced 5-FU resistance can be valuable for the clinical application to predict 5-FU-based chemotherapy response.

4. Based on comparing the ToF-SIMS spectra of low mass ions in normal and patients' sera, we has been establishing the protocol for the chemotherapy response prediction.

5. From the comparative proteome assessment of Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR), heat-shock protein 27 (HSP27) expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased both Herceptin susceptibility of SK-BR-3 HR cells. HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by formation of a Her2HSP27 complex. The present results demonstrate that upregulated HSP27 in human breast cancer cells may reduce Herceptin susceptibility by increasing Her2 protein stability.

목차 Contents

• 표지 ...1
• 제출문 ...3
• 목 차 ...4
• 요약문 ...5
• Summary ...7
• 1. 연구사업의 최종목표 ...9
• 2. 연구사업의 내용 및 결과 ...9
• 3. 연구결과 고찰 및 결론 ...47
• 4. 연구성과 및 목표달성도 ...48
• 5. 연구결과의 활용계획 ...54
• 6. 첨부서류 ...54