Proteomic Profiling of Human Intrahepatic Cholangiocarcinoma Tissues and Characterization of Fatty Acid-Binding Protein 5 : 간내세포암의 프로테옴 프로파일링과 fatty acid-binding protein5의 발현양상

다국어 초록 (Multilingual Abstract)

Background: Intrahepatic cholangiocarcinoma (ICC) is a rare primary malignant liver tumor with an extremely poor prognosis. Although the incidence of ICC continues to increase, little attention has been focused on factors related to its molecular carcinogenesis, such as changes in the expression patterns of oncogenes, tumor suppressor genes, and cell-cycle proteins. The biological behavior of ICC, with respect to its genetic and molecular characteristics, remains to be clarified. When compared with other malignancies, ICC is generally characterized by aggressive proliferation, invasion, and early metastasis. To improve the prognosis of patients diagnosed with ICC, a more thorough understanding of the molecular mechanisms behind its proliferation and progression is necessary.
Methods: To examine the proteomic alterations associated with carcinogenesis of ICC, we compared protein expression profiles in 16 cases of ICC. Tissues from surgically resected ICC samples were compared to adjacent noncancerous bile duct tissues. The proteomic techniques employed in our analysis were proteome separation by two-dimensional electrophoresis, visualization of separated spots by staining, protein excision, tryptic peptide digestion, and protein identification by MALDI-TOF/mass spectrometry.
Results: We identified 151 spots that exhibited a statistically significant change in expression. Of these, 44 spots demonstrated significantly increased intensity and 16 spots manifested significant decrease in ICC tissues. The proteins represented by these spots were identified by MALDI-TOF/mass spectrometry. Subsequent analysis with Western blot also revealed that the expression of FABP5 was increased in cholangiocarcinoma tissues. Furthermore, immunohistochemical data for FABP5 confirmed that their expression was increased in cholangiocarcinoma tissues compared to normal bile duct tissues. Finally, FABP5 expression levels in ICC tissues were clinicopathologically verified in 43 MF-ICC patients, suggesting that increased expression of FABP5 was significantly correlated with tumor size (p=0.047), lymph node metastasis (p = 0.013), angioinvasion (p=0.032), and staging (p=0.007).
Conclusion: The present results showed that the expression of FABP5 was significantly increased in ICC compared with that of corresponding normal bile ducts, and suggested a potential role for FABP5 in tumor progression. Additional functional studies are required to determine the precise role of FABP5 in ICC, and its implication as a potential biomarker and/or therapeutic target in the treatment of ICC.

국문 초록 (Abstract)

2006년 6월부터 2009년 4월까지 3년여 간 경상대학교병원에서 종괴형성형 간내 담관암으로 진단받고 대량 간 절제 및 담도 절제술을 시행한 환자의 암 조직과 정상 담관 조직을 각각 채취하여 단백질을 추출한 후, 이차원 전기영동을 시행하고 말디토프/질량분석기를 이용하여 개개의 단백질을 동정하였다. 젤 상에서 단백질의 발현이 3배 이상 유의한 차이를 나타낸 점적(spot)을 분석한 결과, 새롭게 동정된 단백질 중 fatty acid-binding protein 5(FABP5)는 정상 담관 조직에 비해 암 조직에서 거의 5배 가량 증가하였다. Western blot에서도 전체 16쌍 중 9쌍에서 FABP5는 정상 담관조직에 비해 암 조직에서 유의하게 증가하였다. 한편, 간내 담관암 조직에서의 FABP5의 발현 정도를 확인해 보고자 43례의 종괴형성형 간내담관암 환자의 파라핀 조직을 이용한 면역화학염색을 시행한 결과, 간내담관암 조직에서 FABP5의 발현이 현저히 증가함을 확인할 수 있었다. 후향적으로 분석한 43례의 임상적, 병리학적 특징과 면역 염색 강도에 대한 단변량 분석에서 림프절 전이가 있는 경우 (p = 0.013), 종양크기가 5cm이상 (p = 0.047), 혈관침윤이 있거나 (p = 0.032), 병기가 높은 (p = 0.07) 간내담관암에서 유의하게 그 발현이 증가함을 관찰할 수 있었다. 이는 FABP5 단백질이 간내담관암의 발생과 진행에 관련이 있을 가능성을 강력하게 시사해 주고 있다. 향후 기능적 프로테옴 연구를 통해 간내담관암에서 FABP5 단백질의 발현 및 그 역할기전에 대한 연구가 더 필요할 것으로 보이며, 혈액이나 담즙에서의 연구를 통해 종양 표지자로서의 활용 가능성을 더욱 확실히 할 수 있을 것이라 생각된다.

목차 (Table of Contents)

• INTRODUCTION 1
• Overview 2
• 1. General features of intrahepatic cholangiocarcinoma 2
• 2. Cancer proteomics 5
• 3. Biomarkers in intrahepatic cholangiocarcinoma 8
• Objectives 13
• MATERIALS AND METHODS 14
• Materials 15
• Two-dimensional gel electrophoresis 16
• Protein visualization and image analysis 18
• Protein identification 19
• Western blot analysis 22
• Immunohistochemistry 23
• RESULTS 25
• DISCUSSION 40
• CONCLUSION 49
• REFERENCES 52