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Induction of Apoptosis in HeLa cells by Chloroform Fraction of Citrus grandis Osbeck Leaves and its Compositional Analysis

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Author(s)
김하나
Issued Date
2010
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000004919
Abstract
Dangyuja is a citrus fruit of Jeju Island in Korea, which is known to have a high content of flavonoids with health-related properties. Although recent experimental data have revealed the anticancer potency of some Citrus species, the underlying molecular mechanisms of this apoptotic activity have not yet been studied in detail. In the present study, chloroform fraction (CF) of Dangyuja leaves was investigated on the inhibition of growth on human cervical carcinoma cells. It was found that the exposure of HeLa cells to CF resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner. The CF treatment induced the down regulation of anti-apoptotic Bcl-2 expression which was associated with the proteolytic activation of caspases and the concomitant degradation of poly (ADP ribose) polymerase (PARP) protein. Arrested cell growth and the induction of apoptosis were confirmed by flow cytometry and DNA fragmentation analysis. Moreover, a new and rapid high performance liquid chromatography technique with an UV photodiode-array detector was used to analyze the major components of CF which were identified as sinensetin, nobiletin, tangeretin and 4',5,6,7-tetramethoxyflavone by using electrospray ionisation mass spectrometry (ESI-MS). This is the first report, showing the possible mechanism of antiproliferative effect of Dangyuja leaves for the prevention of cervical cancer in cell culture models. Our results suggested that CF of Dangyuja leaves is excellent sources of functional polymethoxyflavones that may help prevent female cancer and a potentially useful agent for the treatment of certain malignancies.
제주도에서 재배되는 당유자 (Citrus grandis Osbeck)는 많은 양의 플라보노이드 함량을 포함한다. 최근 연구에서 몇몇 감귤류에서 항암 효과를 가진 것으로 밝혀졌지만, apoptosis를 통한 분자수준의 메커니즘은 아직 자세하게 보고되지 않았다.
이번 연구에서는, 당유자 잎의 클로로포름 분획물 (CF)이 human cervical carcinoma cell인 HeLa 세포의 성장억제에 대하여 조사하였다. 그 결과, CF를 처리한 HeLa 세포에서 농도의존적으로 세포 성장 억제와 apoptosis 유도를 확인하였다. CF가 처리된 HeLa 세포에서 anti-apoptotic 단백질인 Bcl-2의 down regulation과 caspase의 기질인 PARP 단백질의 degradation을 확인하였다. Flow cytometry를 통하여 CF가 처리된 HeLa 세포의 arrest와 DNA fragmentation을 확인하였다. LC-MS/MS를 이용하여 CF의 성분을 분석한 결과, polymethoxyflavone 계통인 sinensetin, 4',5,6,7-tetramethoxyflavone, nobiletin과 tangeretin을 동정하였다. 이러한 연구 결과는 처음 보고 되는 것으로, 당유자 잎의 클로로포름 분획물 (CF)이 자궁경부암세포모델에서의 세포 성장 억제 및 항암활성의 메커니즘에 관여하는 것으로 사료된다.
본 연구는 여성 암을 예방하고 종양 치료에 도움을 줄 수 있을 것이라고 사료되는 polymethoxyflavone화합물을 제공하는 훌륭한 자원으로서 당유자 잎 클로로포름 분획물의 우수성을 잘 시사해준다.
Alternative Title
인체 자궁암 세포주 HeLa 세포에서의 당유자 잎 chloroform 분획물의 apoptosis 유도 및 분획물의 성분 분석
Affiliation
제주대학교 대학원
Department
대학원 생명공학과
Advisor
김소미
Awarded Date
2010. 2
Table Of Contents
1.INTRODUCTION 1
2. MATERIALS and METHODS 7
2.1. Materials 7
2.2. Preparation of extracts 8
2.3. Cell culture 9
2.4. Cell viability assay 9
2.5. DNA fragmentation assay 10
2.6. Analysis of morphological changes 10
2.7. Western blot analysis 11
2.8. Flow cytometry analysis 12
2.9. Caspase-3 colorimetric assay 13
2.10. LC-ESI-MS/MS analysis 14
2.10.1. HPLC conditions 16
2.10.2. Gradient 16
2.10.3. Mass spectrometer conditions 17
2.10.4. Software 17
2.11. Statistical analysis 17
3. RESULTS and DISCUSSION 18
3.1. Effect of fractions from Dangyuja leaves on cell viability in human cell line HeLa cells 18
3.2. Effect of CF on DNA laddering 22
3.3 Effect of CF on the cell morphology 24
3.4. Effect of CF on expression of apoptosis-related proteins 26
3.5. Dose-dependent effect of CF on cell cycle distribution 28
3.6. Effect of CF on caspase-3 activity 31
3.7. Chemical characterization of CF by LC-MS/MS 33
4. CONCLUSION 42
5. REFERENCES 43
ACKNOWLEGEMENT 62
Degree
Master
Publisher
제주대학교 대학원
Citation
김하나. (2010). Induction of Apoptosis in HeLa cells by Chloroform Fraction of Citrus grandis Osbeck Leaves and its Compositional Analysis
Type
Dissertation
Appears in Collections:
Faculty of Biotechnology > ETC
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