Effects of Allium senescens L. extract on sorafenib resistant HepG2 cells
- 주제(키워드) HepG2 , Allium senescens L. , sorafenib , PRELI
- 발행기관 고려대학교 대학원
- 지도교수 이승관
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위구분 석사
- 학과 대학원 보건안전융합과학과
- 세부전공 임상검사과학
- 원문페이지 89 p
- 비고 고려대학교 보건과학과 정밀보건과학융합전공 (이중전공)
- UCI I804:11009-000000235484
- DOI 10.23186/korea.000000235484.11009.0001164
- 본문언어 영어
- 제출원본 000046072144
초록/요약
Allium senescens L. known as a perennial aromatic herb is distributed in northern Europe and Asia, especially Ulleung island in south Korea. Allium species have been used for food and medicine for thousands of years. Although Allium senescens L. is effective to blood refreshing, modulation of cholesterol levels in blood, activation of detoxification in liver, there is no research for cancer drug resistance in hepatocarcinoma. An amount of p-Coumaric acid in the extract was evaluated using UHPLC (Ultra High Performance Liquid Chromatography) and the amount was 4.7291 ± 0.06 μg/mL. Unlike protein of relevant evolutionary and lymphoid interest (PRELI) plays as various roles including suppression of apoptosis, activation of differentiation and protection for desiccation in cells, this research documented for PRELI associated with drug resistance. Efficiency of the extract for PRELI, drug transporters, autophagy and mitochondrial apoptosis were evaluated using PCR. With the drug resistance cells, the cellular viability were evaluated at 71% under sorafenib 15μM. Unlike the susceptible cells, PRELI was decreased at 0.86 times in the resistant cells under the p-Coumaric acid. Compared to p-Coumaric acid, the extract down regulated PRELI at 0.51 times. Although Influx transporters (OATPβ1, OCT1) were increased slightly under the extract, the resistant cells were strongly down regulated efflux transporters including ABCB1, ABCC2, ABCG2. Exposed to PRELI knockdown conditions, ABCG2 in the resistant cells were significantly decreased at 0.6 times. Under the extract, the influx protein, OCT1 in the resistant cells was slightly increased at 1.16 times. In addition, the resistant cells exposed to the extract were significantly down regulated autophagic markers including mTOR2, XBP1 and S1P. Moreover, the resistant cells exposed to the extract were significantly up regulated mitochondrial apoptotic marker, BAX. Contrasted to BAX, BCL2 was significantly decreased in the cells. Consequently, the extract seemed to attenuate drug resistance of HepG2 cells through down regulation of PRELI, key protein associated with modulation of expression for the drug transporters, activation of autophagy and mitochondrial apoptosis. Allium senescens L. extract is a useful material to protect drug resistance and to sustain efficacy of sorafenib against liver cancer.
more목차
I. introduction 1
II. Method and Materials 17
1. Natural product 17
2. Chemicals 17
3. Determination of Allium senescens L. contents 18
3-1. HPLC equipment 18
3-2. Preparation of standard solutions 18
3-3. Chromatography analysis of p-Coumaric acid and Allium senescens L. 19
4. In vitro studies 21
4-1. Cell culturing and treatment 21
4-2. The cytotoxicity analyses 22
5. Gene expression analysis 22
5-1. RT-PCR (Reverse Transcription Polymerase Chain Reaction) 22
5-2. The PCR for markers 23
5-3. siRNA (short interfering RNA) transfection 25
6. Statistical analysis 25
Ⅲ. Result 26
1. Quantification of Allium senescene L. and the method assessment 26
2. Determination of HepG2 cell under sorafenib viability by WST assay 34
3. Determination of HepG2 cell under p-Coumaric acid and Allium senescene L. viability by WST assay 36
4. Effect of sorafenib in HepG2 cells on PRELI gene 38
5. Comparison of the effects of p-Coumaric acid and Allium senescens L. on the expression level of PRELI gene in HepG cells 40
6. PCR for drug transporter genes 42
7. Apoptotic and autophagic genes 46
IV. Discussion 49
V. References 56
Ⅵ. 국문초록 72
