Dichloroacetate, a inhibitor of pyruvate dehydrogenase kinase-1, attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer cells
- 주제(키워드) Chemoresistance , Dichloroacetate , 5-Fluorouracil , Gastric neoplasm , Hypoxia , Pyruvate dehydrogenase kinase
- 발행기관 아주대학교
- 지도교수 한상욱
- 발행년도 2013
- 제출일 2013-12-23
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000015888
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In this study, I investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profile of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20 mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20 mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism and the enhancement of apoptosis.
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TABLE OF CONTENTS
ABSTRACT ⅰ
CONTENTS ⅲ
LIST OF FIGURES ⅴ
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 5
1. Human tissue collection and immunohistochemical staining 5
2. Reagent and cells 6
3. Western blotting analysis 8
4. Luciferase assay 9
5. Cell viability test 10
6. Glucose uptake assay 10
7. Lactate production assay 11
8. ATP concentration assay 11
9. Apoptosis assay 12
10. statistical analysis 12
Ⅲ. RESULTS 13
1. The expression of HIF-1α, PDK-1 and p-PDH in human tissues 13
2. Hypoxia-dependent expression of PDK1 gene in vitro 18
3. The synergic effect of DCA on hypoxia-or HIF-1α-induced resistance to 5-FU in vitro 20
4. Measument of metabolic changes after 5-FU and DCA treatment 23
5. The effect of genetic inhibition of PDK-1 on hypoxia-induced 5-FU resistance 27
6. The effect of DCA and 5-FU treatment on the function of PDK-1 and apoptosis under normoxia and hypoxic conditions 30
Ⅳ. DISCUSSION 35
REFERENCES 41
국문요약 50
