Evaluation of the biological activity of two hit compounds and its action mechanisms for drug repositioning
- 주제(키워드) Anhydrous alum , melanogenesis , MITF , CREB , ERK , Pyrimethamine , Autophagy , Senescence , p53
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Jong-Soo Lee, Su dong Kim
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033705
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
I evaluated biological activity of two compounds, anhydrous alum (KAl(SO4)2) and pyrimethamine and elucidated its action mechanisms. Firstly, my research concentrated on investigating the impact of dehydrated alum (KAl(SO4)2) on melanogenesis, the intricate process responsible for the production of melanin. Melanin plays a vital role in skin pigmentation and shielding against UV radiation. In order to explore potential skin- whitening agents, scientists scrutinized the inhibitory effects of high-purity dehydrated alum (KAl(SO4)2) on melanogenesis using B16F1 melanoma cells and delved into its underlying molecular mechanisms. Dehydrated alum (KAl(SO4)2) with a purity exceeding 99%, obtained through the heat treatment of hydrated alum (KAl(SO4)2·12H2O) at 400 °C, successfully diminished melanin content without inducing cytotoxicity. The investigation uncovered that dehydrated alum represses the chief regulator of melanogenesis, microphthalmia-associated transcription factor (MITF), which governs crucial genes in melanogenesis, thereby inhibiting α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. Furthermore, dehydrated alum impeded the phosphorylation of the cAMP response element-binding protein, a co-activator of MITF gene expression, leading to diminished MITF transcription. Notably, dehydrated alum also facilitated extracellular signal-regulated kinase phosphorylation, resulting in compromised nuclear localization of MITF. These findings offer valuable insights into the mode of action of dehydrated alum in B16F1 cells, presenting a promising avenue for cosmetic or therapeutic applications. Secondly, I have been investigated anticancer activity of pyrimethamine, an antiparasitic drug originally used for the treatment of malaria and toxoplasmosis, and its underlying molecular mechanisms. In this study, I found that pyrimethamine had an inhibitory impact on the proliferation of various cancer cells. Effectively, pyrimethamine treatment blocked proliferation of cancer cells along with S phase arrest. Also, pyrimethamine induced phosphorylation of p53 and Chk1 without regard to DNA damage. Thus, with the Pyrimethamine-induced p53 phosphorylation was mediated by Erk1/2 in HCT116 cells. Additionally, pyrimethamine triggered autophagy in a MAP kinase-dependent manner. Moreover, the compound increased the proportion of senescence cells. The multifaceted anticancer effects of pyrimethamine were uncovered in this study to underscore its potential as a promising candidate in the realm of cancer therapeutic strategies. Key words : Anhydrous alum; melanogenesis; MITF; CREB; ERK; Pyrimethamine; Autophagy; Senescence; p53
more목차
CHAPTER 1 1
I. INTRODUCTION 2
II. MATERIALS AND METHODS 5
1. Preparation of Anhydrous Alum 5
2. Cells and Reagents 5
3. Cell Viability Assay 5
4. Melanin Content and Tyrosinase Activity Assays 6
5. RNA Isolation and Quantitative Reverse Transcription PCR (qRT-PCR) 7
6. Western Blot Analysis 7
7. Nuclear and Cytoplasm Subcellular Fractionation 8
8. Immunofluorescence 9
9. Statistical Analysis 9
III. RESULTS 10
1. Pure Anhydrous Alum has no obvious cytotoxicity 10
2. Pure anhydrous alum inhibited melanogenesis 12
3. A-Alum-1 suppresses Tyrosinase gene expression by reducing MITF expression in α-MSH-Induced B16F1 cells 14
4. A-Alum-1 decreases α-MSH-induced MITF expression via inhibiting CREB phosphorylation in B16F1 cells 18
5. A-Alum-1 inhibits nuclear localization of MITF in α-MSH-Induced melanoma cells 20
6. A-Alum-1 upregulates the phosphorylation of Erk1/2 in α-MSH-induced melanoma cells 22
IV. DISCUSSION 26
CHAPTER 2 29
I. INTRODUCTION 30
II. MATERIALS AND METHODS 32
10. Cells and reagents 32
11. Cell Viability Assay 32
12. Clonogenic cell survival assay 33
13. Cell cycle Analysis 33
14. BrdU incorporation 33
15. Western Blot Analysis 34
16. Autophagy assays 35
17. Acridine Orange Assay 35
18. Immunofluorescence 35
19. Measurement of intracellular ROS 36
20. Small interfering RNAs (siRNAs) 36
21. RNA Isolation and Quantitative Reverse Transcription PCR (qRT-PCR) 37
22. Statistical Analysis 37
III. RESULTS 38
8. Pyrimethamine inhibited the proliferation of various cancer cells 38
9. Pyrimethamine induces the phosphorylation of p53 and Chk 1 independently of DNA damage 42
10. The phosphorylation of p53 induced by pyrimethamine in HCT116 cells is regulated by Chk1 and Erk1/2 45
11. Pyrimethamine induced autophagy in various cancer cells 48
12. Autophagy induction by Pyrimethamine was dependent on Erk MAP kinases 51
13. Pyrimethamine induces cellular senescence-like phenotype 53
14. Pyrimethamine induces reactive oxygen species (ROS) 56
IV. DISCUSSION 58
REFERENCES 63
ABSTRACT in Korean (국문 초록) 72
