Novel Anti-cancer Drug Candidates with Src Kinase Inhibitory Property = Src 인산화 효소를 억제하는 새로운 항암제 후보물질 연구

다국어 초록 (Multilingual Abstract)

Critical characteristics of cancer cells are to avoid apoptosis which is a cell death mechanism and to induce metastasis that makes cancer become difficult to cure. Src kinase can be a pharmacological target to regulate cancer because it is a well-known oncogene protein and is highly activated in many types of cancers. Aripiprazole (ARP) is an atypical antipsychotic as a partial agonist of dopamine D2 receptors and 4-isopropyl-2-(1-phenylethyl) aniline (KTH-13-AMP) is an analog that is synthesized based on the molecular structure of KTH-13, which are verified to induce apoptosis in cancer cells. I explored the molecular mechanism underlying anti-cancer activity of ARP and KTH-13-AMP. These agents inhibited the viability of various cancer cells. Also, several apoptotic markers such as apoptotic bodies and chromatin condensation were observed in ARP- or KTH-13-AMP-treated cancer cells. The apoptotic cells stained by AnnexinⅤ and PI were increased by both agents, which implies they can induce apoptosis in cancer cells. In molecular levels, ARP and KTH-13-AMP promoted the activation of capase-3, -9 and -8 and diminished the expression level of Bcl-2. In case of ARP, it induced the protein expression level of p53 unlike KTH-13-AMP. Moreover, ARP suppressed the cell migration in U251 glioma cells. The protein and mRNA expression levels of NF-B-mediated MMP-2 and -9 were downregulated by ARP. The phosphorylation of STAT-3 which is related to cell survival was decreased by treatment with ARP and KTH-13-AMP. In migratory condition, ARP also downregulated Akt/PI3K signaling which induces activation of NF-κB-mediated MMP-2 and -9. Furthermore, it was revealed that these are derived from suppression of Src. indicating Src kinase is the target of both ARP and KTH-13-AMP. In agree with my findings, I conclude that ARP and KTH-13-AMP have high potential to be developed as novel anti-cancer drugs with Src kinase inhibitory property.

목차 (Table of Contents)

• CONTENTS
• List of Abbreviations ⅳ
• List of Figures ⅵ
• List of Tables ⅶ
• Abstract 1
• Part I.
• Anti-cancer activity of aripiprazole by targeting Src kinase.
• 1. INTRODUCTION 4
• 2. MATERIALS AND METHODS 7
• 2. 1. Materials 7
• 2. 2. Cell culture 7
• 2. 3. Cell viability assay 8
• 2. 4. Morphological change test and confocal microscopy 8
• 2. 5. FITC-annexin V/PI staining apoptosis assay 9
• 2. 6. Preparation of cell lysates and immunoblotting analysis 9
• 2. 7. mRNA analysis using semi-quantitative polymerase chain reaction (PCR) 10
• 2. 8. Migration assay 10
• 2. 9. Plasmid transfection and luciferase reporter gene assay 11
• 2.10. in vitro kinase assay 11
• 2.11. Statistical analysis 11
• 3. RESULTS 14
• 3. 1. Aripiprazole induces cytotoxic activity in various cancer cells 14
• 3. 2. Aripiprazole induces apoptosis in U251 glioma cells 14
• 3. 3. Aripiprazole inhibits migration capabilities of U251 glioma cells 22
• 3. 4. Aripiprazole suppresses Src activation 26
• 3. 5. PP2 induces apoptosis and inhibits migration in U251 glioma cells 30
• 4. DISCUSSION 36
• Part II.
• Pro-apoptotic activity of 4-isopropyl-2-(1-phenylethyl) aniline isolated from Cordyceps bassiana
• 1. INTRODUCTION 41
• 2. MATERIALS AND METHODS 43
• 2. 1. Materials 43
• 2. 2. Cell culture 43
• 2. 3. Cell viability assay 44
• 2. 4. Morphological change assay 44
• 2. 5. FITC-annexin V/PI staining apoptosis assay 44
• 2. 6. Preparation of cell lysates and immunoblotting analysis 45
• 2. 7. Statistical analysis 46
• 3. RESULTS 47
• 3. 1. Effect of KTH-13-AMP on the proliferation of cancer cells 47
• 3. 2. Effect of KTH-13-AMP on the induction of apoptosis in C6 glioma cells 51
• 3. 3. Effect of KTH-13-AMP the suppression of cell survival signaling pathway 57
• 4. DISCUSSION 60
• REFERENCES 63
• SUMMARY IN KOREAN 76