유방암세포에서 Retinoic Acid에 의한 Insulin-like growth factor-I 분비조절에 있어서 PKC의 역할 = The role of PKC on the regulation of Insulin-like growth factor-I secretion by Retinoic Acid in the MCF-7 cell

다국어 초록 (Multilingual Abstract)

Retinoic acid (RA) induces growth inhibition and apoptosis in a variety of tumor cells, including MCF-7 cells. IGF-I system has been reported to be associated with the development of cancer. Although MCF-7 cell with RA is to be the major stimuli for the cell growth and apoptosis, the mechanism of IGF-I system (IGF-I secretion and mRNA expression) remains to be elucidated. This study have been found that RA dose- and timedependently decreased the level of secretion and mRNA expression of IGF-I. Moreover, treatment with 10^(-7) M RA for 72 hours activated PKC-δ that is one among PKC-ι, α, λ and δ and decreased the level of secretion and mRNA expression of IGF-I at 72 h by 28.6 ± 4.0 mg/mg (mean ± SD) and 45 ± 16%, respectively (p<0.05). These changes were similar to those of cell viability and insulin receptor substrate-1 (IRS-1) level, and in contrast to the trend of protein kinase C (PKC)-δ activity, which gradually increased following RA treatment. The RA-induced decreases in IRS-1 level, cell viability, and the IGF-I system were recovered by 10^(-8) M rottlerin (a PKC-δ inhibitor) and by the suppression of PKC-δ expression using siRNA. Moreover, the RA-induced increase in the level of the PKC-δ.IRS-1 complex was also blocked by the suppression of PKC-δ activity. Additionally, RA induced ubiquitin-conjugated IRS-1, and we found that MG132 (a ubiquitin-proteasome inhibitor) also recovered the RA-induced decreases in the IRS-1 level, IGF-I system, and cell viability. These results suggest that PKC-δ activity and the ubiquitin-proteasome pathway are involved in the inhibitions of the IGF-I system, cell viability, and the IRS-1 level. Together these results indicate that RA inhibits the IGF-I system, which is related to down-regulation of IRS-1 protein regulated by the ubiquitin-proteasome pathway via PKC-δ in MCF-7 cells.

목차 (Table of Contents)

• ABSTRACT = 1
• 서론 = 3
• 재료 및 방법 = 8
• 1. 재료 = 8
• 3. 세포 배양 = 9
• 3. 세포생존도 검사(MTT) = 10
• 4. 시료의 전 처리 = 10
• 5. IGF-I 추적자 제조 = 11
• 6. IGF-I 방사면역 측정법(IGF-I radioimmunoassay) = 12
• 7. Cell lysis = 12
• 8. Western Blotting = 13
• 9. Immunoprecipitation Assay(IP) = 14
• 10. RNA 검출과 RT-PCR (Reverse transcriptase polymerase- chain-reaction) = 15
• 11. Cell transfection과 small interfering RNA (Si-RNA) = 16
• 12. 통계처리 = 17
• 결과 = 18
• 1) RA 농도와 시간에 따른 IGF-I 분비와 유전자 발현의 효과 = 18
• 2) RA 처리시간에 따른 PKC isoform의 변화 = 19
• 3) RA 처리 농도와 시간에 의한 PKC-δ, IRS-1, IGF-IR 및 세포 생존도의 효과 = 19
• 4) RA에 의한 세포 생존도 및 IRS-1 변동과 PKC-δ 관련성 = 21
• 5) RA에 의한 IGF-I 분비 및 유전자 발현과 PKC-δ 관련성 = 22
• 6) RA에 의한 PKC-δ 및 IRS-1 단백질 변동과 Ubiquitin-proteasome pathway 상호 관련성 = 22
• 7) RA에 의한 PKC-δ 활성이 IGF-I 분비 및 유전자 발현 변동과 Ubiquitin-proteasome pathway 상호 관련성 = 25
• 고찰 = 26
• 결론 = 33
• Legend for figures = 35
• 참고문헌 = 52