사람 신경모세포종에서 fenretinide에 의한세포자멸사의 유도기전
Fenretinide Induced Apoptosis in Human Neuroblastoma Cell Lines
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사람의 신경모세포종 세포주에 대한 합성 retinoid의 일종인 fenretinide의 세포성장 억제효과 및 세포자 멸사를 확인하고, 세포신호전달 과정을 알아보고자 하였다. Fenretinide의 신경모세포종 세포주에 대한 세포독성 정도와 세포 자멸사를 확인하기 위하여 MTT assay, cytoplasmic DNA fragmentation, TUNEL 염색, Western blot analysis 등을 시행하였다. MTT assay결과에서 신경모세포종은 10 μM 농도의 fenretinide을 처리한 후 72시간 배양하였을 때의 세포생존 율은 대조군에 비하여 CHP134는 47%, IMR32는 12%, SH-SY5Y는 53%를 보여 세포독성 효과를 나타내었으며 PC12에서는 fenretinide의 세포독성이 보이지 않았다. Fenretinide 처리 후 시간에 따라 cytoplasmic DNA fragmentation과 PARP 단백질 분해가 증가하였으므로 신경모세포종에 대한 fenretinide의 세포독성은 세포자멸사 과정의 유도에 인한 것을 확인하였다. Fenretinide에 의하여 SH-SY5Y의 경우에만 Bad가 시간이 지남에 따라 발현의 감 소함이 확인되었으나 CHP134와 IMR32에서는 Bad의 변화를 관찰할 수 없었으며 Bcl-x의 발현 변화도 관찰되지 않았다. Fenretinide에 의한 MAP kinase의 활성변화 결과는 CHP134에서 JNK 인산화 감소와 p38 인산화 증가, IMR32에서 p38 인산화 감소, SH-SY5Y에서 ERK의 인산화 증가 등 다양한 변화를 보였으며 그 외에는 큰 변화 가 없었다. ERK의 억제제인 PD98059, p38 억제제 SB203580, PI3 kinase 억제제 LY294002, protein tyrosine kinase 억제제 genistein 등을 전처리하였으나 fenretinide에 의한 PARP 단백질의 단백분해에는 변화가 없었다 이상의 결과들로부터 fenretinide에 의한 신경세포암에서의 세포증식 억제는 세포자멸사 기전이 유도되어 나타 나는 현상이며. 이는 MAP kinase 신호전달 과정이나 PI3 kinase, protein tyrosine kinase 등의 신호전달 과정과는 무 관하게 PARP의 단백분해를 통한 세포자멸사임을 확인할 수 있었다.
Abstract
Retinoids play an important role in growth, reproduction and differentiation. Recently, retinoids have been used to both protect and treat from various animal models of carcinogenesis. In this study the effect of N-(4-hydroxyphenyl) retinamide (fenretinide) on viability of human neuroblastoma cell lines were evaluated. For the evaluation of apoptosis of human neuroblastoma cell lines by fenretinide. MTT assay, cytoplasmic DNA fragmentation, TUNEL stain, and Western blot analysis were performed. In MTT assay, fenretinide inhibited the proliferation of CHP134, IMR32 and SH-SY5Y but not in PC12 cells. Cytoplasmic DNA fragmentation was induced by treament of fenretinide (10 μM) for 48 h in IMR32 cells. PARP cleavage was detected by Western blot analysis after 16 h of treatment of fenretinide in CHP134, IMR32 and SH-SY5Y. These fenretinide effects on growth inhibition and increased apoptosis followed to the time dependent manner. The fenretinide treatment did not affect the phosphorylation of MAP kinases (ERK, JNK, p38). There was no change of Bcl-x and Bad expression after treatment of fenretinide (1 μM) in neroblastoma cell lines. Pretreatement of PD98059, SB203580, LY294002, or genistein also did not affect fenretinide-induced PARP cleavage in neuroblastoma cell lines. From these results, the fenretinide-induced apoptosis is due to the PARP cleavage which occured MAP kinase signal cascades independently.
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