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PC12 세포에서 CoCl2 유발 세포자멸에 대한 Bcl-2 family와 caspase의 역할

Bcl-2 Family and Caspases are Involved in CoCl2-Induced Apoptosis of PC12 Cells

김원재|정지연|박희주|김선헌
대한체질인류학회|2006.06|해부·생물인류학 (Anatomy & Biological Anthropology)|제19권 제2호|pp. 137-148|DOI 10.11637/aba.2006.19.2.137

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ApoptosisBcl-2 family미토콘드리아MitochondriaCoCl2CaspasePC12 세포PC12 cells세포자멸
주제분류해부학

초록

허혈성/저산소 상태에서 신경손상을 유발하는 세포고사가 발생되는데, 이러한 신경세포자멸 기전 및 치료 방법이 아직 정립되어 있지 않다. CoCl2는 활성산소종(ROS)을 생산하는 등, 저산소 환경과 유사한 조건을 초래하 는 것으로 알려져 있다. 본 연구의 목적은 PC12 세포에서 CoCl2에 의한 저산소 상태에서 세포자멸기전에 대한 Bcl-2와 caspase들의 역할을 구명하는데 있다. 세포생장력은 MTT 방법으로 측정하였고, DNA 절편화 및 세포자멸 소체는 DNA laddering과 propidium iodide (PI) 염색법으로 조사하였다. Bcl-2와 Bax 발현정도는 RT-PCR법, Bid, Fas/APO-1 발현과 미토콘드리아에서 세포질 로 분비된 cytochrome c는 Western blot으로 분석하였으며, caspase-3와 caspase-9 활성은 spectrophotometer 그리고 caspase-8의 활성은 fluorospectrometer에 의해 측정하였다. CoCl2 투여는 PC12 세포수를 시간과 농도 의존적으로 감소시켰고, DNA 절편화 현상과 세포자멸 소체를 유도하 였다. 또한, 미토콘드리아에서 세포질로 유리되는 cytochrome c양이 증가되었고, caspase-9와 -3의 활성이 증가되었 다. 이러한 결과는 CoCl2 투여한 세포에서 유도된 세포자멸기전에 미토콘드리아가 매개되었음을 보여주었다. 한편, CoCl2 투여에 의해 Fas/APO-1의 발현이 증가되었고, caspase-8 활성이 증가된 결과로 CoCl2 유도된 세포자멸 기전 에 death receptor 매개 신호경로와 연관되었음을 알 수 있었다. 또한, Bcl-2 family에 대한 RT-PCR 분석결과, 세포 자멸를 억제하는 Bcl-2 발현은 감소되었으나, 세포자멸을 자극하는 Bax발현은 증가되었으며, 한편 pro-Bid 발현은 감소되었다. 따라서, 본 실험의 결과들은 PC12 세포에서 Bcl-2 family와 caspase들이 미토콘드리아와 death receptor를 매개하 는 두 신호경로를 통해서 CoCl2 유도 세포자멸에 중요한 역할을 함을 시사하였다.

Abstract

Hypoxic/ischemic condition induces the neuronal apoptotic events, consequently resulting in neuronal damages. Cobalt chloride (CoCl2) could mimic the hypoxic condition including the production of reactive oxygen species (ROS). This study aimed to investigate the roles of Bcl-2 family and caspases as central regulators of apoptosis, in CoCl2- induced apoptosis of PC12 cells. Cell viability was determined by MTT assay and DNA fragmentation was detected by DNA laddering. The expression levels of Bcl-2, Bax, Bid, cytochrome c and Fas/APO-1 were determined by RT-PCR or Western blotting analysis in CoCl2-treated PC12 cells. Caspase-9 and caspase-3 activities were assessed using spectrophotometry and caspase-8 activity was measured with fluorospectrocytometry. Administration of CoCl2 decreased viability of cells in a dose- and time-dependent manner. Furthermore, fragmentation of the genomic DNA and apoptotic bodies were induced in CoCl2-treated PC12 cells. Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated, whereas Bax, pro-apoptotic molecule, was upregulated in CoCl2-treated cells. Treatment of CoCl2 augumented the release of cytochrome c into the cytoplasm and increase of caspase-8, -9, and -3 activities. In addition, CoCl2 upregulated Fas and downregulated pro-Bid, which are known to be correlated with death receptor-mediated apoptotic signaling pathway. Therefore, these results suggest that Bcl-2 family and caspase play crucial roles in CoCl2-induced apoptosis through mitochondria- and death receptor-dependent pathways in PC12 cells.

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