Effects of epigallocatechin gallate on CoCl_(2)-induced apoptosis in PC12 cells = PC12 세포에서 CoCl_(2) 유발 세포자멸사에 대한 epigallocatechin-gallate의 역할

다국어 초록 (Multilingual Abstract)

Neuronal apoptotic events, consequently resulting in neuronal cell death, are occurred in hypoxic/ischemic condition. This cell death has been shown to be accompanied with the production of reactive oxygen species (ROS), which can attack cellular components such as nucleic acids, proteins and phospholipid. However, the underlying mechanisms of apoptosis induced in hypoxic/ischemic condition and its treatment methods are unsettled. Cobalt chloride (CoCl_(2)) has been known to mimic hypoxic condition including the production of ROS. Epigallocatechin gallate (EGCG). a green tea polyphenol, has diverse pharmacologial activities in cell growth and death. This study was aimed to investigate the apoptotic mechanism by CoCl_(2) and effects of EGCG on CoCl_(2)-induced apoptosis in PC12 cells.
Administration of CoCl_(2) decreased cell survival in dose- and time-dependent manners and induced genomic DNA fragmentation. Treatment with 100 µM EGCG for 30 min before PC12 cells were exposed to 150 µM CoCl_(2), being resulted in the cell viability and DNA fragmentation being rescued. CoCl_(2) caused morphologic changes such as cell swelling and condensed nuclei, whereas EGCG attenuated morphologic changes by CoCl_(2). EGCG suppressed the apoptotic peak and a loss of Δψ_(m) induced by CoCl_(2). CoCl_(2) decreased Bcl-2 expression but Bax expression was not changed in CoCl_(2)-treated cells. EGCG attenuated the Bcl-2 underexpression by CoCl_(2). CoCl_(2) augumented the cytochrome c release from mitochondria into cytoplasm and increased caspase-8, -9 and caspase-3 activity, a marker of the apoptotic executing stage. EGCG ameliorated the incruement in caspase-8, -9 and -3 activity, and cytochrome c release by CoCl_(2). NAC (N-acetyl-cysteine), a scavenger of ROS, attenuated CoCl_(2)-induced apoptosis in consistent with those of EGCG.
These results suggest, that CoCl_(2) induces apoptotic cell death through both mitochondria- and death receptor-dependent pathway and EGCG has neuroprotective effects against CoCl_(2)-induced apoptosis in PC12 cells.

국문 초록 (Abstract)

신경세포자멸사는 저산소 및 허혈환경에서 일어나며 이러한 세포죽은 reactive oxidant species (ROS) 생성을 동반함이 알려져있다. 그러나, 저산소 및 허혈환경에서 일어나는 세포자멸사의 기전 및 그 치료방법은 아직 정립되어 있지 않다. CoCl_(2)는 ROS를 생성하는 등 저산소환경과 유사한 조건을 초래하는 것으로 알려져 있다. Epigallocatechin gallate (EGCG)는 녹차의 polyphenol 성분으로서 세포성장과 죽음에 다양한 약리학적 효과를 나타냄이 알려져 있다. 본 연구는 PC12 세포에서 CoCl_(2)에 의한 세포자멸사기전을 밝히고 이에 미치는 EGCG의 효과를 조사하는데 목적이 있다. Cell viability는 MTT 측정으로 조사되었고, DNA fragmentation은 DNA laddering으로 조사되었다. Bcl-2와 Bax발현 정도는 RT-PCR로, caspase-3와 -9의 활성은 spectrophotometer, caspase-8의 활성은 flow cytometry에 의해 측정되었다. 미토콘드리아에서 세포질로 분비된 cytochrome c는 western blot으로, 분해된 DNA 양과 미토콘드리아 세포막전위 (Δψ_(m))는 FACScan으로 조사되었다.
CoCl_(2)투여로 PC12 세포수는 용량 및 시간 의존형태로 감소하였고, genomic DNA fragmentation이 발생하였다. CoCl_(2)투여로 야기된 cell viability의 감소와 DNA fragmentation은 EGCG 전처치에 의해 억제되었다. CoCl_(2)은 세포용적팽창과 condensed nuclei 같은 형태적 변화를 일으켰으며, apoptotic peak, Δψ_(m)감소 및 cytochrome c 유리를 야기하였다. EGCG는 CoCl_(2)에 의한 세포형태변화, apoptotic peak, Δψ_(m)소실 및 cytochrome c 유리를 억제시켰다. CoCl_(2)는 Bcl-2 발현을 감소시켰지만, Bax 발현에는 영향을 미치지 않았다. EGCG는 CoCl_(2)에 의해 야기된 Bcl-2 발현 감소를 억제시켰다. CoCl_(2)는 caspase-3, -8, 그리고 -9의 활성을 증가시켰으며, EGCG는 CoCl_(2)에 의한 세포자멸사를 억제시켰다.
본 실험결과는 PC12 세포에서 CoCl_(2)가 미토콘드리아 의존 및 death receptor의존 기전으로 세포자멸사를 일으키며, EGCG는 세포자멸사기전을 억세지킴으로 신경보호기능을 가짐을 시사하였다.

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