합성 chenodeoxycholic acid 유도체 HS-1200이유도한 사람흑색종세포 세포자멸사 연구
Synthetic Chenodeoxycholic Acid Derivative HS-1200-induced Apoptosis of Human Melanoma Cells
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담즙산과 합성담즙산유도체가 여러 종류의 암세포에 세포자멸사(apoptosis)를 유도하며, 항암효과가 있다 고 알려져 있다. 또한 합성 chenodeoxycholic acid (CDCA) 유도체가 여러 가지 암세포에 유도한 세포자멸사 연구 들이 보고되어 왔다. 하지만 아직까지 사람흑색종세포에 합성 CDCA 유도체가 유도한 세포자멸사 연구는 보고되 지 않았다. 그래서 본 연구는 합성 CDCA 유도체인 HS-1199와 HS-1200이 사람흑색종세포(G361 세포)에 세포자 멸사 효과와 세포자멸사 기작을 밝혀내기 위해서 수행되었다. 합성 CDCA에 처리된 G361 세포의 생존율을 확인하기 위해서 MTT 방법을 사용하였고, 세포자멸사 유도 검증 은 DNA 전기영동법과 Hoechst 염색을 이용하였다. 세포자멸사에 관계하는 단백질의 발현 변화와 세포 내에서 이 동을 밝혀내기 위해서 Western bot 분석과 면역형광염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체 막 전위 변화를 측정하였다. 합성 CDCA 유도체로 처리된 G361 세포에서 caspase-3, DFF, PARP의 파괴, caspase-3 (HS-1200 only), PARP, DFF의 분절화, DNA 조각남(HS-1200 only), 핵 응축, proteasome 활성화의 감소, 사립체막전위(MMP)의 감소, 그리 고 cytochrome c와 AIF의 사립체에서 세포질로의 유리와 같은 다양한 세포자멸사의 증거를 보였다. 두 개의 합성 CDCA 유도체 중에서 HS-1200이 HS-1199보다 더욱 강한 세포자멸사 효과를 보였다. 본 연구는 CDCA 유도체인 HS-1200이 사람흑색종세포에서 proteasome, 사립체 그리고 caspase 경로을 통해서 세포자멸사를 유도하는 것을 증명하였다. 이러한 결과는 HS-1200이 사람흑색종의 새로운 치료적 전략으로 응용 될 수 있다고 생각한다.
Abstract
Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. It wasn’t discovered those materials have apoptosis-inducing effects on G361 human melanoma cells. The present study was done to examine the synthetic bile acid derivatives, HS-1199 and HS- 1200, induced apoptosis on G361 cells and such these apoptosis events. The viability of G361 cells was assessed by the MTT assay. Induction of apoptosis was confirmed by DNA electrophoresis and Hoechst staining. Westen blot analysis and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. Tested G361 cells showed several lines of apoptotic manifestation such as activation of caspase-3, DFF and PARP, DNA degradation (HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential, and the release of cytochrome c and AIF to cytosol. Between two synthetic derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199 did. Taken collectively, we here demonstrated for the first time that synthetic CDCA dedrivatives induce apoptosis of human melanoma cells through the proteasome, mitochondria and caspase pathway. Therefore our data provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human melanoma cells from its powerful apoptosis-inducing activity.
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