천연물질Chios gum mastic과합성chenodeoxycholic acid유도체HS-1200의병용처리가사람골육종세포에미치는세포자멸사효과에대한연구
Apoptotic Effect of Co-Treatment with a Natural Product,Chios Gum Mastic, and a Synthetic Chenodeoxycholic Acid Derivative,HS-1200, on Human Osteosarcoma Cells
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Chios gum mastic(CGM)은 그리이스 지역에서만 서식하는 Pistacia lenticulus 나무의 줄기와 잎에서 추출 한 수지상의 천연 추출물이다. 합성 chenodeoxycholic acid (CDCA) 유도체가 여러 가지 암세포에 유도한 세포자멸 사 연구들이 보고되어져 왔다. 본 연구는 천연물질인 CGM과 합성 CDCA 유도체인 HS-1200의 병용처리가 사람 골육종세포에 효과적인 상승 세포자멸사 효과가 있는지를 알기 위해서 수행되었다. CGM과 HS-1200의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 MTT 법을 시행하였고, 세포자멸사의 유도와 증가를 확인하기 위해서 Hoechst 염색법과 DNA 전기영동법을 사용하였다. 병용처리 때, 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위해서 Western bot 분 석과 면역형광염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 병용처리 된 사람골육종세포는 단독처리 된 사람골육종세포에서 거의 관찰할 수 없었던 많은 핵 응축, DNA 조 각남, 사립체막 전위와 proteasome 활성도의 감소, DNA 양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, caspase-7, caspase-3 그리고 PARP의 활성화와 같은 세포자멸사 증거를 보였다. 24시간 동안 40 μg/mL CGM과 25 μM HS-1200을 각기 단독처리 한 결과에서는 세포자멸사를 유도 못했으나, 병용처리한 결과에는 아주 탁월한 세포자멸사의 유도를 보였다. 이러한 병용처리 결과는 사람골육종의 새로운 치 료적 전략으로 응용될 수 있다고 생각한다.
Abstract
Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently it reported that CGM induced apoptosis in a few cancer cells in vitro. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of HOS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and translocation of apoptosis-related proteins in co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change In this study, HOS cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation whereas each single treated HOS cells did not. Although the single treatment of 40 μg/mL CGM or 25 μM HS-1200 for 24 h did not induce apoptosis, the cotreatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of CGM and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.
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