Eugenol에 의해 세포자멸사를 보이는 세포 배양된 다양한암세포들에서의 phospho-ser 15-p53의 이동
Translocation of Phospho-ser 15-p53 in Eugenol-inducedApoptosis of in vitro Cultured Cancer Cells
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신이(Magnoliae flos)나 정향(clove) 같은 풀잎의 주성분인 eugenol (4-allyl-2-methoxyphenol)은 p53 단백 질 경로를 통해서 비만세포의 세포자멸사(apoptosis)를 유도한다고 알려져 있다. 본 연구는 eugenol의 생체내에서 의 효과와 다른 유형의 p53 단백질을 발현하는 여러 가지의 암세포에서의 eugenol 유도 세포자멸사의 분자생물학 적인 기전을 알기 위해서 수행되었다. 배안 비만세포에 대한 eugenol의 효과를 알기 위해서 쥐을 이용한 아나필락시스 실험을 시행하였고 세포자멸 사를 일으키는 비만세포를 관찰하기 위해서는 TUNEL 염색법을 사용하였다. 그리고 여러 가지의 암세포들에 eugenol을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이저주사현미경 검경, 사립체분리 등을 시행하 였다. 생체내에서의 실험에서 eugenol은 창자간막(mesentery)의 비만세포에 세포자멸사를 유도하여, 비만세포의 밀집 도를 감소시켰다. 비록 eugenol이 p53의 발현을 증가시키지는 못했지만, eugenol은 p53을 사립체로 이동시키고, 이 동된 후 연이어서 cytochrome c를 사립체에서 세포질로 유리시키는 것이 관찰되었다. 그리고 eugenol은 wild type p53을 가지고 있는 암세포들에서는 phospho-ser 15-p53을 증가시켰으나, mutant p53 혹은 p53 결핍 암세포들에서 는 phospho-ser 15-p53의 증가는 없었다. 더 나아가서 wild type p53을 가지고 있는 암세포에서 사립체로 이동한 p53이 ser 15에 인산화되는 것이 관찰되었다. 본 연구는 eugenol이 wild type p53을 가진 암세포에서 phospho-ser 15-p53의 세포내 위치이동을 통해 세포자멸 사를 유도하다는 증거를 제시하였다. 더 나아가서 wild type p53을 가진 암세포의 항암효과는 phospho-ser 15-p53 의 의존적 방법을 통한 세포자멸사를 유도하는 eugenol의 약리학적 효과가 관계할 수 있다는 것을 추정할 수 있 다.
Abstract
Eugenol (4-allyl-2-methoxyphenol), a major ingredient of herbs such as clove and Magnoliae Flos, is known to induce apoptosis in mast cells via p53 pathway. This study was undertaken to examine the in vivo effect of eugenol and the molecular mechanism underlying eugenol-induced apoptosis in several cancer cells with different p53 status. Effect of eugenol on mesenteric mast cells was tested using a rat anaphylaxis model. And TUNEL staining was conducted to observe the cells undergoing apoptosis. Several cancer cells were treated with eugenol, and Western blotting, immunocytochemistry, confocal microscopy and mitochondrial fractionation were conducted. Eugenol induced apoptosis in mast cells of mesentery in vivo, decreasing the density of mast cells. Although eugenol did not increase the expression level of p53, it caused the translocation of p53 into mitochondria and subsequent release of cytochrome c. Eugenol increased the level of phospho-ser 15-p53 in several cancer cells with wild type p53 but not in the cells with mutant p53 or p53 deficient cancer cell. In cancer cells with wild type p53, p53 translocated into mitochondria was phosphorylated on ser 15. In conclusion, eugenol induces apoptosis in cancer cells with wild type p53 via the translocation of phospho-ser 15- p53. Furthermore our data suggest that the anticancer effect on cancer cells with wild type p53 may be involved with the pharmacological effect of eugenol regulating apoptosis via a phospho-ser 15-p53 dependent fashion.
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