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Apoptotic Effect of Co-Treatment with Chios Gum Mastic and Eugenol on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

사람혀편평세포암종세포에서 Chios gum mastic과 eugenol의 병용처리가 미치는 세포자멸사 효과에 관한 연구

  • Sohn, Hyeon-Jin (Department of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Yea, Byeong-Ho (Department of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Kim, In-Ryoung (Department of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Park, Bong-Soo (Department of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Jeong, Sung-Hee (Department of Oral Medicine, School of Dentistry, Pusan National University) ;
  • Ahn, Yong-Woo (Department of Oral Medicine, School of Dentistry, Pusan National University) ;
  • Ko, Myung-Yun (Department of Oral Medicine, School of Dentistry, Pusan National University)
  • 손현진 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 예병호 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 김인령 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 박봉수 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 정성희 (부산대학교 치의학전문대학원 구강내과학교실) ;
  • 안용우 (부산대학교 치의학전문대학원 구강내과학교실) ;
  • 고명연 (부산대학교 치의학전문대학원 구강내과학교실)
  • Received : 2011.07.15
  • Accepted : 2011.08.20
  • Published : 2011.09.30

Abstract

Eugenol (4-allyl-2-methoxyphenol) is a natural phenolic constituent extensively used in dentistry as a component of zinc oxide eugenol cement and is applied to the mouth environment. Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and natural phenolic compound, eugenol on SCC25 human tongue squamous cell carcinoma cell line. To investigate whether the co-treatment with eugenol and CGM compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by Hoechst staining, TUNEL staining and DNA hypoploidy. Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and the translocation of apoptosis-related proteins in co-treatment. In this study, co-treatment of with eugenol and CGM on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, the increase and decrease of Bax and Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-3, caspase-6 caspase-7, caspase-9, PARP, Lamin A/C and DFF45 (ICAD) whereas each single treated SCC25 cells did not show or very slightly these patterns. Although the single treatment of 40 ${\mu}g$/ml CGM and 0.5 mM eugenol for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that combination therapy with CGM and eugenol could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.

Chios gum mastic (CGM)은 그리이스 키오스 섬에서만 자생하는 Pistiacia lentiscus L. var. Chia. 의 잎과 줄기로부터 추출되어진 식물성 수지이며, 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 위궤양, 십이지장궤양 등의 민간 치료약재로서 사용되어져 왔다. 정향나무에서 추출하는 페놀화합물인 eugenol은 zinc oxide eugenol의 구성성분으로 치과치료를 위해 많이 사용되고 있다. 본 연구는 사람혀편평세포암종세포(SCC25 cells)에서 천연물질인 CGM과 eugenol을 병용처리한 후 세포자멸사 효과가 있는지를 알아보기 위해 수행하였다. CGM과 eugenol의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위하여 MTT법을 시행하였고, 세포자멸사의 유도와 증가를 알기 위하여 Hoechst 염색법, TUNEL 염색법, DNA hypoploidy법을 사용하였다. 그리고 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위하여 Western blot 분석과 면역형광염색법을 수행하였다. 본 연구에서는 CGM과 eugenol이 병용처리된 SCC25 세포에서 핵의 농축, DNA분절, Bax의 증가와 Bcl-2의 감소, DNA양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, caspase-3, caspase-6, caspase-7, caspase-9, PARP, Lamin A/C 그리고 DFF45 (ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 반면에 CGM과 eugenol이 각각 단독 처리된 SCC25 세포에서는 세포자멸사 현상이 미미하였다. 24시간 동안 40 ${\mu}g$/ml의 CGM과 0.5 mM의 eugenol을 각기 단독처리 한 결과에서는 세포자멸사를 거의 유도하지 못했으나, 병용처리 한 결과에는 아주 탁월한 세포자멸사의 유도를 보였다. 그러므로 본 실험결과는 사람구강편평세포암종 환자들에게 CGM과 eugenol의 병용요법이 새로운 치료전략으로서의 가능성을 줄 수 있다고 생각한다.

Keywords

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