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인체 폐암 세포주 A549에서 Litsea populifolia 추출물의 항산화 및 항암활성 분석

Anti-oxidative and Anti-cancer Activities of Ethanol Extract of Litsea populifolia

  • 진수정 (동의대학교 블루바이오소재개발센터) ;
  • 오유나 (동의대학교 블루바이오소재개발센터) ;
  • 정현영 (동의대학교 블루바이오소재개발센터) ;
  • 윤희정 (동의대학교 공과대학 바이오응용공학부) ;
  • 박정하 (동의대학교 공과대학 바이오응용공학부) ;
  • 권현주 (동의대학교 블루바이오소재개발센터) ;
  • 김병우 (동의대학교 블루바이오소재개발센터)
  • Jin, Soojung (Blue-Bio Industry Regional Innovation Center, Dong-eui University) ;
  • Oh, You Na (Blue-Bio Industry Regional Innovation Center, Dong-eui University) ;
  • Jeong, Hyun Young (Blue-Bio Industry Regional Innovation Center, Dong-eui University) ;
  • Yun, Hee Jung (Division of Applied Bioengineering, College of Engineering, Dong-eui University) ;
  • Park, Jung-ha (Division of Applied Bioengineering, College of Engineering, Dong-eui University) ;
  • Kwon, Hyun Ju (Blue-Bio Industry Regional Innovation Center, Dong-eui University) ;
  • Kim, Byung Woo (Blue-Bio Industry Regional Innovation Center, Dong-eui University)
  • 투고 : 2019.04.03
  • 심사 : 2019.06.05
  • 발행 : 2019.06.30

초록

본 연구에서는 인체 폐암 세포인 A549를 사용하여 Litsea populifolia 에탄올 추출물(EELP)의 항산화 및 항암활성과 그 분자적 기전에 관하여 연구하였다. 먼저 EELP의 DPPH 라디칼 소거활성을 측정한 결과, $IC_{50}$$11.71{\mu}g/ml$로 유의적인 항산화활성을 보였다. 또한 EELP가 인체폐암세포주인 A549와 정상 폐세포인 IMR90의 세포증식에 미치는 영향을 알아본 결과, 정상세포의 생존율에는 거의 영향을 끼치지 않은 반면, EELP 농도의존적으로 A549 세포의 성장이 저해되었으며, 세포 주기 변화를 분석한 결과 EELP에 의해 A549 세포의 강력한 G1 arrest가 유도되는 것을 확인하였다. EELP에 의해 유도되는 G1 arrest는 세포주기 조절 인자인 Cyclin D1, Cyclin E, Cyclin-dependent kinase인 CDK2와 CDK6의 mRNA 발현 감소와 더불어 단백질 발현 감소와 연관되어 있었다. 또한 EELP 처리에 의한 CDK/Cyclin complex의 발현 저해는 DNA 손상에 의해 활성화되는 CHK2의 활성화 형태인 p-CHK2의 발현 증가에 따른 p53 인산화에 따른 활성화와 CDK 활성화 효소인 CDC25A 탈인산화효소의 인산화에 따른 저해에 의해 나타나는 결과로 사료된다. 이러한 결과들로부터 EELP는 두가지 경로인 p53-의존성과 p53-비의존성(ATM/CHK2/CDC25A/CDK2) 경로를 통해 A549의 G1 arrest를 유도하여 세포 증식을 억제하는 것으로 사료된다. 본 연구결과는 EELP가 폐암에 대한 새로운 항암활성 소재로서 사용될 수 있는 가능성을 시사하며, 또한 EELP의 세포주기 조절에 의한 항암기전을 이해하고 향후 지속적 연구를 하는 데 있어서 귀중한 기초자료로 사용될 수 있을 것이다.

Litsea populifolia, a plant species of the Lauraceae family, is widely distributed in the tropical and subtropical areas of Asia. The phylogenetic relationships and botanical characteristics of L. populifolia have been reported; however, its anti-oxidative and anti-cancer activities remain unclear. In this study, we evaluated the anti-oxidative and anti-cancer effects of ethanol extracts of L. populifolia (EELP) together with the molecular mechanism of its anti-cancer activity in human lung adenocarcinoma A549 cells. EELP showed significant anti-oxidative effects with a 50% inhibitory concentration at $11.71{\mu}g/ml$, which was measured by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. EELP exhibited cytotoxic activity and induced cell cycle arrest at the G1 phase in A549 cells in a dose-dependent manner, whereas EELP did not have the cytotoxic effect on the normal human lung cell line IMR90. Treatment with EELP also resulted in a decreased expression of G1/S transition-related molecules-including cyclin-dependent kinase (CDK) 2, CDK6, cyclin D1, and cyclin E-both for the transcription and translation levels. EELP-induced G1 arrest was associated with the phosphorylation of checkpoint kinase 2 (CHK2), p53, cell division cycle 25 homolog A (CDC25A), and the reduction of CDC25A expression in A549 cells. Collectively, these results suggest that EELP may exert an anti-cancer effect by cell cycle arrest at the G1 phase through both p53-dependent and p53-independent (ATM/CHK2/CDC25A/CDK2) pathways in A549 cells.

키워드

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Fig. 1. Effects of EELP on cell growth and morphology in human lung carcinoma A549 cells.

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Fig. 2. G1 arrest of cell cycle in EELP-treated A549 cells.

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Fig. 3. Transcriptional repression of G1 checkpoint proteins by EELP in A549 cells.

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Fig. 4. Downregulation of G1 checkpoint proteins by EELP in A549 cells.

Table 1. Primer sets for RT-PCR analysis

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Table 2. Total phenolic contents (TPC) and DPPH activity of ethanol extract of Litsea populifolia

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Table 3. Cell cycle distribution of ethanol extract of Litsea popu-lifolia

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