Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Extract from Artemisia annua Linné Induces Apoptosis through the Mitochondrial Signaling Pathway in HepG2 Cells

HepG2 간암세포에서 미토콘드리아 경로를 통한 개똥쑥 추출물의 Apoptosis 유도 효과

  • Kim, Bo Min (Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University) ;
  • Kim, Guen Tae (Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University) ;
  • Kim, Eun Ji (Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University) ;
  • Lim, Eun Gyeong (Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University) ;
  • Kim, Sang-Yong (Department of Food Science & Bio Technology, Shinansan University) ;
  • Kim, Young Min (Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University)
  • 김보민 (한남대학교 생명나노대학 생명시스템과학과) ;
  • 김근태 (한남대학교 생명나노대학 생명시스템과학과) ;
  • 김은지 (한남대학교 생명나노대학 생명시스템과학과) ;
  • 임은경 (한남대학교 생명나노대학 생명시스템과학과) ;
  • 김상용 (신안산대학교 식품생명과학과) ;
  • 김영민 (한남대학교 생명나노대학 생명시스템과학과)
  • Received : 2016.08.04
  • Accepted : 2016.09.13
  • Published : 2016.12.31
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Abstract

The Akt/mammalian target of the rapamycin (mTOR) pathway is activated in the majority of human cancers. Activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy. In this study, we evaluated the apoptotic effect of ethanol extract of Artemisia annua L. through down-regulation of Akt signal pathways and the mitochondrial pathway in hepato-carcinoma cells (HepG2). A. annua extract is known as a medicinal herb that is effective against cancer. We evaluated anti-proliferative activity by MTT-based viability assay and apoptotic effect by Annexin-V/PI staining, mitochondrial membrane potential (MMP), and caspase-3/7 activity as determined by flow cytometry. A. annua treatment led to loss of MMP, resulting in cytochrome c-inducible activation of caspase-3/7. Treatment with A. annua extract reduced activities of Akt/mTOR/anti-apoptotic proteins (such as Bcl-2 and $Bcl-X_L$), leading to increased activation of tumor suppressor p53 and pro-apoptotic proteins (such as Bax and Bak). We applied LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR) to determine the relationship between signal transduction of proteins associated with apoptosis. LY294002 and rapamycin significantly reduced cell viability and increased apoptosis. These results indicate that Bcl-2 and caspase-3 are key regulators in A. annua extract-induced apoptosis in HepG2 cells and are controlled through the Akt/mTOR signaling pathway.

Akt 및 mTOR는 세포 생존에 필수적인 경로로 세포 성장과 증식 등에서 중요한 역할을 하는 것으로 알려져 있다. 본 연구에서는 항암 및 항균 효과가 있는 것으로 알려진 개똥쑥(Artemisia annua L.)에 의한 HepG2 간암세포의 apoptosis 유도 효과를 확인하였다. 본 연구 결과에 의하면 개똥쑥 추출물의 처리 농도가 증가함에 따라 HepG2 세포의 생존율은 억제되었으며, 이는 apoptosis 유도 효과에 의한 것임을 세포의 형태적 변화와 flow cytometry를 통해 확인하였다. 그리고 mitopotential assay와 caspase-3/7 activity assay, western blotting으로 Bcl-2 family 단백질을 확인함으로써 apoptosis 경로 중 내인성 경로(intrinsic pathway)에 의해 apoptosis가 일어남을 알 수 있었다. 이러한 효과는 Akt/mTOR의 활성 저해와 연관이 있었으며 Akt/mTOR의 저해제인 LY294002/rapamycin을 개똥쑥 추출물과 병행처리하였을 경우 개똥쑥 추출물에 의한 apoptosis 효과를 더욱 증대시켰다. 따라서 Akt/mTOR의 저해는 개똥쑥 추출물의 apoptosis 효과를 상승시켰으며 이에 따라 미토콘드리아의 기능 손상과 caspase 활성의 증가를 통해 이루어짐을 확인하였다.

Keywords

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