Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Relationship between Reactive Oxygen Species and Adenosine Monophosphate-activated Protein Kinase Signaling in Apoptosis Induction of Human Breast Adenocarcinoma MDA-MB-231 Cells by Ethanol Extract of Citrus unshiu Peel

진피 추출물에 의한 인간유방암 MDA-MB-231 세포의 apoptosis 유도에서 ROS 및 AMPK의 역할

  • Kim, Min Yeong (Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • HwangBo, Hyun (Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • Ji, Seon Yeong (Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • Hong, Su-Hyun (Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • Choi, Sung Hyun (Department of System Management, Korea Lift College) ;
  • Kim, Sung Ok (Department of Food Science and Biotechnology, College of Engineering, Kyungsung University) ;
  • Park, Cheol (Department of Molecular Biology, College of Natural Sciences, Dong-eui University) ;
  • Choi, Yung Hyun (Department of Biochemistry, Dong-eui University College of Korean Medicine)
  • 김민영 (동의대학교 한의과대학 생화학교실) ;
  • 황보현 (동의대학교 한의과대학 생화학교실) ;
  • 지선영 (동의대학교 한의과대학 생화학교실) ;
  • 홍수현 (동의대학교 한의과대학 생화학교실) ;
  • 최성현 (한국승강기대학교 승강기시스템관리과) ;
  • 김성옥 (경성 대학교 공과대학 식품응용공학부) ;
  • 박철 (동의대학교 자연과학대학 분자생물학과) ;
  • 최영현 (동의대학교 한의과대학 생화학교실)
  • Received : 2019.01.09
  • Accepted : 2019.02.11
  • Published : 2019.04.30
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Abstract

Citrus unshiu peel extracts possess a variety of beneficial effects, and studies on their anticancer activity have been reported. However, the exact mechanisms underlying this activity remain unclear. In the current study, the apoptotic effect of ethanol extract of C. unshiu peel (EECU) on human breast adenocarcinoma MDA-MB-231 cells and related mechanisms were investigated. The results showed that the survival rate of MDA-MB-231 cells treated with EECU was significantly inhibited in a concentration-dependent manner, which was associated with the induction of apoptosis. EECU-induced apoptosis was associated with the activation of caspase-8 and caspase-9, which initiate extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3, a representative effect caspase. EECU suppressed the expression of the inhibitor of apoptosis family of proteins, leading to an increased Bax/Bcl-2 ratio and proteolytic degradation of poly (ADP-ribose) polymerase. EECU also enhanced the loss of the mitochondrial membrane potential and cytochrome c release from the mitochondria to the cytosol, along with truncation of Bid. In addition, EECU activated AMP-activated protein kinase (AMPK), and compound C, an AMPK inhibitor, significantly weakened EECU-induced apoptosis and cell viability reduction. Furthermore, EECU promoted the generation of reactive oxygen species (ROS), which acted as upstream signals for AMPK activation as pretreatment of cells, with the antioxidant N-acetyl cysteine reversing both EECU-induced AMPK activation and apoptosis. Collectively, these findings suggest that EECU inhibits MDA-MB-231 adenocarcinoma cell proliferation by activating intrinsic and extrinsic apoptotic pathways, which was mediated through ROS/AMPK-dependent pathways.

한의학에서는 진피(陳皮)라고 칭하는 귤의 껍질(Citrus unshiu peel) 추출물은 항산화, 항염증 및 항균 특성을 포함한 다양한 약리학적 효능을 갖는 것으로 알려져 있다. 최근 그들의 항암 활성에 대한 가능성이 보고되었지만 정확한 기전 연구는 여전히 미비한 실정이다. 본 연구에서는 인간 유방암 MDA-MB-231 세포를 대상으로 진피 에탄올(EECU, ethanol extract of C. unshiu peel) 추출물의 항암 효능을 평가하고 그에 따른 기전 연구를 수행하였다. 본 연구의 결과에 의하면 EECU에 의한 MDA-MB-231 세포의 증식억제는 세포사멸(apoptosis) 유도와 관련이 있었다. EECU에 의한 apoptosis는 caspase-8, -9 및 -3의 활성화와 IAPs 계열의 발현 감소에 따른 PARP의 분해와 Bax : Bcl-2 비율의 증가와 연관이 있었다. 또한 EECU는 Bid의 truncation과 함께 미토콘드리아 막 잠재력의 감소와 세포질로 cytochrome c의 이동을 촉진시켰다. 아울러 EECU는 AMPK 및 ACC의 인산화를 촉진시켰으나, AMPK 효소 활성의 저해제는 EECU에 의한 apoptosis 유도와 생존력 저하를 현저하게 억제하였다. 부가적으로 EECU는 AMPK 활성화의 상류 신호로 작용하는 활성산소종(ROS)의 생성을 촉진시켰으며, 강력한 항산화제인 NAC는 EECU에 의한 AMPK의 활성화와 apoptosis를 역전시켰다. 결론적으로 EECU는 ROS/AMPK 의존적인 내인성 및 외인성 apoptosis 경로를 활성화시킴으로써 MDA-MB-231 세포 증식을 억제하였음을 알 수 있었다.

Keywords

SMGHBM_2019_v29n4_410_f0001.png 이미지

Fig. 1. Inhibition of cell viability and induction of apoptosis by EECU in MDA-MB-231 cells.

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Fig. 2. Activation of caspases and inhibition of IAP family proteins expression by EECU in MDA-MB-231 cells.

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Fig. 3. Effects of EECU on the levels of DR-related and Bcl-2 family proteins in MDA-MB-231 cells.

SMGHBM_2019_v29n4_410_f0004.png 이미지

Fig. 4. Effects of EECU on the levels of MMP values and cytochrome c expression in MDA-MB-231 cells.

SMGHBM_2019_v29n4_410_f0005.png 이미지

Fig. 5. Involvement of AMPK activation in EECU-induced apoptosis in MDA-MB-231 cells.

SMGHBM_2019_v29n4_410_f0006.png 이미지

Fig. 6. ROS-dependent activation of AMPK by EECU in MDA-MB-231 cells.

SMGHBM_2019_v29n4_410_f0007.png 이미지

Fig. 7. A suggested schematic model for EECU-induced apoptosis in MDA-MB-231 cells.

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