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

김민영; 황보현; 지선영; 홍수현; 최성현; 김성옥; 박철; 최영현

doi:10.5352/jls.2019.29.4.410

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

생명과학회지 = Journal of life science, v.29 no.4 = no.228, 2019년, pp.410 - 420

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)

초록
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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.

Abstract ▼ AI-Helper

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.

주제어

표/그림 (7)

그림 Fig. 1. Inhibition of cell viability and induction of apoptosis by EECU in MDA-MB-231 cells.
그림 Fig. 2. Activation of caspases and inhibition of IAP family proteins expression by EECU in MDA-MB-231 cells.
그림 Fig. 3. Effects of EECU on the levels of DR-related and Bcl-2 family proteins in MDA-MB-231 cells.
그림 Fig. 4. Effects of EECU on the levels of MMP values and cytochrome c expression in MDA-MB-231 cells.
그림 Fig. 5. Involvement of AMPK activation in EECU-induced apoptosis in MDA-MB-231 cells.
그림 Fig. 6. ROS-dependent activation of AMPK by EECU in MDA-MB-231 cells.
그림 Fig. 7. A suggested schematic model for EECU-induced apoptosis in MDA-MB-231 cells.

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제안 방법

Effects of EECU on the levels of MMP values and cytochrome c expression in MDA-MB-231 cells. (A) After 24 hr incubation with the indicated concentrations of EECU, the cells were stained with JC-1 dye, and were then analyzed on a flow cytometer, in order to evaluate the changes in MMP. The data were expressed as the mean ± SD of three independent experiments (*p<0.
unshiu peel (EECU) against MDA-MB-231 human breast adenocarcinoma cancer cells. In this study, we found for the first time that AMPK activation by EECU triggers apoptotic cell death in MDAMB-231 cells, and suggest that ROS production is involved in AMPK activation by EECU.

데이터처리

In each treatment group, the statistical significance was compared and verified using a one-way ANOVA or Student t-test method (p<0.05 or p<0.01).

이론/모형

The effect of EECU on MDA-MB-231 cell viability was analyzed by MTT assay. Fig.

성능/효과

We therefore investigated the possible relevance of ROS production in EECU-induced AMPK activation. As noted in the results, the accumulation of ROS by treatment with EECU disappeared in cultured MDA-MB-231 cells in medium containing anti-oxidant NAC, and markedly attenuated EUCC-induced phosphorylation of AMPK and ACC. Moreover, EECU-induced apoptosis was effectively prevented by blocking ROS production, and the reduced cell viability was also significantly restored to the control level.
In conclusion, the present results demonstrate that EECU induces apoptosis in MDA-MB-231 adenocarcinoma cells through intrinsic and extrinsic pathways by increasing DRrelated regulators, MMP loss, the cytosolic release of cytochrome c, and ROS generation, combined with an increase in the Bax/Bcl-2 ratio and Bid truncation. EECU also promoted AMPK activation, and the inhibition of AMPK activity blocked EECU-induced activation of caspase, and prevented the induction of apoptosis.
7). It also demonstrated that EECU activates AMPK in a ROS-dependent manner, and contributes to EECU-mediated suppression of MDA-MB-231 cell viability.
Our immunoblotting results demonstrated that EECU remarkably enhanced the phosphorylation of AMPKα (Thr 172), as well as ACC (Ser 79), with increasing EECU concentration (Fig. 5A), indicating that they were converted to the activated state.
Because the activation of caspase cascades plays a key role in two representative apoptosis-inducing pathways [5, 23], we next investigated whether caspase activation is involved in the induction of apoptosis by EECU in MDA-MB-231 cells. Our immunoblotting results showed that expression of pro-caspase-8, an initiator caspase of the extrinsic apoptosis pathway, was apparently decreased with increasing EECU concentration, while expression of active-caspase-8 was increased. Although no expression was observed of the activecaspase-9 or active-caspase-3, an initiator caspase of the extrinsic apoptosis pathway and a typical effector caspase, respectively, the expression of their pro-forms was suppressed, depending on the EECU treatment concentration (Fig.
2 indicated that the possibility that two apoptotic pathways were activated in the induction of apoptosis by EECU, so we next investigated the effect of EECU on the expression of DR-related and Bcl-2 family proteins. The immunoblotting data indicated that the expressions of Fas, Fas-associated protein with death domain (FADD), DR4, DR5, and TNF-related apoptosis-inducing ligand (TRAIL) were concentration-dependently increased by EECU treatment, even though the expression of Fas ligand (FasL) was not changed (Fig. 3), suggesting that EECU might regulate the extrinsic pathway. Among the Bcl-2 family proteins, anti-apoptotic Bcl-2 expression was remarkably reduced by EECU treatment, but the expression of pro-apoptotic Bax was increased to some extent.
unshiu peel, and showed that EECU could inhibit the proliferation of MDA-MB-231 adenocarcinoma cells, and induce apoptosis. The principal finding of this study is that both extrinsic and intrinsic apoptosis pathways can be activated to induce apoptosis of MDA-MB-231 cells by EECU (Fig. 7). It also demonstrated that EECU activates AMPK in a ROS-dependent manner, and contributes to EECU-mediated suppression of MDA-MB-231 cell viability.
To determine whether EECU treatment led to growth inhibition due to apoptosis induction, the apoptosis rate was measured by a flow cytometer. The results showed that compared with the untreated control group, EECU markedly enhanced the percentage of apoptotic cells (Fig. 1C), indicating that EECU suppressed cell viability by inducing apoptosis in MDA-MB-231 cells.
Activation of AMPK requires phosphorylation by upstream AMPK kinase, and regulates the activity of various downstream targets that regulate cell fate [2, 3]. Therefore, the relation of AMPK pathway to EECU-induced apoptosis in MDA-MB-231 cells was investigated, and it was found that EECU promotes phosphorylation of AMPK and its downstream target ACC. However, blockade of AMPK activation by compound C, an AMPK inhibitor, significantly prevented EECU-induced apoptosis and decreased cell viability, which was accompanied by the reduction of caspase activation and PARP degradation.

후속연구

unshiu Peel, the apoptotic effect of WECU in MDAMB-231 cells seems to be due to the overall effect of various substances contained in WECU. Therefore, further investigation of the relevance of other intracellular signaling pathways and identification of key active compounds in the WECU should be undertaken in the future.

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