$2{\beta}$, $3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid처리에 의한 인간 간암세포주 HepG2의 apoptosis 유도

유기현,이종민,황보전,송명종,양혜정,백남인,김성훈,김대근,권병목,박미현,정인식,Yoo, Ki-Hyun,Lee, Jong-Min,HwangBo, Jeon,Song, Myoung-Chong,Yang, Hye-Joung,Baek, Nam-In,Kim, Soung-Hoon,Kim, Dae-Keun,Kwon, Byoung-Mok,Park, Mi-Hyun,Chung, 한국응용생명화학회 2006 Applied Biological Chemistry (Appl Biol Chem) Vol.49 No.4

Triterpenoid를 포함하고 있는 $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid를 애기마름으로부터 분리하였다. 이것은 pentacyclic triterpenes의 공통 구조를 가지며 amyrin ursolic acid 그룹에 속해 있다. 본 연구에서는 이 화합물의 독성 영향을 인간 간암 세포주인 HepG2에서 조사하였다. $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid는 처리한 양에 비례하여 HepG2 세포주에서 독성을 보였다. 그리고 Confocal microscopy 결과는 $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid를 HepG2 세포에 처리한 시간에 비례하여 녹색 형광의 증가를 보여주었다. $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid는 또한 HepG2 세포의 sub-G1 cell population 뿐만 아니라 DNA 분절(fragmentation) 현상의 증가를 보여 주었다. 이러한 결과는 $22{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid가 HepG2 세포에서 apoptosis를 통한 세포 사멸 유도를 의미한다. [ $2{\beta},\;3{\alpha}$ ], 23-trihydroxyrus-12-ene-28-oic acid was isolated from Trapa pseudoincisa S. et Z. It has a common structure of pentacyclic triterpenes and belongs to the amyrin ursolic acid group. The cytotoxic effect of this compound was investigated in human hepatoma cell line HepG2. $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid showed dose-dependent cytotoxicity in HepG2 cells. Confocal microscopy data showed that green fluorescence was increased in $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid treated-HepG2 cells in a time-dependent manner. $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid also increased the sub-G1 cell population of HepG2 cells as well as ladder-like DNA fragmentation. Taken together, our results indicate that $2{\beta},\;3{\alpha}$, 23-trihydroxyrus-12-ene-28-oic acid induced apoptosis in HepG2 cells.

자금정(紫金錠)이 간암세포주 HepG2의 세포고사 및 세포주기에 미치는 영향

조영기,전지영,신용진,설재균,이재화,원진희,문구,Cho, Young-Kee,Jeon, Ji-Young,Shin, Yong-Jeen,Seol, Jae-Kyun,Rhee, Jae-Hwa,Won, Jin-Hee,Moon, Goo 대한한방내과학회 2007 大韓韓方內科學會誌 Vol.28 No.4

Objectives : Jageum-Jung is used as an anti-cancer agent in oriental medicine, but the mechanism by which it induces cell death in cancer cells is still unclear. The purpose of this study was to investigate the effects of Jageum-Jung on apoptosis and cell cycle arrest in HepG2 hepatoma cells. Methods : Various cancer cell lines including HepG2, C6 glioma, SH-SY5Y, PANC-1, and MCF-7 cells, were used. Apoptosis was determined by DAPI nuclei staining and flow cytometry in HepG2 cells treated with various concentrations (from 25 to 200 ${\mu}g/ml$) of $H_2O$ extract of Jageum-Jung (JGJ) for 48 hrs. Expression of cell cycle arrest mediators including Rb, p53, p21, cyclin B1, cdk4, and cyclin E proteins were measured by Western blot analysis. To estimate intracellular hydrogen peroxide levels and intracellular nitric oxide levels, HepG2 cells were stained with DCFH-DA dye and DAF dye, subjected on flow cytometric analysis. Results : 1. Jageum-Jung decreased the viability of HepG2 cells in a dose-dependent manner. 2. Jageum-Jung induced the catalytic activation of caspase-3 in HepG2 cells. 3. Jageum-Jung increased the intracellular hydrogen peroxide and NO in HepG2 cells. 4. Jageum-Jung increased the expression of Rb, p53 and p21 in HepG2 cells. 5. Jageum-Jung induced the expression of cyclin B1, cdk4, and cyclin E in HepG2 cells. Conclusions : Taken together, we suggest that Jageum-Jung exhibits cytotoxic effects on HepG2 cells, causing apoptosis and cell cycle arrest. The results showed that Jageum-Jung may do so by regulating the expression of specific target molecules that promote efficient apoptotic cell death following $G_2$/M phase arrest in a dose-dependent manner.

유근피(楡根皮) 추출액(抽出液)이 HeoG2 간암세포(肝癌細胞)에 미치는 항암효과(抗癌效果) 및 기전(機轉)에 대(對)한 연구(硏究)

최수덕,박용권,김강산,강병기,한상일,Choi, Su-Deock,Park, Young-Kweon,Kim, Gang-San,Kang, Byung-Ki,Han, Sang-Il 대한한방내과학회 2000 大韓韓方內科學會誌 Vol.21 No.2

Objectives : The effects of aqueous extracts of Cortex ulmi pumilae (a traditional medicine for cancer treatment in oriental medicine) on the induction of apoptotic cell death were investigated in human liver origm hepatoma cell lines, HepG2. Methods : The death of HepG2 cells was markedly induced by the addition of extracts of Cortex ulmi pumilae in a dose-dependent manner. The apoptotic characteristic ladder pattern of DNA strand break was not observed in cell death of HepG2. In addition, it was not shown nucleus chromatin condensation and fragmentation under hoechst staining. However, by the using annexin V staining assay, externalizations of phosphatidylserine in HepG2 cell which were treated with Cortex ulmi pumilae extracts were detected in the early time (at 9 hr after extract treatment). Furthermore, LDH release was not detected in this early stage. Therefore, Cortex ulmi pumilae extracts-induced cell death of HepG2 cells is mediated by apoptotic death signal processes. Result : The activity of caspase 3-like proteases remained in a basal level in HepG2 cells which treated with the extract of Cordyceps sinensis. However, it was markedly increased in HepG2 cells which treated with two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) which were differently extracted (respectively, 2.3 and 3.3 fold). On a while, the phosphotransferase activities of JNK1 was markedly induced in HepG2 cells which were treated with two extracts of Cortex ulmi pumilae. On the contrary, the activation of transcriptional activator, activating protein1(AP-1) and NF-kB were severely decreased by these two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K). In addition, antioxidants (GSH and NAC) and intracellular $Ca2^+$ level regulator (Bapta/AM and Thapsigargin) did not affect Cortex ulmi pumilae extracts-induced apoptotic death of HepG2 cells. Conclusions : In conclusion, our results suggest that two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) induces the apoptotic death of human liver origin hepatoma HepG2 cells via activation of caspase 3-like proteases as well as JNK1, and inhibition of transcriptional activators, AP-1 and $NK-{\kappa}B$.

전나무 잎 추출물이 알코올 대사 효소활성과 H₂O₂ 및 에탄올로 유도된 HepG2 세포에서의 세포사멸에 미치는 영향

박미화(Mi Hwa Park),최영주(Young Ju Choi),김윤세(Yoon Se Kim),정경임(Kyung Im Jung) 한국식품영양과학회 2022 한국식품영양과학회지 Vol.51 No.6

본 연구에서는 전나무 잎 추출물이 알코올 분해 활성(ADH, ALDH)에 미치는 영향을 알아보고, H₂O₂ 및 알코올로 손상된 간세포의 apoptosis 과정에서 전나무 잎 추출물이 미치는 영향을 확인하기 위해 H₂O₂와 에탄올을 이용하여 HepG2 인간 간암 세포주에 산화스트레스를 유도한 후 항산화 효과와 간세포 보호 효과를 확인하였다. 알코올 분해의 1차 효소인 ADH 활성과 1차 대사산물인 아세트알데하이드를 분해하는 ALDH 활성에 대한 전나무 잎 추출물의 효과를 확인한 결과, 두 가지 효소 모두 전나무 잎 추출물에 농도 의존적으로 증가하였으며(P<0.05), 1 mg/mL 농도에서의 ADH 및 ALDH 활성은 각각 155.53% 및 130.89%로 나타났다. 산화적 스트레스 및 알코올성 간 손상으로부터 전나무잎 추출물의 효과를 검증하기 위하여 H₂O₂ 및 에탄올로 스트레스를 유도한 HepG2 세포에 전나무 잎 추출물을 처리한 결과 H₂O₂ 및 에탄올 단독 처리구에 비해 세포생존율은 유의적으로 증가하였다(P<0.05). H₂O₂ 및 에탄올로 유도된 HepG2 세포의 ROS level은 전나무 추출물에 농도 의존적으로 현저하게 감소하였다(P<0.05). 전나무 잎 추출물이 H₂O₂ 및 에탄올로 유도된 HepG2 간세포의 apoptosis에 미치는 영향을 알아보기 위해 Bax, Bcl-2 및 caspase-3 유전자의 단백질 발현 정도를 측정하였다. H₂O₂ 및 에탄올로 처리된 배지에서 배양한 HepG2 세포에서의 pro-apoptotic gene인 Bax 유전자 발현은 증가하였으나 전나무 잎 추출물의 처리에 의해 발현이 감소하였다(P<0.05). 또한 H₂O₂ 및 에탄올로 처리된 배지에서 배양한 HepG2 세포에서의 anti-apoptotic gene인 Bcl-2 유전자의 발현을 측정한 결과 H₂O₂로 간 손상을 유도했을 때 Bcl-2의 발현은 전나무 잎 추출물의 처리에 의해 증가하였으나(P<0.05), 에탄올로 간독성을 유도했을 때는 전나무 잎 추출물 처리에 의한 발현량 변화는 나타나지 않았다. Caspase-3 단백질 발현을 측정한 결과, H₂O₂ 및 에탄올로 간독성을 유도했을 때는 발현이 증가했으며, 전나무 잎 추출물 처리 시 발현을 감소시켜 산화적 손상 및 apoptosis 보호 효과를 확인하였다. 따라서 본 연구 결과 전나무 잎 추출물은 알코올 분해 활성과 함께 산화적 스트레스 및 알코올성 간독성으로 인한 ROS level을 낮추고 산화적 스트레스 및 알코올에 의한 apoptosis를 효과적으로 억제하여 간독성에 대한 보호 효과가 있는 것으로 판단된다. This study investigated the protective effect of the Abies holophylla leaf extract on the alcohol metabolism enzyme activity and against H₂O₂ and ethanol-induced oxidative stress in HepG2 cells. The effect of Abies holophylla leaf extract on alcohol metabolism was determined by measuring the generation of reduced nicotinamide adenine dinucleotide (NADH) by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). ADH and ALDH activities of Abies holophylla leaf extract were increased in a dose-dependent manner (P<0.05), and were determined to be 155.53% and 130.89% at 1 ㎎/mL concentration, respectively. Exposure to the Abies holophylla leaf extract resulted in decreased levels of reactive oxygen species production in H₂O₂ and ethanol-induced HepG2 cells (P<0.05). Moreover, H₂O₂-induced HepG2 cells treated with the Abies holophylla leaf extract showed reduced expression of the pro-apoptotic protein Bax and increased expression of the anti-apoptotic protein Bcl-2 (P<0.05). Furthermore, Abies holophylla leaf extract treatment significantly decreased the protein expression of Bax in ethanol-induced HepG2 cells (P<0.05). However, the protein expression of Bcl-2 remained unaltered. Treatment with the Abies holophylla leaf extract also reduced the caspase-3 protein expression in H₂O₂ and ethanol-exposed HepG2 cells. In conclusion, our results indicate that the Abies holophylla leaf extract exerts a cytoprotective effect against H₂O₂ and ethanol-induced cell damage. We believe that the Abies holophylla leaf extract has the potential to be developed as a natural hepatoprotective agent.

Millettia erythrocalyx 에탄올 추출물의 항산화 활성 및 항암 활성에 관한 연구

진수정(Soojung Jin),오유나(You Na Oh),손유리(Yu Ri Son),최선미(Sun Mi Choi),권현주(Hyun Ju Kwon),김병우(Byung Woo Kim) 한국생명과학회 2018 생명과학회지 Vol.28 No.1

Millettia erythrocalyx는 콩과(Fabaceae)에 속하는 식물로 중국, 태국, 인도 등 열대·아열대 지역에 분포하며, 항바이러스 활성을 보유하고 있다는 보고가 있으나 항산화능과 항암활성 등에 관한 연구는 보고된 바가 없다. 따라서 본 연구에서는 M. erythrocalyx의 에탄올 추출물(EEME)을 사용하여 항산화능을 측정하고, 인체간암세포주인 HepG2에 대한 항암활성과 그 분자적 기전에 관하여 분석하였다. 먼저 DPPH radical scavenging activity를 통해 분석한 결과, EEME의 IC50는 2.74 μg/ml로 뛰어난 항산화능을 보유하였음을 확인하였다. 또한 EEME 농도 의존적으로 HepG2 세포의 성장을 억제하였다. EEME의 HepG2 세포 사멸 효과의 기전을 분석하기 위하여 세포주기를 분석한 결과, EEME 농도의존적으로 SubG1 세포가 증가하였으며, Annexin V 염색과 DAPI 염색을 통해 apoptotic 세포 및 apoptotic body가 증가됨을 확인하였다. 또한 apoptosis 관련 단백질들의 발현변화를 분석한 결과, EEME 처리에 의해 사멸수용체인 Fas와 pro-apoptotic 단백질인 Bax의 발현이 증가되었으며, caspase-3, -8, -9가 활성화되고 최종적으로 PARP가 분해되어 apoptosis가 유도되었음을 확인하였다. 이러한 결과들로부터 EEME는 내인성 및 외인성 경로를 통한 apoptosis 유도에 의하여 HepG2 세포의 증식을 억제하는 항암활성을 보유하였음을 확인하였다. Millettia erythrocalyx, a species of plant in the Fabaceae family, is widely distributed in the tropical and subtropical regions of the world, such as the Indies, China, and Thailand. The antiviral activity of flavonoids from M. erythrocalyx has been reported; however, the antioxidative and anticancer activities of M. erythrocalyx remain unclear. In this study, we evaluated the antioxidative and anticancer effects of ethanol extract of M. erythrocalyx (EEME) and the molecular mechanism of its anticancer activity in human hepatocellular carcinoma HepG2 cells. EEME exhibited significant antioxidative effects, with a concentration at 50% inhibition (IC50) value of 2.74 μg/ml, as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay; moreover, it inhibited cell proliferation in a dosedependent manner in HepG2 cells. Cell cycle analyses showed that EEME induced HepG2 cell accumulation in the subG1 phase in a dose-dependent manner. EEME also induced apoptosis of HepG2 cells, with increases in apoptotic cells and apoptotic bodies, as detected by Annexin V and 4,6-diamidino-2-phenylindole (DAPI) staining, respectively. Treatment with EEME resulted in increased expression of First apoptosis signal (Fas), a death receptor, and Bcl-2-associated X protein (Bax), a proapoptotic protein, and the activation of caspase-3, 8, and 9, resulting in the cleavage of poly (Adenosine diphosphate-ribose) polymerase (PARP). Collectively, these results suggest that EEME may exert an anticancer effect in HepG2 cells by inducing apoptosis via both the intrinsic and extrinsic pathways.

HepG2 세포에서 Isoeugenol의 PCSK9 억제 효능

최효경(Hyo-Kyoung Choi),정민유(Min-Yu Chung) 한국식품영양과학회 2020 한국식품영양과학회지 Vol.49 No.9

본 논문에서는 isoeugenol의 PCSK9 억제능 관련 메커니즘을 규명하였다. HepG2 세포를 지방 부족 환경으로 만들고 isoeugenol을 처리하여 PCSK9과 LDLR, 그리고 전사인자의 단백질 및 유전자 발현 조절을 측정하였다. 독성 없는 농도의 isoeugenol(~100 μM)은 LDLR의 단백질 발현 조절에 관여하지 않았지만, PCSK9의 단백질 및 유전자 발현을 감소시켰다. Isoeugenol은 PCSK9 조절에 독립적으로 관여하는 전사인자인 HNF1α의 유전자 및 단백질 조절에는 관여하지 않았다. 따라서 isoeugenol은 SREBP2를 통해 PCSK9과 LDLR의 유전자 발현을 억제하였음을 알 수 있었다. 또한 DLPS로 지방 고갈이 유도된 HepG2 세포에 스타틴(rosuvastatin 0.2 μM)과 isoeugenol을 동시 처리했을 때, isoeugenol은 스타틴에 의해 증가한 PCSK9의 발현을 농도의존적으로 감소시켰다. 고콜레스테롤혈증 동물 모델에서도 isoeugenol의 이러한 PCSK9 억제 효능이 관찰되는지에 대한 추후 연구가 필요하다. 본 연구를 통하여 isoeugenol 은 PCSK9 억제 활성을 나타내며 스타틴 치료와 병행하여 그 한계점을 보완할 수 있는 소재로서 제안될 수 있다. Hypercholesterolemia is a major cause of a number of different chronic diseases, including atherosclerosis. Statins for the treatment of hypercholesterolemia induce transactivation of low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 interferes with LDL cholesterol and its receptor binding, promoting LDLR degradation and thereby increasing circulating LDL cholesterol. In the current study, HepG2 cells were treated with 10% delipidated serum (DLPS) in the presence or absence of isoeugenol. Lipid depletion increased both LDLR and PCSK9 protein expression levels in HepG2 cells, and isoeugenol inhibited PCSK9 in HepG2 cells. The objective of this study was to investigate the mechanism by which isoeugenol inhibits PCSK9 and subsequently increases LDLR protein expression HepG2 cells under lipid depletion conditions. A non-toxic level of isoeugenol reduced PCSK9 protein expression without affecting LDLR protein expression. Lipid depletion enhanced gene expression of LDLR, PCSK9, and their transcription factors SREBP2 and HNF1α in HepG2 cells. Isoeugenol significantly attenuated LDLR, PCSK9, and SREBP2 gene expression (P<0.0001), but not HNF1α. Consistently, isoeugenol also markedly reduced SREBP2 protein expression, but not HNF1α. This suggests that isoeugenol-mediated SREBP2 downregulation reduced LDLR and PCSK9 gene expression, and attenuated PCSK9 expression contributed to maintenance of LDLR protein expression in HepG2 cells under lipid depletion conditions. In DLPS-treated HepG2 cells, isoeugenol dose-dependently reduced statin-mediated elevation of PCSK9 protein expression. This study suggests isoeugenol as a potential statin co-treatment material and PCSK9 inhibitor.

Effect of NADPH Oxidase Inhibition on Heme Oxygenase-1 Expression in Human Hepatoma Cell Line HepG2

이상권(Sang Kwon Lee),김강미(Kang Mi Kim),박광훈(Kwang Hoon Park),박영철(Young Chul Park) 한국생명과학회 2011 생명과학회지 Vol.21 No.11

CoPP는 다양한 세포에서 HO-1의 유전자 발현과 활성을 증가시키는 강력한 유도제로 알려져 있다. HO-1는 세포 및 조직의 손상을 보호한다는 연구가 활발히 진행되고 있으나, 그 작용 기전에 대해서는 아직 잘 모르고 있다. 본 논문에서는 porphyrin 계열의 CoPP의 자극에 의해 유도되는 HO-1 유전자 발현에서 NADPH oxidase의 활성이 미치는 영향을 인간 간암세포주 HepG2에서 조사하였다. 배양 중인 HepG2 세포에서 CoPP는 HO-1의 발현을 농도의존적으로 증가시키는 것을 확인하였다. NADPH oxidase 저해제로 잘 알려져 있는 DPI를 전처리한 후 CoPP로 자극한 세포에서는 HO-1의 발현이 강력하게 억제되는 것으로 나타났다. DPI의 이런 억제 효과가 HO-1의 전사 조절인자 Nrf2의 활성에도 영향을 줄 수 있기 때문에 DPI를 전처리 한 후 CoPP 자극에 의한 Nrf2의 핵으로의 이동을 분석하였다. 그 결과, DPI는 CoPP에 의해 유도되는 Nrf2의 핵으로의 이동과 세포 내 존재하는 양을 감소시키는 것을 확인하였다. 다른 HO-1 발현 유도제로 알려져 있는 hemin에 의한 자극의 경우에도 DPI는 HepG2 세포의 HO-1의 발현을 억제하는 효과를 나타내었다. 그리고, p47<SUP>phox</SUP>에 대한 siRNA를 사용하여 효과적으로 p47<SUP>phox</SUP> 유전자 발현을 knockdown 시켜서 NADPH oxidase의 활성을 억제시키는 방법을 사용하였다. 그 결과, p47<SUP>phox</SUP> silencing한 세포에 CoPP를 처리한 경우는 control siRNA를 처리한 세포와 비교할 때 HO-1 발현이 현저히 감소됨을 관찰할 수 있었다. 마지막으로, 세포 내 ROS 생성을 억제하는 GSHmee가 처리된 세포에서는 CoPP나 hemin이 Nrf2의 활성을 증가시키지 못하였고, 그 결과 HO-1의 발현을 유도하지 못하는 것을 알 수 있었는데, 이는 ROS가 CoPP나 hemin에 의한 HO-1 유전자 발현 과정에 중요한 역할을 한다는 것을 의미한다. 이를 종합해 볼 때, 인간 간암세포주 HepG2에서 CoPP나 hemin의 자극에 의한 HO-1 유전자의 발현에는 NADPH oxidase의 활성이 요구된다는 것을 알 수 있고, 그 활성은 세포 내 ROS를 생성시키는 것으로 역할을 한다고 여겨진다. Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. In this study, we investigated the role of NADPH oxidase on the expression of HO-1 in human liver hepatoma cell line HepG2. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, markedly inhibited HO-1 expression and the nuclear translocation of transcription factor Nrf2 in cobalt protoporphyrin (CoPP) or hemin-treated HepG2 cells. Similarly, the knockdown of p47<SUP>phox</SUP>, a cytosolic factor for NADPH oxidase activity, by siRNA inhibited the CoPP-induced expression of HO-1. In addition, GSHmee, an intracellular anti-oxidant, blocked the expression of HO-1 in CoPP-treated cells. Based on these results, we conclude that the blockage of NADPH oxidase with DPI or p47<SUP>phox</SUP> siRNA inhibits CoPP-induced HO-1 expression in HepG2 cells, and also suggest that the expression of HO-1 in CoPP-induced HepG2 cells is associated with increase of intracellular ROS by NADPH oxidase activity.

Machaerium cuspidatum 메탄올 추출물의 항산화 및 항암활성에 관한 연구

진수정 ( Soojung Jin ),오유나 ( You Na Oh ),박현진 ( Hyun-jin Park ),권현주 ( Hyun Ju Kwon ),김병우 ( Byung Woo Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.4

Machaerium cuspidatum은 Fabaceae과 legume속에 속하는 캐노피 덩굴식물(canopy liana)로, 열대 우림 지역에 분포하는 식물이다. 본 연구에서는 Machaerium cuspidatum 메탄올 추출물(MEMC)의 항산화능을 확인하고, 항암 활성 및 그 기전을 인체 폐암세포 A549, 인체간암세포 HepG2를 사용하여 분석하였다. 먼저 DPPH를 이용하여 MEMC의 radical 소거능을 분석한 결과, 소거능 50%의 MEMC 농도(IC₅₀)는 1.66 μg/ml이었으며, MEMC가 추출물인 것을 감안할 때 효과적인 항산화능을 보유하고 있음을 알 수 있었다. 또한 MEMC는 A549, HepG2 및 HT29에 대해 농도의존적으로 세포 사멸 효과를 보였으며, 세포 형태 변화를 유도하였다. A549와 HepG2를 사용하여 세포주기를 분석한 결과, MEMC 처리 농도가 증가할수록 apoptotic 세포를 의미하는subG1기의 세포가 증가하였다. 따라서 MEMC에 의한 A549 및 HepG2의 apoptosis 유도를 Annexin V/7AAD 염색으로 확인한 결과, A549 및 HepG2에서 MEMC 농도의존적으로 Annexin V 양성 세포의 비율이 증가하였으며, DAPI 염색 결과 MEMC 농도의존적으로 A549와 HepG2의 apoptotic body가 증가하였다. 특히 MEMC에 의한 apoptosis는 p53과 Bax의 발현증가 및 Bcl-2의 발현감소와 연관되어 있으며, caspase-3, -8, -9의 활성화와 poly ADP ribose polymerase (PARP)의 단편화를 일으키는 것을 확인하였다. 이러한 결과 들로부터 MEMC는 외인성 및 내인성 경로를 통한 apoptosis 유도에 의해 A549와 HepG2의 증식을 억제시키는 것으로 사료된다. Machaerium cuspidatum, a canopy liana, is a species of genus legume in the Fabaceae family and contributes to the total species richness in the tropical rain forests. In the present study, we investigated the antioxidative and anti-cancer effects of M. cuspidatum and its mode of action. The methanol extract of M. cuspidatum (MEMC) exhibited anti-oxidative activity with an IC₅₀ value of 1.66 μg/ml, and this was attributable to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity. MEMC also exhibited a cytotoxic effect and induced morphological changes in a dose-dependent manner in several cancer cell lines including human lung adenocarcinoma A549 cells, human hepatocellular carcinoma HepG2 cells, and human colon carcinoma HT29 cells. Moreover, MEMC treatment induced the accumulation of subG1 population, which is indicative of apoptosis in A549 and HepG2 cells. MEMC-induced apoptosis was confirmed by the increase in Annexin V-positive apoptotic cells and apoptotic bodies using Annexin-V staining and DAPI staining, respectively. Further investigation showed that MEMC-induced apoptosis was associated with the increase in p53 and Bax expression, and the decrease in Bcl-2 expression. In addition, MEMC treatment led to proteolytic activation of caspase-3, 8, and 9 and degradation of poly-ADP ribose polymerase (PARP). Taken together, these results suggest that MEMC may exert a beneficial anti-cancer effect by inducing apoptosis via both the extrinsic and intrinsic pathways in A549 and HepG2 cells.

Fabrication of self-assembled RGD layer for cell chip to detect anticancer drug effect on HepG2 cells

Waleed Ahmed El-Said,Cheol-Heon Yea,Hyunhee Kim,최정우 한국물리학회 2009 Current Applied Physics Vol.9 No.2

HepG2 cells have been immobilized on nanoscale self-assembled synthetic oligopeptide modified chip surface and subsequently used for anticancer drug screening. Nanoscale controlled self-assembled peptide layer was investigated by AFM (Atomic Force Microscopy). The immobilization of HepG2 cells on nanoscale controlled surface was investigated by using Raman spectroscopy. HepG2 cells were grown on peptide modified gold surface acting as working electrode. The AFM investigation of the oligopeptide modified surface showed excellent agreement with the nanoscale nature of the peptide modification, and the voltammetric response of HepG2 cells on this surface towards an anticancer drug showed a linear relationship with the cell number. As an application, electrochemical detection of anticancer drug effect of HepG2 cells was shown. These results indicate that RGD (Arg-Gly-Asp) peptide self-assembled layer mediated the cell immobilization technique and the voltammetric signal analysis system can be applied to construct a cell chip for diagnosis, drug detection, and on-site monitoring. HepG2 cells have been immobilized on nanoscale self-assembled synthetic oligopeptide modified chip surface and subsequently used for anticancer drug screening. Nanoscale controlled self-assembled peptide layer was investigated by AFM (Atomic Force Microscopy). The immobilization of HepG2 cells on nanoscale controlled surface was investigated by using Raman spectroscopy. HepG2 cells were grown on peptide modified gold surface acting as working electrode. The AFM investigation of the oligopeptide modified surface showed excellent agreement with the nanoscale nature of the peptide modification, and the voltammetric response of HepG2 cells on this surface towards an anticancer drug showed a linear relationship with the cell number. As an application, electrochemical detection of anticancer drug effect of HepG2 cells was shown. These results indicate that RGD (Arg-Gly-Asp) peptide self-assembled layer mediated the cell immobilization technique and the voltammetric signal analysis system can be applied to construct a cell chip for diagnosis, drug detection, and on-site monitoring.

HepG2 cells as an in vitro model for evaluation of cytochrome P450 induction by xenobiotics

최종민,오수진,이상윤,임지혜,오정민,류창선,곽휘찬,이지윤,강건욱,김상겸 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.5

Although various in vitro assays have beendeveloped to evaluate the cytochrome P450 (CYP)-inducingpotential of drug candidates, there is a continuing needfor the development of a reliable model in drug discovery. The objective of the present study was to compare CYPinduction by chemicals in HepG2 cells with Huh7, NKNT-3, and reverted NKNT-3 cells. HepG2 cells showed moresimilarity to human liver than the other cell lines in comparisonsof the expression of cellular proteins. In evaluationof basal CYP activity, Huh7 cells exhibited the highestCYP1A2 and CYP3A4 activity, and HepG2 cells showedthe highest CYP2B6 activity. The inducibility of CYP1A2,CYP2B6, and CYP3A4 by prototypical inducers wasdetermined using enzyme assay, immunoblot analysis, andreal-time PCR. Among the cells tested, HepG2 cells werehighly responsive to CYP inducers, such as 3-methylcholanthrenefor CYP1A2 and phenobarbital for CYP2B6 andCYP3A4. Moreover, HepG2 cells were responsive to variousCYP1A2, CYP2B6, and CYP3A4 inducers as determinedusing fluorogenic and LC–MS/MS substrates. Thus,HepG2 cells may be comparable to human hepatocytes forthe evaluation of CYP induction or slightly less sensitive. These results suggest HepG2 cells as a cell-based model in screening for CYP inducers in drug discovery