• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.993-1001
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• 2016

Type 2 diabetes mellitus patients are at increased risk of many forms of malignancies, especially of the pancreas, colon and hepatocellular cancer. Unfortunately, little is known of the possible interaction between antidiabetic drugs and anticancer agents. The present study investigates the influence of metformin (MET) and sitagliptin (SITA) on the in vitro anticancer activity of the microtubule depolymerization inhibitor agent epothilone A (EpoA). Hepatocellular liver carcinoma cell line (HepG2) viability and apoptosis were determined by the MTT test and by double staining with PO-PRO-1 and 7-aminoactinomycin D, respectively, after treatment with EpoA, metformin or sitagliptin. The levels of nuclear factor NF-${\kappa}B$ and p53 were evaluated in the presence and absence of inhibitors. While EpoA and MET inhibited HepG2 cell proliferation, SITA did not. EpoA and SITA induced higher p53 levels than MET. All tested drugs increased the level of NF-${\kappa}B$. Only MET enhanced the proapoptotic effect of EpoA. The EpoA+MET combination evoked the highest cytotoxic effect on HepG2 cells and led to apoptosis independent of p53, decreasing the level of NF-${\kappa}B$. These findings support the link between NF-${\kappa}B$ and p53 in the modulation of apoptotic effects in HepG2 cells treated by EpoA. Our studies indicate that the combination of EpoA and MET applied in subtoxic doses has a stronger cytotoxic effect on liver cancer cells than each of the compounds alone. The therapeutic advantages of the combination of EpoA with MET may be valuable in the treatment of patients with diabetes mellitus type 2 (T2DM) and liver cancer.

• 대한한방내과학회지
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• 제27권1호
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• pp.149-165
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• 2006

Objectives: This study was done to evaluate the effects of Saengkankunbi-tang on cell-viability, proliferation, cell-cycle, apoptosis and DNA replication on HepG2 cell and to find out by which molecular-biological mechanism by which Saengkankunbi-tang operates. Methods : The MTT assay, cell counting assay, [3H]-thymidine incorporation assay, flow cytometric analysis, tryphan blue exclusion assay, western blot analysis, quantative RT-PCR were taken. Results : Saengkankunbi-tang had no effect on proliferation, cell-cycle and DNA replications of HepG2 cells, while it improved cell viability and reduced apoptosis, and it activated Akt and NFKB. But, it did not produce an effect on cell viability and apoptosis when P13K/Akt pathway was blocked by LY294002 nor when $NF{\kapa}B$ activation was blocked by DN-$I{\kapa}B$. Conclusion : These results suggests that Saengkankunbi-tang improves cell viability and reduces apoptosis of HepG2 cells, by activating $NF{\kapa}B$ through PI3K/Akt pathway.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제15권1호
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• pp.35-48
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• 1993

The in vitro predictive tests in cancer chemotherapy of cancer cell lines to anticancer drugs were determined using novel dye exclusion assay [NDEA], [3H] thymidine incorporation, and clonogenic assay [CA>. Antitumor effect of Bleomycin, Cis-platin, Vinblastine, Methotrexate to HEp-2, B16 cell lines using rapid assays was compared with [CA> in this study. In dye exclusion assay of B l6 cell line, cancer cells were sensitive to Bleomycin at all concentrations, to Vinblastine at the level of peak plasma concentration [PPC], ${\times}1/10$ [PPC](P<0.05). And Bleomycin revealed relatively good cytotoxicity than that of CDDP and vinblastine at ${\times}10$[PPC], (P<0.05). HEp-2 cells were resistive to methotrexate at the level of ${\times}100$[PPC] (P<0.05) In [3H] thymidine incorporation assay, B 16 cells were sensitive to Bleomycin, CDDP, Vinblastine at the level of [PPC], ${\times}10$ [PPC](P<0.01). Dose-dependent drugs of bleomycin, CDDP were more sensitive than Vinblastine at high concentration (P<0.05). In clonogenic assay, HEp-2 cell line was sensitive to three drugs of all concentrations except ${\times}10$ [PPC] of CDDP. B 16 cell line was sensitive to all drugs(P<0,01). In comparison of chemosensitivity tests among three assays, the results were correlated(${\gamma}=0.99$, P<0.05).

• 생명과학회지
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• 제13권3호
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• pp.324-332
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• 2003

최근 Phenobarbital에 의해서 발현되는 CYP2B유전자의 발현촉진 매개인자로서 Constitutive Androstane Receptor (CAR)가 최근에 규명되었다. 사람의 간암세포인 HepG2 cell line에 CAR유전자를 transfection시켰을 경우 PBRU의 활성을 촉진시켰다. 지금까지 연구된 CAR의 역할은 주로 간세포 내에서 cytochrome P4502B 유전자의 발현을 촉진하는 Phenobarbital의 매개체로서 알려져 있다. 다세포동물에서 각각의 분화된 세포는 독특한 유전자의 발현 Pattern을 갖고 있어서 어느 특정한 세포에서 일어나는 유전자의 발현특징이나 작용기전 혹은 기능이 다른 종류의 세포에서는 일어나지 않거나 상이한 기전에 의해서 조절된다. 이러한 세포간 특이성을 이용하여 유전자 발현에 관여하는 인자들을 규명하는데 기초적 자료로 이용될 수 있다. 따라서 본 연구의 목적은 CYP2B유전자의 발현이 일어나지 않는 콩팥세포 line (COS)에서 CAR 단백질 수용체가 PBRU의 활성촉진에 영향을 미치는지의 여부를 조사하고자 하였다. Control 상태의 Hep G2와 COS 배양세포에서는 CAR 단백질의 발현이 거의 나타나지 않았다. mCAR1-GFP를 transfection 한 후 CAR antibody를 이용하여 immunoblot을 시행하였을 경우, Hep G2 세포에서는 발현이 비교적 약하게 나타났지만, COS 세포에서는 강한 mCAR1-GFP 단백질의 발현을 보였다. 한편, GFP antibody를 이용하여 immunoblot을 시행하였을 경우에는 COS 세포에서 강한 mCAR1-GFP 단백질의 발현을 나타내었을 뿐만 아니라 Hep G2 세포에서도 명백히 단백질의 발현을 관찰할 수 있었다. 또한, Hep G2와 COS세포 모두에서 endogenous RXR의 발현이 일어남을 확인하였고 RXR expression plasmid를 transfection시켰을 때 두 세포 모두에서 단백질의 발현이 현저하게 증가되었다. Constitutive Androstane Receptor (CAR)에 의한 CYP2B의 PBRU 활성효과를 다르게 분화된 세포에서 차이가 일어나는지를 비교하기 위하여 CAR에 의해 매개되는 PBRU의 transactivation효과를 Hep G2와 COS세포에서 조사하였다. Hep G2 세포에서는 transfection된 CAR의 발현에 의해 firefly luciferase 보고단백질의 활성이 약 12배 증가하였다. CAR 발현유전자를 15 ng transfection하였을 때 주어진 보고유전자의 양에 대하여 최대반응을 나타내었고 CYP2B1PBRU가 제거된 CYP2C1 promotor/firefly luciferase를 보고유전자로 사용하였을 때는 CAR에 의한 luciferase의 활성이 나타나지 않았다. Hep G2와는 달리, COS세포에서는 transfection된 CAR의 발현이 PBRU에 의한 firefly luciferase보고단백질의 발현에 영향을 주지 못하였다. 이러한 결과들은 분화된 세포의 종류에 따라서 constitutive androstane receptor의 CYP2BPBRU 활성효과가 다르게 나타날 수 있음을 제시할 뿐만 아니라, 간세포에서 Phenobarbital에 의한 PBRU의 활성유도에 영향을 주는 endogenous 매개 인자들 중 CAR와 RXR과는 다른 전사조절인자들이 필요로 하며 이러한 인자들의 발현이 콩팥 세포에서는 다르게 존재함을 시사한다.

• 한국식품영양과학회지
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• 제33권6호
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• pp.936-940
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• 2004

본 연구는 된장 메탄올 추출물의 항암효과를 검토하기 위해서 다른 콩 관련 발효식품과 원재료인 콩과 밀가루의 메탄올추출물과 비교하면서 여러 인체 암세포들의 성장 억제 실험과DNA 합성 저해 실험을 행하였다. AGS 인체 위암세포의 경우 첨가농도 200 $\mu\textrm{g}$/mL에서 콩된장과 70% 콩된장 메탄올 추출물은 각각 80%, 78%의 저해효과를 가졌으며 청국장 메탄올 추출물은 65%, 일본의 미소 메탄올 추출물은 54%의 저해효과를 보였다. Hep 3B 인체 간암세포에 대한 증식억제 효과 결과에서도 콩된장과 70% 콩된장의 경우, 첨가농도 200 $\mu\textrm{g}$/mL에서 각각 77%, 73%의 저해효과를 가지는 반면, 청국장, 일본의 미소는 각각 60%, 56%의 억제효과를 보였고 콩과 콩/밀가루의 경우 각각 56%, 40%의 저해효과를 나타내었다. HT-29 인체 대장암세포에서도 이상의 간암, 위암세포의 결과와 유사하게 증식억제효과를 보여 콩된장과 70% 된장 메탄올 추출물이 각각 86%, 87%의 저해효과를 나타내면서 다른 콩관련 발효식품들과 원재료 콩에 비해 큰 활성을 보였다. 또한 DNA 합성 저해 실험에서 AGS 인체 위암세포는 된장 메탄올 추출물 100 $\mu\textrm{g}$/mL, 200 $\mu\textrm{g}$/mL 투여시에는 각각 65%, 76%의 DNA 합성 저해 효과가 관찰되었고, 반면 콩의 메탄올 추출물의 경우 첨가농도 100 $\mu$/mL, 200 $\mu$/mL 때 각각 47%, 68%의 DNA 합성 저해 효과를 가졌으며 된장의 경우보다 낮은 저해 효과를 나타내었다. Hep 3B 인체 간암세포는 된장 메탄올 추출물 100 $\mu\textrm{g}$/mL, 200 $\mu\textrm{g}$/mL 투여시 각각 43%, 59%의 DNA합성 저해 효과를 나타내었다. 이상의 연구 결과들로부터 된장이 다른 콩 발효식품 및 원재료 콩보다 높은 암세포 성장 억제 효과와 DNA 합성 저해 효과를 가지는 것은 콩만으로 3개월 간 발효시킨 된장의 우수성과 함께 발효과정을 거치는 동안 원재료인 콩에서는 없었던 혹은 함량이 적은 성분들이 생성되거나 증가되어 항암효과를 나타내는 것으로 추정되어진다.

• Molecules and Cells
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• 제40권3호
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• pp.202-210
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• 2017

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

• Journal of Microbiology and Biotechnology
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• 제25권3호
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• pp.413-417
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• 2015

Recently, we isolated HY253, a novel decahydrofluorene analog with a molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis, which is known as Dokwhal (獨活), a traditional medicinal herb. Moreover, we previously reported its cytotoxic activity on cancer cell proliferation in human lung cancer A549 and cervical cancer HeLa cells. The current study aimed to evaluate its detailed molecular mechanisms in cell cycle arrest and apoptotic induction in human hepatocellular carcinoma HepG2 cells. Flow cytometric analysis of HepG2 cells treated with $60{\mu}M$ HY253 revealed appreciable cell cycle arrest at the G1 phase via inhibition of Rb phosphorylation and down-regulation of cyclin D1. Furthermore, using western blots, we found that up-regulation of cyclin-dependent kinase inhibitors, such as p21^CIP1 and p27^KIP1, was associated with this G₁ phase arrest. Moreover, TUNEL assay and immunoblottings revealed apoptotic induction in HepG2 cells treated with $60{\mu}M$ HY253 for 24 h, which is associated with cytochrome c release from mitochondria, via down-regulation of anti-apoptotic Bcl-2 protein, which in turn resulted in activation of caspase-9 and -3, and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, we suggest that HY253 may be a potent chemotherapeutic hit compound for treating human liver cancer cells via up-regulation and activation of the p53 gene.

• 대한한의학회지
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• 제26권1호
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• pp.46-58
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• 2005

Objective : This study on Sipimikwanjung-tang was undertaken to evaluate its antioxidant capacities and antiperoxidation activities in rat liver tissues. Sipimikwanjung-tang which has been one of the prescriptions in sasang constitutional medicine is usually applied for the therapy of various liver diseases. It is elucidated that Sipimikwanjung-tang has antioxidants on liver tissue of rat and the cytotoxic effects on human hepatoblastoma Hep G2 cells. Methods: Sipimikwanjung-tang extract in antioxidant effects of Hep G2 cells is evaluated by MTT assay, DAPI staining, DNA fragmentation assays and FACS can analysis. Results: Sipimikwanjung-tang induced apoptosis in Hep G2 cells, and induced G1 and G2M arrest of the cell cycle as well as a significant increase in PARP and caspase-3 activity. It induced an increase in $H_2O_2$ generation and the subsequent $NF-{\kappa}B$ activation and also induced cell apoptosis through the caspase-3-dependent pathways in the low concentration of Sipimikwanjung-tang extracts. However, the high dose of Sipimikwanjung-tang extract in Hep G2 cells inhibited $TGF-{\beta}l-induced$ apoptosis via increase in cellular $H_2O_2$, formation and $NF-{\kappa}B$ activation in human hepatoblastoma Hep G2 cells. Conclusion: From this study, the possibility that Sipimikwanjung-tang extracts apply to antioxidant and apoptotic treatment of disease is revealed.

• 생약학회지
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• 제38권2호통권149호
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• pp.176-180
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• 2007

Five liglans, sauchinone (1), di-0-methyltetrahydrofuriguaiacin B (2), manassantin A (3), manassantin B (4) and saucerneol B (5), have been isolated from the MeOH extract of Saururus chinesis roots. The evaluation for protective effect of compounds 1-5 against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells was conducted. Compounds 1,2, and 5 showed significant protective effects with the EC$_{50}$ values of74.2${\pm}$0.9, 111.3${\pm}$0.8,64.3${\pm}$0.8 ${\mu}$M, respectively. Silybin, one of the well-known hepatoprotective agents, used as a positive control, and also showed protective effect with an EC$_{50}$ value of 86.2${\pm}$0.5 ${\mu}$M.

• Journal of Nutrition and Health
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• 제27권9호
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• pp.910-923
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• 1994

The effects of individual fatty acids, differing in their degree of unsaturation(18:0, 18:1, 18:2 and 18:3) on the biosynthesis and secretion and lipids were investigated in Hep-G2 cells. Synthesis of apolipoprotein was measured by the incorporation of 3H-leucine into apolipoprotein(d<1.21g/ml) and synthesis of lipids was measured by the incorporation of 3H-glycerol and 14C-acetate into various lipid classes. Inclusion of 1.0mM of each fatty acids into the culture medium significantly increased the synthesis of total apolipoprotein and Apo B(p<0.05). However, addition of fatty acid did not affect the synthesis of cellular and medium protein. Among different fatty acids tested, oleic acid had the greatest effect on Apo B synthesis. While stearic, linoleic and linolenic acid, all had similar effects. The secretion of triglyceride into the medium markedly increased in all fatty acid groups being 5-6 times over the albumin control. The triglyceride secretion was the highest int he oleic acid group. The secretion of phospholipid and cholesterol also increased with triglyceride output. A positive relationship existed between the output of lipoprotein-triglyceride and Apo B. Since the synthesis of Apo B was significantly increased when various fatty acids were included into the culture medium, part of the apparently stimulated synthesis of the apolipoprotein may be in response to the increased formation and secretion of lipoprotein lipids.