• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5343-5348
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• 2015

Promoter hypermethylation of the runt-related transcription factor 3 (RUNX3) gene is associated with increased risk of hepatocellular carcinoma (HCC). Oxidative stress plays a vital role in both carcinogenesis and progression of HCC. However, whether oxidative stress and RUNX3 hypermethylation in HCC have a cause-and-effect relationship is not known. In this study, plasma protein carbonyl and total antioxidant capacity (TAC) in patients with hepatitis B virus (HBV)-associated HCC (n=60) and age-matched healthy subjects (n=80) was determined. RUNX3 methylation in peripheral blood mononuclear cells (PBMC) of subjects was measured by methylation-specific PCR. Effect of reactive oxygen species (ROS) on induction of RUNX3 hypermethylation in HCC cells was investigated. Plasma protein carbonyl content was significantly higher, whereas plasma TAC was significantly lower, in HCC patients than healthy controls. Based on logistic regression, increased plasma protein carbonyl and decreased plasma TAC were independently associated with increased risk for HCC. PBMC RUNX3 methylation in the patient group was significantly greater than in the healthy group. RUNX3 methylation in hydrogen peroxide ($H_2O_2$)-treated HepG2 cells was significantly higher than in untreated control cells. In conclusion, increase in oxidative stress in Thai patients with HBV-associated HCC was demonstrated. This oxidative increment was independently associated with an increased risk for HCC development. RUNX3 in PBMC was found to be hypermethylated in the HCC patients. In vitro, RUNX3 hypermethylation was experimentally induced by $H_2O_2$. Our findings suggest that oxidative stress is a cause of RUNX3 promoter hypermethylation in HCC cells.

• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8623-8629
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• 2014

Background: As an important component of the NDC80 kinetochore complex, NUF2 is essential for kinetochore-microtubule attachment and chromosome segregation. Previous studies also suggested its involvement in development of various kinds of human cancers, however, its expression and functions in human hepatocellular carcinoma (HCC) are still unclear. Materials and Methods: In the present study, we aimed to test the hypothesis that NUF2 is aberrant in human HCCs and associated with cell growth. Results: Our results showed significantly elevated expression of NUF2 in human HCC tissues compared to adjacent normal tissues, and high expression of NUF2 in HCC cell lines. Using lentivirus-mediated silencing of NUF2 in HepG2 human HCC cells, we found that NUF2 depletion markedly suppressed proliferation and colony formation capacity in vitro, and dramatically hampered tumor growth of xenografts in vivo. Moreover, NUF2 silencing could induce cell cycle arrest and trigger cell apoptosis. Additionally, altered levels of cell cycle and apoptosis related proteins including cyclin B1, Cdc25A, Cdc2, Bad and Bax were also observed. Conclusions: In conclusion, these results demonstrate that NUF2 plays a critical role in the regulation of HCC cell proliferation and apoptosis, indicating that NUF2 may serve as a potential molecular target for therapeutic approaches.

• Asian Pacific Journal of Cancer Prevention
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• 제15권7호
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• pp.3219-3226
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• 2014

Chemotherapy continues to be a mainstay of cancer treatment, although drug resistance is a major obstacle. Lipid metabolism plays a critical role in cancer pathology, with elevated ether lipid levels. Recently, alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipid synthesis, was shown to be up-regulated in multiple types of cancer cells and primary tumors. Here, we demonstrated that silencing of AGPS in chemotherapy resistance glioma U87MG/DDP and hepatic carcinoma HepG2/ADM cell lines resulted in reduced cell proliferation, increased drug sensitivity, cell cycle arrest and cell apoptosis through reducing the intracellular concentration of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe) and prostaglandin E2 (PGE2), resulting in reduction of LPA receptor and EP receptors mediated PI3K/AKT signaling pathways and the expression of several multi-drug resistance genes, like MDR1, MRP1 and ABCG2. ${\beta}$-catenin, caspase-3/8, Bcl-2 and survivin were also found to be involved. In summary, our studies indicate that AGPS plays a role in cancer chemotherapy resistance by mediating signaling lipid metabolism in cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• 제14권8호
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• pp.4533-4537
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• 2013

Objective: To use microarray chip technology for screening of stem cell radiation related miRNAs in laryngeal squamous cell carcinoma; study and explore the relationship of miRNAs with radiosensitivity of laryngeal squamous cells. Method: After conventional culture and amplification of the laryngeal squamous carcinoma cell line Hep-2, CD 133+ cells were screened out with combination of isolated culture of stem cell microspheres and FACS for preparation of laryngeal cancer stem cells. After radiation treatment, miRNAs of laryngeal squamous carcinoma stem cells before and after radiation were enriched and purified. After microarray hybridization with mammalian miRNA and scanning of fluorescence signal, the miRNAs of laryngeal squamous carcinoma stem cells before and after radiation was subject to differential screening and clustering analysis. Real-time quantitative RT-PCR was used to verify part of the differentially expressed miRNAs. Results: 70 miRNAs related to laryngeal cancer stem cell radiation with 2-fold difference in expression were screened out, in which 62 were down-regulated and 8 were up-regulated. Fluorescent quantitative RT-PCR results were consistent with miRNAs chip results. Conclusion: Some miRNAs may be involved in self-regulation with laryngeal squamous carcinoma stem cell radiation.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.3109-3116
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• 2013

The majority of hepatocellular carcinoma (HCC) patients have a poor prognosis with current therapies, and new approaches are urgently needed. We have developed a novel therapeutic cancer vaccine platform based on tumor cell derived autophagosomes (DRibbles) for cancer immunotherapy. We here evaluated the effectiveness of DRibbles-pulsed dendritic cell (DC) immunization to induce anti-tumor immunity in BALB/c mouse HCC and humanized HCC mouse models generated by transplantation of human HCC cells (HepG2) into BALB/c-nu mice. DRibbles were enriched from H22 or BNL cells, BALB/c-derived HCC cell lines, by inducing autophagy and blocking protein degradation. DRibbles-pulsed DC immunization induced a specific T cell response against HCC and resulted in significant inhibition of tumor growth compared to mice treated with DCs alone. Antitumor efficacy of the DCs-DRibbles vaccine was also demonstrated in a humanized HCC mouse model. The results indicated that HCC/DRibbles-pulsed DCs immunotherapy might be useful for suppressing the growth of residual tumors after primary therapy of human HCC.

• 대한한의학회지
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• 제24권2호
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• pp.1-11
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• 2003

Objectives : The aim of this study was to characterize the effect of Injinchunggan-tang on $TGF-{\beta}1-induced$ hepatic fibrosis. Methods : mRNA and protein expression levels of $TGF-{\beta}1$ in Injinchunggan-tang-treated HepG2 cells were compared to untreated cells using quantitative RT-PCR and ELISA assay, respectively. mRNA expression levels of the TGF-1 pathway genes (TR-1, TR-II, Smad2, Smad3, Smad4, and PAI-1) and fibrosis-associated genes (CTGF, fibronectin, and collagen type 1) were evaluated by quantitative RT-PCR. The effect of Injinchunggan-tang on cell proliferation of T3891 human fibroblast was evaluated using [$^3H$]thymidine incorporation assay. Results : Expression of $TGF-{\beta}1$ mRNA and protein was inhibited by Injinchunggan-tang in a dose- and time-dependent manner. Whereas $TGF-{\beta}1-mediated$ induction of PAI-1 was suppressed by Injinchunggan-tang, expression of the $TGF-{\beta}1$ pathway genes such as TR-1, TR-II, Smad2, Smad3, and Smad4 was not affected by Injinchunggan-tang treatment. Injinchunggan-tang was found to inhibit $TGF-{\beta}1-induced$ cell proliferation of T3891 human fibroblast, and also abrogated $TGF-{\beta}1-mediated$ transcriptional up-regulation of CTGF, fibronectin, and collagen type I. Conclusions : This study strongly suggests that the liver cirrhosis-suppressive activity of Injinchunggan-tang may be derived at least in part from its inhibitory effect on $TGF-{\beta}1$ functions, such as blockade of $TGF-{\beta}1$ stimulation of fibroblast cell proliferation and fibrosis-related gene expression as well as expression of $TGF-{\beta}1$ itself.

• 식품산업과 영양
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• 제12권2호
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• pp.51-57
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• 2007

한국산 복령과 후박의 이용성 확대를 위하여 각각 추출물의 ABTS와 DPPH라디칼의 소거능, 환원력의 항산화능 효과 및 인체암 세포주의 항암활성에 대하여 조사하였다. 복령과 후박을 버섯균사체로 발효시킨 한약 추출액의 항산화활성(ABTS와 DPPH 라디칼소거, 환원력)은 시료 추출물의 농도가 증가할수록 비례적으로 증가하였으며, DPPH 라디칼 소거활성은 ABTS보다 비교적 높게 나타났고, 원료후박 추출물($21.16{\sim}24.68%$)은 원료복령 추출물($45.46{\sim}49.63%$)에 비하여 약 2배 이상 높았다. 복령 시료 추출물의 농도별에 따른 환원력은 원료 복령($0.55{\sim}0.63$)에 비하여 동충하초, 팽이버섯 및 큰 느타리 균사체로 발효시킨 시료($0.50{\sim}0.62$)의 흡광도로 서로 비슷한 증가를 나타내었고, 후박 시료 추출물의 농도별에 따른 환원력은 원료 후박 추출물($0.98{\sim}1.06$)이 3가지의 버섯 균사체 발효후박 추출물($0.76{\sim}1.01$)에 비하여 약간 높았다. 자궁경부암세포(HeLa)와 대장암세포(HT-29)는 원료복령과 후박의 추출물이 각각의 발효한약에 비하여 저해활성이 높았다. 간암세포(HepG2)는 $200{\mu}g/assay$ 농도에서 팽이버섯 균사체 발효복령의 추출물이 가장 높았는데, 원료복령과 동충하초 및 큰 느타리에 비하여 각각 1.79, 1.35, 1.03배 높은 저해활성을 나타내었다. 그리고 팽이버섯과 동충하초 균사체를 배양한 발효후박에서 각각 $11.39{\sim}53.92%$, $10.71{\sim}50.21%$ 범위였으며, 원료후박에 비하여 $200{\mu}g/assay$ 농도에서 각각 2.21배, 2.05배 높은 저해활성을 나타내었다. 유방암세포(MCF-7)는 발효복령 추출물의 저해활성이 팽이버섯 균사체($58.35{\sim}72.87%$)에서 가장 높았으며, 큰 느타리버섯($61.04{\sim}67.66%$)과 동충하초($39.74{\sim}66.40%$) 및 원료복령($50.32{\sim}69.24%$)은 서로 비슷하였다.

• BMB Reports
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• 제43권12호
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• pp.818-823
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• 2010

Plk 1 is overexpressed in many human malignancies including laryngeal carcinoma. However, its therapeutic potential has been never examined in laryngeal carcinoma. In the present study, a simple cellular morphology-based strategy was firstly proposed for rapidly screening the effective siRNAs against Plk1. Furthermore, we investigated the effects of Plk1 depletion via a novel identified effective siRNA against Plk1, Plk1 siRNA-607, on human laryngeal carcinoma Hep-2 cells. The results indicated that Plk1 siRNA-607 transfection resulted in a significant inhibition in Plk1 expression in cells, and subsequently caused a dramatic mitotic cell cycle arrest followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth and viability of the laryngeal carcinoma cells. Taken together, our present study not only suggests a simple strategy for rapidly screening effective siRNAs against Plk1 but also implicates that Plk1 may serve as a potential therapeutic target in human laryngeal carcinoma.

• BMB Reports
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• 제46권12호
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• pp.588-593
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• 2013

Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis.

• BMB Reports
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• 제46권6호
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• pp.322-327
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• 2013

The ATP-binding cassette transporters ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. To identify cis-regulatory elements of the two genes, we have cloned and analyzed twenty-three evolutionary conserved region (ECR) fragments using the CMV-luciferase reporter system in HepG2 cells. Two ECRs were found to be responsive to the Liver-X-Receptor (LXR). Through elaborate deletion studies, regions containing putative LXREs were identified and the binding of $LXR{\alpha}$ was demonstrated by EMSA and ChIP assay. When the LXREs were inserted upstream of the intergenic promoter, synergistic activation by $LXR{\alpha}/RXR{\alpha}$ in combination with GATA4, $HNF4{\alpha}$, and LRH-1, which had been shown to bind to the intergenic region, was observed. In conclusion, we have identified two LXREs in ABCG5/ABCG8 genes for the first time and propose that these LXREs, especially in the ECR20, play major roles in regulating these genes.