• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8595-8601
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• 2014

Background: miR-200a expression is frequently altered in numerous cancers. The aim of the present study was to determine the role of microRNA-200a in advanced ovarian carcinomas. Materials and Methods: We measured miR-200a expression in 72 matched normal ovarian tissues and advanced ovarian carcinomas, and also two ovarian carcinoma cell lines (SKOV3 and SKOV3.ip1 - the latter being more invasive and metastatic than the parental SKOV3) by stem-loop real-time RT-PCR based on TaqMan microRNA assay using U6 as a reference. Levels of miR-200a expression were compared by disease stage, tumor grade, histology, and lymph node involvement. To evaluate the role of microRNA-200a, cell proliferation and invasion of SKOV-3 and SKOV-3.ip1 were analyzed with miR-200a inhibitor/mimic transfected cells. Results: Of 72 paired samples, 65 cancer tissues overexpressed microRNA-200a greater than two fold in comparison with matched normal epithelium. Specifically, patients with lymph node metastasis showed significant elevation. The level correlated with clinicopathological features, including high tumor grade, late disease stage, most notably with lymph node metastasis, but not with tumor histology. In addition, SKOV-3.ip1 cells also overexpressed miR-200a compared with SKOV-3, and miR-200a inhibitor transfected SKOV-3.ip1 cells showed significant reduction in cellular proliferation and invasion, while a miR-200a mimic stimulated the opposite behavior. Conclusions: We provide definitive evidence that miR-200a is up-regulated in a significant proportion of advanced ovarian carcinomas, and that elevated miR-200a expression facilitates tumor progression. Our findings support the notion that miR-200a is an onco-microRNA for ovarian cancer, and elevation is a useful potential diagnostic indicator. This study also provides a solid basis for further functional analysis of miR-200a in advanced ovarian cancer.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.17-24
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• 2013

This study examined the expression of inhibitor of DNA-binding (Id) proteins and vascular endothelial growth factor (VEGF) in the ovary according to female age using a mice model as the first step in investigating the potential role of Ids and VEGF in ovarian aging. C57BL inbred female mice of three age groups (6-9, 14-16, and 23-26 weeks) were injected with 5 IU pregnant mare's serum gonadotropin (PMSG) in order to synchronize the estrus cycle. After 48 h, ovarian expression of Ids and VEGF was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. Ovarian apoptosis was examined by ovarian expression of Bcl-2 and Bcl-xL. Expression of Id-1 and VEGF was decreased with advancing female age, but not Id-2, Id-3, and Id-4. In particular, their expressions were significantly decreased in aged mice of 23-26 weeks compared with the young mice of 6-9 weeks (p < 0.05). In contrast, ovarian apoptosis was greatly increased in the aged mice compared to the young mice. This result suggests that Id-1 may have an implicated role in ovarian aging by associating with VEGF.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.279-287
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• 2013

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.

• 동의생리병리학회지
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• 제19권3호
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• pp.722-728
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• 2005

The DNA topoismerase I inhibitor ${\beta}-lapachone$, the product of a lapacho tree (Tabebuia avellanedae) from South America, activates a novel apoptotic response in a number of cell lines. In the present report, we investigated the effects of ${\beta}-lapachone$ on the growth of human lung in human non-small-cell-lung-cancer A549 cells. Upon treatment with ${\beta}-lapachone$, a concentration-dependent inhibition of cell viability and cell proliferation was observed as measured by hemocytometer counts and MTT assay. The ${\beta}-lapachone-treated$ cells developed many of the hallmark features of apoptosis, including membrane shrinking, condensation of chromatin and DNA fragmentation. These apoptotic effects of ${\beta}-lapachone$ in A549 cells were associated with a marked induction of pro-apoptotic Bax expression, however the levels of anti-apoptotic Bcl-2 expression were decreased in a dose-dependent manner. Accordingly, elevated amount of cyclin-dependent kinase inhibitor p21 expression accompanied by up-regulation of tumor suppressor p53 was observed. By RT-PCR analyses, decrease in gene expression level of telomerase reverse transcriptase and telomeric repeat binding factor were also observed. Thus, these findings suggest that ${\beta}-lapachone$ may be a potential anti-cancer therapeutics for the control of human lung cancer cell model.

• Molecular & Cellular Toxicology
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• 제2권3호
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• pp.159-165
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• 2006

To determine the anticancer effect of D-amygdalin (D-mandelinitrole-${\beta}$-D-gentiobioside) in human chronic myeloid leukemia cells K562, we profiled the gene expression between amygdalin treatment and control groups. Through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of D-amygdalin was $57.79{\pm}1.83%$ at the concentration of 5 mg/mL for 24 h. We performed cDNA microarray analysis and compared the gene expression profiles between D-amygdalin (5 mg/mL, 24 h) treatment and control groups. Among the genes changed by D-amygdalin, we paid attention to cell cycle-related genes, and particularly cell cycle regulator genes; because arrest of cell cycle processing was ideal tactic in remedy for cancer. In our data, expressions of cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B), ataxia telangiectasia mutated (includes complementation groups A, C, and D) (ATM), cyclin-dependent kinase inhibitor 1C (p57, Kip2) (CDKN1C), and CHK1 checkpoint homolog (CHEK1, formally known as CHK1) were increased, while expressions of cyclin-dependent kinase 2 (CDK2), cell division cycle 25A (CDC25A), and cyclin E1 (CCNE1) were decreased. The pattern of these gene expressions were confirmed through RT-PCR. Our results showed that D-amygdalin might control cell cycle regulator genes and arrest S phase of cell cycle in K562 cells as the useful anticancer drug.

• The Korean Journal of Physiology and Pharmacology
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• 제19권5호
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• pp.401-411
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• 2015

Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications. Recently, several studies have demonstrated that allergen-induced airway remodeling and ovalbumin-induced asthma is mediated by AR. Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Whether AR is involved in pathogenesis of pulmonary fibrosis and whether epalrestat attenuates pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. The expression of AR, TGF-${\beta}_1$, ${\alpha}$-SMA and collagen I was analyzed by immunohistochemisty, real-time PCR or western blot. In vivo, epalrestat treatment significantly ameliorated the bleomycin-mediated histological fibrosis alterations and blocked collagen deposition concomitantly with reversing bleomycin-induced expression up-regulation of TGF-${\beta}_1$, AR, ${\alpha}$-SMA and collagen I (both mRNA and protein). In vitro, epalrestat remarkably attenuated proliferation of pulmonary fibroblasts and expression of ${\alpha}$-SMA and collagen I induced by TGF-${\beta}_1$, and this inhibitory effect of epalrestat was accompanied by inhibiting AR expression. Knockdown of AR gene expression reversed TGF-${\beta}_1$-induced proliferation of fibroblasts, up-regulation of ${\alpha}$-SMA and collagen I expression. These findings suggest that AR plays an important role in bleomycin-induced pulmonary fibrosis, and epalrestat inhibited the progression of bleomycin-induced pulmonary fibrosis is mediated via inhibiting of AR expression.

• The Korean Journal of Physiology and Pharmacology
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• 제20권4호
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• pp.347-355
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• 2016

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha ($TNF-{\alpha}$) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of $TNF-{\alpha}$ on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe $CM-DCFH_2-DA$. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. $TNF-{\alpha}$ induced LOX-1 expression in a dose- and time- dependent manner in endothelial cells. $TNF-{\alpha}$ induced ROS formation, phosphorylation of $NF-{\kappa}B$ p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. $NF-{\kappa}B$ inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited $TNF-{\alpha}-induced$ LOX-1 expression. CPT and NAC suppressed $TNF-{\alpha}-induced$ LOX-1 expression and phosphorylation of $NF-{\kappa}B$ p65 and ERK in rat aorta. These data suggested that $TNF-{\alpha}$ induced LOX-1 expression via ROS activated $NF-{\kappa}B/ERK$ pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

• International Journal of Oral Biology
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• 제35권4호
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제28권6호
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• pp.434-439
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• 2002

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. OSCC generally has a poor prognosis due to its tendency towards a local invasion and subsequent metastasis, which is mediated by multiple proteolytic enzymes and angiogenesis. Soy products contain high levels of isoflavonoids, including the tyrosine kinase inhibitor, genistein, which has been identified as a potent inhibitor of cell proliferation and in vitro angiogenesis. The purpose of this in vitro study is to evaluate the anti-cancer effect of genistein with respect to the angiogenesis and basement membrane invasion in OSCC. The highly invasive OSCC cell line, HSC-3 cells were cultured in the presence of $10{\mu}M$ genistein for 24h. To evaluate the effects of genistein on the invasiveness and the gelatinolytic activity, in vitro invasion assay and zymography were performed. In order to evaluate the effect on the VEGF and bFGF mRNA expression, RT-PCR and northern hybridization reaction, and chemiluminescence detection were applied. The in vitro invasion assay showed that the genistein treatment reduced the cellular invasion through the artificial basement membrane and significant difference between the control group and the genistein treated group was shown in MMP-2 activity. Especially, the 62 kDa activated form of MMP-2 in the control group was 1.8 times higher than that in the genistein treated group. The results of the northern blot analyses indicated that VEGF mRNA expression in the genistein treated group was significantly down regulated. This study showed that genistein inhibits angiogenesis and reduces basement membrane invasion in OSCC. It seems to support the possibility of genistein as an anti-cancer agent.

• Molecules and Cells
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• 제37권11호
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• pp.795-803
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• 2014

To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.