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Urinary cells can be an ideal source for generating hiPSCs and progenitors, as they are easily accessible, non-invasive, and universally available. We generated human induced pluripotent stem cells (hiPSCs) from the urinary cells of a healthy donor using a Sendai virus-based gene delivery method. The generated hiPSC line, KSCBi001-A, has a normal karyotype (46,XY). The pluripotency and capacity of multilineage differentiation were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from National Stem Cell Bank, Korea National Institute of Health.

Amyloid-β (Aβ) peptide plays a major role in the pathogenesis of Alzheimer's disease (AD), and is generated by β- and γ-secretase-mediated proteolytic processing of amyloid-β protein precursor (AβPP). In the present study, we investigated the effect of 118 natural compounds on Aβ production in the medium of HEK293 cells stably expressing human AβPP695 (HEK293-AβPP) using Aβ42 sandwich ELISA to find natural compounds that can modulate Aβ production. We found that a coumarin derivative of citrus fruits, auraptene, increased Aβ production. Treatment of HEK293-AβPP cells and rat primary cortical neurons with auraptene significantly increased the secretion of Aβ40, Aβ42, and the Aβ42/40 ratio. However, auraptene did not change the protein levels of the AβPP processing enzymes, a disintegrin and metalloproteinases 10 (ADAM10, α-secretase), β-site AβPP cleaving enzyme-1 (BACE-1, β-secretase), and presenilin 1 (PS1, γ-secretase component). Auraptene increased the activity of γ-secretase but not that of α- and β-secretase. Furthermore, auraptene enhanced γ-secretase-mediated production of Aβ from AβPP or AβPP-C99, but not through α- and β-secretase. Auraptene also phosphorylated c-Jun N-terminal kinase (JNK), and pretreatment with the JNK inhibitor, SP600125, reduced auraptene-induced γ-secretase activity. Overall, our results suggest that auraptene-mediated activation of JNK may contribute to the production of Aβ by promoting γ-secretase activity.

The cytoplasmic C-terminal domain of amyloid-β protein precursor (AβPP) binds to several proteins that regulate the trafficking and processing of AβPP and affects amyloid-β (Aβ) production. We previously reported that levels of AT-motif binding factor 1 (ATBF1) are increased in the brains of 17-month-old Tg2576 mice compared with wild-type controls, and that Aβ42 increases ATBF1 expression, inducing death in primary rat cortical neurons. Here, we show that ATBF1 levels are increased in the cytoplasm of hippocampal neurons in Alzheimer's disease (AD) brains compared with non-AD brains. Furthermore, cotransfection of human embryonic kidney (HEK293T) and human neuroblastoma (SH-SY5Y) cells with ATBF1 and AβPP695 increased steady-state levels of AβPP via the binding of ATBF1 to the AβPP cytoplasmic domain (amino acids 666-690), resulting in increased Aβ production and cellular and soluble AβPP (sAβPP) levels without affecting the activity or levels of AβPP processing enzymes (α-, β-, or γ-secretase). Conversely, knockdown of endogenous ATBF1 reduced levels of cellular AβPP, sAβPP, and Aβ in HEK293 cells overexpressing human AβPP695. Our findings provide insight into the dynamics of AβPP processing and Aβ production, and suggest that ATBF1 is a novel AβPP binding protein that may be a suitable therapeutic target for AD.

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.

Metformin is a major oral anti-diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK-dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC-zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin-induced phosphorylation of AS160/PKC-zeta. In addition, a colorimetric absorbance assay showed that insulin-induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC-zeta but not with GLUT4. The C-terminal-deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub-cellular localization, while wild-type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild-type Rab4 co-localized with PKC-zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK-AS160-PKC-zeta and modulates insulin-mediated GLUT4 translocation.

Although the altered expression of integrin alpha4 is known to be associated with transformation or metastasis in several human cancers, the information on cholangiocarcinoma (CC) is still poor. In this study, we investigated the promoter methylation status of integrin alpha4 gene in CC.A total of 29 CC, 19 adjacent non-tumor-containing tissue and 15 normal liver specimens were used for identification of gene methylation status by methylation-specific polymerase chain reaction.The frequency of DNA methylation was 55.17% (16 of 29) in the CC specimens (P0.001). Also, transcripts of the integrin alpha4 gene were decreased in all CC tissues in which there was DNA methylation of the integrin alpha4 gene. In addition, the downregulated expression of integrin alpha4 in CC cells with hypermethylation of the integrin alpha4 gene was restored by treatment with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Moreover, we found that DNA methylation of integrin alpha4 was detected in all CC tissues obtained from patients with LN metastasis (7/7). Furthermore, phosphorylation of paxillin, cell migration-related molecule, was regulated by silencing of integrin alpha4.Taken together, these results suggest that loss of the integrin alpha4 gene is caused by aberrant DNA methylation of the 5'-CpG island site of the gene, and methylation of the integrin alpha4 gene can be a useful marker of metastasis of CC.

Intrahepatic cholangiocarcinoma (ICC) arises from epithelial cells in the intrahepatic bile duct. Until now, only few reports have been available concerning the genetic changes during the progression of ICC. In this study, we analyzed chromosomal aberrations in 19 frozen ICC samples using comparative genomic hybridization. The common chromosomal gains were observed in 8q22 approximately qter (11 cases, 58%), 5p14 approximately pter (32%), 2q33 approximately qter (26%), 7p (26%), 17q21 approximately q22 (26%), 18q12 approximately q21 (26%), and 19q13.1 (26%). DNA amplification was identified in nine tumors (47%). Chromosomal loss was found in Y (60%), 1p34 approximately pter (37%), 4q(32%), 18q21 approximately qter (32%) 19p (32%), X (32%), 5q11 approximately q14 (26%), 8p(26%), 9p (26%), and 17p (26%). Chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of ICC.

ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and reverses cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR), retinoid X receptor (RXR), and peroxisome proliferator-activated receptors (PPARs). To identify natural compounds that can upregulate ABCA1 expression, we developed a reporter assay using U251-MG (human glioma cell line) cells that stably express a human ABCA1 promoter-luciferase and performed a cell-based high-throughput screening of 118 natural compounds. Using this system, we identified honokiol, a compound extracted from Magnolia officinalis, as an activator of the ABCA1 promoter. We found that honokiol also increased ABCA1 mRNA and protein expression levels in a dose-dependent manner in U251-MG cells without significant cell death and also increased ABCA1, ABCG1 and apolipoprotein E (apoE) expression levels in THP-1 macrophages. PPAR antagonists did not diminish the induction of ABCA1 expression by honokiol in U251-MG cells. Cotreatment of the cells with honokiol and T0901317 (synthetic LXR ligand) further increased the ABCA1 expression level, whereas cotreatment with 9-cis retinoic acid had no additive effect compared with treatment with honokiol alone. We also found that honokiol has binding affinity to RXRbeta. In this study, we identified for the first time honokiol as an upregulator of ABCA1 expression, which is mediated by the binding of honokiol to RXRbeta as a ligand.

Aberrant DNA methylation of 5'-CpG islands located within gene promoters has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. To ascertain the mechanism of gene promoter hypermethylation in cholangiocarcinoma (CC), we investigated promoter methylation status of the candidate genes ID4, DLC-1, and SFRP1 in 41 CCs, 19 adjacent nontumor tissues, and 15 normal liver tissues using methylation-specific PCR (MSP). The frequencies of DNA methylation were: 57.5% (23 of 40) for ID4, 14.3% (5 of 35) for DLC-1, and 63.4% (26 of 41) for SFRP1, respectively. In contrast, a low frequency of methylation was detected in nontumor tissues. In addition, hypermethylated status of these genes was detected in three kinds of CC cell lines. Moreover, the downregulated expression of these genes in these cells was restored by treatment with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Taken together, these results suggest that aberrant DNA methylation may contribute to the tumorigenesis of cholangiocarcinoma.

AM251, a cannabinoid antagonist, has various biological activities. In this study, we found that AM251 suppressed the viability of hepatoma HepG2 cells and also increased phosphorylation of JNK (c-jun N-terminal kinase) and ATF3 (activating transcription factor 3). In addition, AM251 phosphorylated AMPK (AMP-activated protein kinase) in a time and dose-dependent manner. Inhibition of AMPK blocked AM251-induced JNK/ATF3 phosphorylation. Expression of AMPK or treatment with AICAR (5-aminoimidazole-4-carboxy-amide-1- d -ribofuranoside), an AMPK activator, activated the JNK/ATF3 pathways. Together, these results suggest that AM251 may have anti-tumor effects in hepatoma through activation of the AMPK–JNK–ATF3 signal pathway.

Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is one of the major components of honeybee propolis. In the present study, we investigated the metabolic effects of CAPE in skeletal muscle cells and found that CAPE stimulated glucose uptake in differentiated L6 rat myoblast cells and also activated AMPK (AMP-activated protein kinase). In addition, the inhibition of AMPK blocked CAPE-induced glucose uptake, and CAPE activated the Akt pathway in a PI3K (phosphoinositide 3-kinase)-dependent manner. Furthermore, CAPE enhanced both insulin-mediated Akt activation and glucose uptake. In summary, our results suggest that CAPE may have beneficial roles in glucose metabolism via stimulation of the AMPK pathway.

To gain insight into the differential mechanism of gene promoter hypermethylation in acute and chronic leukemia, we identified the methylation status on one part of 5'CpG rich region of 8 genes, DAB2IP, DLC-1, H-cadherin, ID4, Integrin alpha4, RUNX3, SFRP1, and SHP1 in bone marrows from acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) patients. Also, we compared the methylation status of genes in AML and CML using methylation-specific PCR (MSP). The frequencies of DNA methylation of ID4, SFRP1, and SHP1 were higher in AML patients compared to those in CML patients. In contrast, no statistical difference between AML and CML was detected for other genes such as DLC-1, DAB2IP, H-cadherin, Integrin alpha4, and RUNX3. Taken together, these results suggest that these methylation-controlled genes may have different roles in AML and CML, and thus, may act as a biological marker of AML.

Intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm of the biliary epithelium,is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of ICC are not well understood and only a few cytogenetic studies of ICC have been published. Recently, technique of degenerate oligonucleotide primed (DOP)-PCR comparative genomic hybridization (CGH) permits genetic imbalances screening of the entire genome using only small amounts of tumor DNA. In this study chromosomal aberrations in 33 Korean ICC were investigated by DOP-PCR CGH. The common sites of copy number increases were 20q (67%), 17 (61%), 11q11-q13 (42%), 8p12- qter (39%), 18p (39%), 15q22-qter (36%), 16p (36%), 6p21 (30%), 3q25-qter (27%), 1q41-qter (24%), and 5p14-q11.2 (24%). DNA amplification was identified in 16 carcinomas (48%). The frequent sites of amplification were 20q, 17p, 17q23-qter, and 7p. The most frequent sites of copy number decreases were 1p32-pter (21%) and 4q (21%). The recurrent chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of ICC.