Hee-Jung Cho

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.

Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells.

BackgroundThe studies on cancer-stem-cells (CSCs) have attracted so much attention in recent years as possible therapeutic implications. This study was carried out to investigate the gene expression profile of CSCs in human lung adenocarcinoma A549 cells.ResultsWe isolated CSCs from A549 cell line of which side population (SP) phenotype revealed several stem cell properties. After staining the cell line with Hoechst 33342 dye, the SP and non-side population (non-SP) cells were sorted using flow cytometric analysis. The mRNA expression profiles were measured using an Affymetrix GeneChip® oligonucleotide array. Among the sixty one differentially expressed genes, the twelve genes inclusive three poor prognostic genes; Aldo-keto reductase family 1, member C1/C2 (AKR1C1/C2), Transmembrane 4 L six family member 1 nuclear receptor (TM4SF1), and Nuclear receptor subfamily 0, group B, member 1 (NR0B1) were significantly up-regulated in SP compared to non-SP cells.ConclusionThis is the first report indicating the differences of gene expression pattern between SP and non-SP cells in A549 cells. We suggest that the up-regulations of the genes AKR1C1/C2, TM4SF1 and NR0B1 in SP of human adenocarcinoma A549 cells could be a target of poor prognosis in anti-cancer therapy.

Gintonin, Newly Identified Compounds from Ginseng, Is Novel Lysophosphatidic Acids-Protein Complexes and Activates G Protein-Coupled Lysophosphatidic Acid Receptors with High Affinity

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G-protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s) were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 > LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitor Y-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.

Single-step extraction followed by LC for determination of (fluoro)quinolone drug residues in muscle, eggs, and milk

In this study, a simplified method for the extraction and determination of seven fluoroquinolone residues (danofloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, ofloxacin, and sarafloxacin) and three quinolones (oxolinic acid, flumequine, and nalidixic acid), in porcine muscle, table eggs, and commercial whole milk, which required no cleanup step, was devised. This procedure involves the extraction of analytes from the samples via liquid-phase extraction, and the subsequent quantitative determination was accomplished via LC-fluorescence detection. Analyte separation was successfully conducted on an XBridge-C(18) column, with a linear gradient mobile phase composed of acetonitrile and 0.01 M oxalic acid buffer at pH=3.5. The one-step liquid-liquid extraction method evidenced good selectivity, precision (RSDs=0.26-15.07%), and recovery of the extractable analytes, ranging from 61.12 to 115.93% in matrices. The LOQs ranged from 0.3 to 25 microg/kg. A survey of ten samples purchased from local markets was conducted, and none of the samples harbored fluoroquinolone residues. This method is an improvement over existing methodologies, since no additional cleanup was necessary.

Pharmacokinetic properties and antitumor efficacy of the 5-fluorouracil loaded PEG-hydrogel

BackgroundWe have studied the in vitro and in vivo utility of polyethylene glycol (PEG)-hydrogels for the development of an anticancer drug 5-fluorouracil (5-FU) delivery system.MethodsA 5-FU-loaded PEG-hydrogel was implanted subcutaneously to evaluate the drug retention time and the anticancer effect. For the pharmacokinetic study, two groups of male rats were administered either an aqueous solution of 5-FU (control group)/or a 5-FU-loaded PEG-hydrogel (treated group) at a dose of 100 mg/kg. For the pharmacodynamic study, a human non-small-cell lung adenocarcinoma (NSCLC) cell line, A549 was inoculated to male nude mice with a cell density of 3 × 106. Once tumors start growing, the mice were injected with 5-FU/or 5-FU-loaded PEG-hydrogel once a week for 4 weeks. The growth of the tumors was monitored by measuring the tumor volume and calculating the tumor inhibition rate (IR) over the duration of the study.ResultsIn the pharmacokinetic study, the 5-FU-loaded PEG-hydrogel gave a mean residence time (MRT) of 8.0 h and the elimination half-life of 0.9 h; these values were 14- and 6-fold, respectively, longer than those for the free solution of 5-FU (p < 0.05). In the pharmacodynamic study, A549 tumor growth was significantly inhibited in the 5-FU-loaded PEG-hydrogel group in comparison to the untreated group beginning on Day 14 (p < 0.05-0.01). Moreover, the 5-FU-loaded PEG-hydrogel group had a significantly enhanced tumor IR (p < 0.05) compared to the free 5-FU drug treatment group.ConclusionWe suggest that 5-FU-loaded PEG-hydrogels could provide a useful tool for the development of an anticancer drug delivery system.

Effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; 25 µg/ml) and methotrexate (MTX; 50 µg/ml), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptasepolymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

In Vitro inhibitory potential of decursin and decursinol angelate on the catalytic activity of cytochrome P-450 1A1/2, 2D15, and 3A12 isoforms in canine hepatic microsomes

Danggui is one of the most popular herbal medicines consumed by patients in different clinical settings in Asian countries. In this study, the two major pyranocoumarin compounds extracted from the Korean Angelica gigas root decursin (DC) and decursinol angelate (DA) were examined in vitro with regard to their abilities to inhibit hepatic CYP1A1/2, CYP2D15, and CYP3A12 catalytic activities in canine liver microsomes. The two components were capable of inhibiting CYP1A1/2, CYP2D15, and CYP3A12 catalytic activities, but the potencies varied. DC and DA selectively and noncompetitively inhibited CYP1A1/2 activity, with Ki values of 90.176 and 67.560 μM, respectively. On the other hand, they exhibited slight inhibitory effects on CYP2D15 and CYP3A12 with Ki values of 666.180 and 872.502 μM, 990.500 and 909.120 μM (1’hydroxymidazolam, MDZ1’H), and 802.800 and 853.920 μM (4-hydroxymidazolam, MDZ4H), respectively. Additionally, they showed increased inhibition after preincubation, which suggests the involvement of a mechanism-based inhibition. In sum, this in vitro data should be heeded as a signal of possible in vivo interactions. The use of human liver preparations would considerably strengthen the practical impact of the data generated from this study.

Development and validation of a liquid chromatography method with electrospray ionization tandem mass spectrometry for the determination of brotizolam residues in beef and commercial whole milk

In this work, a liquid chromatography-tandem mass spectrometric detection technique was developed and validated for the determination of brotizolam residues in beef muscle and commercial whole milk. This procedure involves the extraction of the analyte from the samples via liquid-solid extraction, and caffeine was used as an internal standard. The analyte was successfully separated on an XTerra-C(18) column, with a mobile phase composed of 0.01% formic acid in acetonitrile and 1 mm ammonium formate-0.01% formic acid in water. The one-step extraction method evidenced good selectivity, precision (RSD = 9.87-26.47%), and the recovery of the extractable analyte was 92.61-115.98% in the matrices. The limits of quantification ranged between 0.4 and 0.5 µg/kg. The developed method is simple since it requires no additional cleanup procedures.

Pharmacokinetics and milk distribution characteristics of orbifloxacin following intravenous and intramuscular injection in lactating ewes

The purpose of the current investigation is to elucidate the pharmacokinetic profiles of orbifloxacin (OBFX) in lactating ewes (n = 6) following intravenous (i.v.) and intramuscular (i.m.) administrations of 2.5 mg/kg W. In a crossover study, frequent blood, milk, and urine samples were drawn for up to 48 h after the end of administration, and were then assayed to determine their respective drug concentrations through microbiological assay using Klebsiella pneumoniae as the test micro-organism. Plasma pharmacokinetic parameters were derived from plasma concentration-time data using a compartmental and noncompartmental analysis, and validated a relatively rapid elimination from the blood compartment, with a slope of the terminal phase of 0.21 +/- 0.02 and 0.19 +/- 0.06 per hour and a half-life of 3.16 +/- 0.43 and 3.84 +/- 0.59 h, for i.v. and i.m. dosing, respectively. OBFX was widely distributed with a volume of distribution V((d(ss))) of 1.31 +/- 0.12 L/kg, as suggested by the low percentage of protein binding (22.5%). The systemic body clearance (Cl(B)) was 0.32 +/- 0.12 L/h x kg. Following i.m. administration, the maximum plasma concentration (C(max)) of 1.53 +/- 0.34 microg/mL was reached at t(max) 1.25 +/- 0.21 h. The drug was completely absorbed after i.m. administration, with a bioavailability of 114.63 +/- 11.39%. The kinetic milk AUC(milk)/AUC(plasma) ratio indicated a wide penetration of orbifloxacin from the bloodstream to the mammary gland. OBFX urine concentrations were higher than the concurrent plasma concentrations, and were detected up to 30 h postinjection by both routes. Taken together, these findings indicate that systemic administration of orbifloxacin could be efficacious against susceptible mammary and urinary pathogens in lactating ewes.

Determination of the esculetin contents of medicinal plants by liquid chromatography-tandem mass spectrometry.

We developed a LC-MS/MS method for the determination of esculetin contents in medicinal plants. The analysis was performed using multiple reaction monitoring in negative mode, and an XBridge™ C(18) column (2.1 × 100 mm, 3.5 µm) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r(2)  > 0.9993). The limits of detection and quantitation were 0.02 and 0.07 ng/mL, respectively. The intra-day and inter-day precisions were 1.5-6.8 and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini Cortex showed the highest content of esculetin (761-5475 mg/kg). In Mori Folium and Artemisiae Capillaris Herba, 5.2-21.5 and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive and reproducible. Contrary to expectations based on traditional medical knowledge, although Artemisiae Capillaris Herba contains a large amount of esculetin, it appears from this study that Fraxini Cortex contains a greater amount. The pharmacological effects of esculetin isolated from medicinal plants should be investigated as part of new medicines development.