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.

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.

Blockade of interleukin-6 receptor suppresses the proliferation of H460 lung cancer stem cells.

IL-6/6R signaling is closely associated with tumor growth and poor prognosis. Although there is evidence that interleukin-6 receptor (IL-6R)-mediated signaling promotes the growth and malignancy of cancer, the role of IL-6R in cancer stem cells (CSCs) is poorly defined. This study investigated the role of IL-6R in the proliferation of CSCs. Sphere-forming cells were isolated from the H460 non-small cell lung cancer (NSCLC) cell line and identified as CSCs using confocal microscopy, RT-PCR and WST-1 assay. The H460 spheres demonstrated the typical characteristics of CSCs, including CD133 expression, upregulation of Nanog, self-renewal, and drug resistance to methotrexate (MTX) and fluorouracil (5-FU). The release of IL-6R and its ligand, IL-6, were quantitatively determined and compared between CSCs and non-CSCs. The concentration of soluble IL-6R (sIL-6R) was remarkably high in CSCs compared to that in non-CSCs. Furthermore, significant upregulation of the IL-6R gene was also observed in the CSCs. The growth of CSCs was significantly inhibited by transfection with IL-6R small-interfering RNA (siRNA), as well as with the IL-6R monoclonal antibody (mAb). In addition, blocking both IL-6R and IL-6 using siRNA or mAbs intensified the inhibition of CSC proliferation. These findings indicate that IL-6R is present in CSCs and has an important role in the proliferation of CSCs in the H460 lung cancer cell line. Therefore, we suggest that IL-6R is both a viable target for the development of CSC-directed lung cancer therapeutics and a potential CSC marker in NSCLC.

Functional expression and characterization in Xenopus laevis oocytes of the ABCG2 transporter derived from A549 human lung adenocarcinoma cells.

We cloned the ATP-binding cassette sub-family G member 2 (ABCG2) transporter, the most recently identified among several major human multidrug-resistance pumps, from A549 human lung adenocarcinoma cells in order to characterize its function and substrate specificity. In a previous report, we confirmed that a stem cell-like side population of A549 cells highly expressed the ABCG2 gene and had a unique ability to resist the anticancer drug methotrexate (MTX). In this study, ABCG2 cDNA was cloned by RT-PCR and converted into cRNA by an in vitro transcription system for expression in Xenopus laevis (X. laevis) oocytes. The transcribed cRNA of the ABCG2 gene was injected into the oocytes under the absence of cofactors or heterologous partner proteins or some lipids from the media. A high expression of ABCG2 was observed on the oocyte surface by immunofluorescence and confocal laser microscopy. We tested the functional effect of ABCG2 expression on drug efflux by directly injecting MTX into X. laevis oocytes. The drug concentration within the oocytes was quantified with LC-MS/MS; the analysis showed that the accumulation of MTX was significantly decreased in the X. laevis oocytes expressing ABCG2 compared with the control oocytes not expressing ABCG2. These findings show that the ABCG2 protein has an important role in the efflux of MTX through the cell membrane of X. laevis oocytes. Therefore, it might be that ABCG2, abundantly expressed in the stem cell population of A549 cells, can modulate resistance to MTX in lung cancer therapy.

Determination of Oxyclozanide in Beef and Milk using High-Performance Liquid Chromatography System with UV Detector.

This study was developed and validated for the determination of oxyclozanide residue concentrations in beef and commercial milk, using high-performance liquid chromatography system. Oxyclozanide was successfully separated on a reverse phase column (Xbridge-C(18), 4.6×250 mm, 5 µm) with a mobile phase composed of acetonitrile and 0.1% phosphoric acid (60:40, v/v%). This analytical procedure involved a deproteinization process using acetonitrile for beef and 2% formic acid in acetonitrile for commercial milk, dehydration by adding sodium sulfate to the liquid analytical sample, and a defatting process using n-hexane; after these steps, the extract was exposed to a stream of nitrogen dryness. The final extracted sample was dissolved in the mobile phase and filtered using a 0.45 µm syringe filter. This method had good selectivity and recovery (70.70±7.90-110.79±14.95%) from the matrices. The LOQs ranged from 9.7 to 9.8 µg/kg for beef and commercial milk. The recoveries met the standards set by the CODEX guideline.