MDR-1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB-V1 cells

AbstractThe relevance of MDR-1 gene expression to the multidrug resistance phenotype was investigated. Drug-resistant cells, KB-V1 and MCF7/ADR, constantly expressed mRNA of the MDR-1 gene and were more resistant to vinblastine and adriamycin than drug-sensitive cells, KB-3–1 and MCF7. The drug efflux rate of KB-V1 was the same as KB-3–1 although the MDR-1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB-3–1 than KB-V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P-glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR-1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms

Enhanced cardiac expression of two isoforms of matrix metalloproteinase-2 in experimental diabetes mellitus.

BackgroundDiabetic cardiomyopathy (DM CMP) is defined as cardiomyocyte damage and ventricular dysfunction directly associated with diabetes independent of concomitant coronary artery disease or hypertension. Matrix metalloproteinases (MMPs), especially MMP-2, have been reported to underlie the pathogenesis of DM CMP by increasing extracellular collagen content.PurposeWe hypothesized that two discrete MMP-2 isoforms (full length MMP-2, FL-MMP-2; N-terminal truncated MMP-2, NTT-MMP-2) are induced by high glucose stimulation in vitro and in an experimental diabetic heart model.MethodsRat cardiomyoblasts (H9C2 cells) were examined to determine whether high glucose can induce the expression of the two isoforms of MMP-2. For the in vivo study, we used the streptozotocin-induced DM mouse heart model and age-matched controls. The changes of each MMP-2 isoform expression in the diabetic mice hearts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical stains were conducted to identify the location and patterns of MMP-2 isoform expression. Echocardiography was performed to compare and analyze the changes in cardiac function induced by diabetes.ResultsQuantitative RT-PCR and immunofluorescence staining showed that the two MMP-2 isoforms were strongly induced by high glucose stimulation in H9C2 cells. Although no definite histologic features of diabetic cardiomyopathy were observed in diabetic mice hearts, left ventricular systolic dysfunction was determined by echocardiography. Quantitative RT-PCR and IHC staining showed this abnormal cardiac function was accompanied with the increases in the mRNA levels of the two isoforms of MMP-2 and related to intracellular localization.ConclusionTwo isoforms of MMP-2 were induced by high glucose stimulation in vitro and in a Type 1 DM mouse heart model. Further study is required to examine the role of these isoforms in DM CMP

Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis

Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required

In situ rumen degradability characteristics of rice straw, soybean curd residue and peppermint (Mentha piperita) in Hanwoo steer (Bos Taurus coreanae)

This study was intended to evaluate in situ rumen degradability characteristic of soybean curd residue (SCR) and peppermint compared to rice straw, which are used as a functional feed source for beef cattle for high quality beef production. Two steers were fitted with rumen and duodenum cannulae and in situ degradable and nylon bags were used to assess digestion of rice straw, SCR and peppermint in three replicated experiments. The highly effective dry matter (DM) degradability of peppermint was attributable to the high rate of the a and b fractions. Dry matter disappearance rate of SCR after 48 h was higher (p < 0.05) than that of peppermint. Based on DM disappearance, SCR showed the highest digestibility (p < 0.05). Crude protein disappearance (%) was higher for SCR and peppermint than rice straw (p < 0.05), and neutral detergent fibre disappearance (%) was higher for SCR (p < 0.05). Soybean curd residue and peppermint have great nutritive values as feed sources for ruminants, and using SCR and peppermint would have possibilities for efficient and functional livestock production.Keywords: Beef cattle, digestibility, effective degradability, soybean curd residue, peppermintAfrican Journal of Biotechnology Vol. 12(14), pp. 1710-171

Enhancement of angiogenic and vasculogenic potential of endothelial progenitor cells by haptoglobin

AbstractEndothelial progenitor cells (EPCs) were transfected with the haptoglobin (Hp) gene to investigate the effect of Hp on cell function. Hp potentiated the gene expression of various pro-angiogenic factors in the EPCs. The Hp-modified EPCs also increased in vitro tube formation on Matrigel compared with control cells. In hindlimb ischaemia models, Hp–EPCs showed a greater ability for improving blood perfusion and recovery from ischaemic injury. These results indicate that Hp improves EPC function in neovasculogenesis, which suggests that ex vivo modification of EPCs with the Hp gene can be applied to the treatment of vascular damage

5-lipoxygenase mediates docosahexaenoyl ethanolamide and N-arachidonoyl-L-alanine-induced reactive oxygen species production and inhibition of proliferation of head and neck squamous cell carcinoma cells

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Abstract Background Endocannabinoids have recently drawn attention as promising anti-cancer agents. We previously observed that anandamide (AEA), one of the representative endocannabinoids, effectively inhibited the proliferation of head and neck squamous cell carcinoma (HNSCC) cell lines in a receptor-independent manner. In this study, using HNSCC cell lines, we examined the anti-cancer effects and the mechanisms of action of docosahexaenoyl ethanolamide (DHEA) and N-arachidonoyl-L-alanine (NALA), which are polyunsaturated fatty acid (PUFA)-based ethanolamides like AEA. Methods and Results DHEA and NALA were found to effectively inhibit HNSCC cell proliferation. These anti-proliferative effects seemed to be mediated in a cannabinoid receptor-independent manner, since the antagonist of cannabinoid receptor-1 (CB1) and vanilloid receptor-1 (VR1), two endocannabinoid receptors, did not reverse the ability of DHEA and NALA to induce cell death. Instead, we observed an increase in reactive oxygen species (ROS) production and a decrease of phosphorylated Akt as a result of DHEA and NALA treatment. Antioxidants efficiently reversed the inhibition of cell proliferation and the decrease of phosphorylated Akt induced by DHEA and NALA; inhibition of 5-lipoxygenase (5-LO), which is expected to be involved in DHEA- and NALA-degradation pathway, also partially blocked the ability of DHEA and NALA to inhibit cell proliferation and phosphorylated Akt. Interestingly, ROS production as a result of DHEA and NALA treatment was decreased by inhibition of 5-LO. Conclusions From these findings, we suggest that ROS production induced by the 5-LO pathway mediates the anti-cancer effects of DHEA and NALA on HNSCC cells. Finally, our findings suggest the possibility of a new cancer-specific therapeutic strategy, which utilizes 5-LO activity rather than inhibiting it

Red Ginseng Extract Attenuates Kainate-Induced Excitotoxicity by Antioxidative Effects

This study investigated the neuroprotective activity of red ginseng extract (RGE, Panax ginseng, C. A. Meyer) against kainic acid- (KA-) induced excitotoxicity in vitro and in vivo. In hippocampal cells, RGE inhibited KA-induced excitotoxicity in a dose-dependent manner as measured by the MTT assay. To study the possible mechanisms of the RGE-mediated neuroprotective effect against KA-induced cytotoxicity, we examined the levels of intracellular reactive oxygen species (ROS) and [Ca2+]i in cultured hippocampal neurons and found that RGE treatment dose-dependently inhibited intracellular ROS and [Ca2+]i elevation. Oral administration of RGE (30 and 200 mg/kg) in mice decreased the malondialdehyde (MDA) level induced by KA injection (30 mg/kg, i.p.). In addition, similar results were obtained after pretreatment with the radical scavengers Trolox and N, N′-dimethylthiourea (DMTU). Finally, after confirming the protective effect of RGE on hippocampal brain-derived neurotropic factor (BDNF) protein levels, we found that RGE is active compounds mixture in KA-induced hippocampal mossy-fiber function improvement. Furthermore, RGE eliminated 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and the IC50 was approximately 10 mg/ml. The reductive activity of RGE, as measured by reaction with hydroxyl radical (•OH), was similar to trolox. The second-order rate constant of RGE for •OH was 3.5–4.5×109 M−1·S−1. Therefore, these results indicate that RGE possesses radical reduction activity and alleviates KA-induced excitotoxicity by quenching ROS in hippocampal neurons