Paclitaxel can induce tumor necrosis factor (TNF) and interleukin-1 gene expression, similar to lipopolysaccharides. Since lipopolysaccharide-induced expression of TNF is related to activation of NF-B, we determined whether NF-B could be activated by paclitaxel. In the human lung adenocarcinoma cell line A549, paclitaxel activated NF-B in a dose-dependent manner with maximal activation after 2-4 h. Since paclitaxel could upregulate TNF and interleukin-1 secretion and subsequent NF-B activation could be caused by these cytokines, the effect of two other groups of anticancer drugs including vinca alkaloids (vinblastine and vincristine) and anthracyclines (daunomycin and doxorubicin), neither of which induce TNF or interleukin-1 gene expression, were examined. Like paclitaxel, vinblastine, vincristine, daunomycin, and doxorubicin each caused activation of NF-B. Therefore, it is unlikely that activation of NF-B caused by these agents or by paclitaxel is mediated via cytokine up-regulation. Paclitaxel, a diterpene compound, was originally isolated from the stem bark of Taxus brevifolia and shown to have antiproliferative activity against various cultured cells as well as antineoplastic activity in tumor patients (1). These effects of paclitaxel appear to be related to its ability to bind to tubulin, to promote microtubule assembly, and to stabilize microtubules by bundle formation (2-4). Recently, Ding et al. (5). have found paclitaxel to exhibit cell cycle-independent, endotoxin-like effects on murine macrophages. Paclitaxel, like endotoxin (lipopolysaccharide; LPS), 1 causes murine macrophages to downregulate TNF receptors and initiate synthesis and secretion of TNF. In a similar fashion, paclitaxel can induce expression of both IL-1␣ and IL-1␤ (6). In addition, paclitaxel also induces tyrosine phosphorylation of microtubule associated protein kinases (7). Likewise, paclitaxel enhances ␥-interferon induction of nitric oxide synthase and secretion of nitric oxide (6), a macrophage tumoricidal factor. The pathways linking these responses to paclitaxel are believed to be similar to those causing such responses to LPS (5). Thus, an intracellular target affected by paclitaxel might be involved in actions of LPS in macrophages and other cells. Determination of which intracellular molecule paclitaxel and LPS affect in common could provide further insight into the actions of LPS on mammalian cells and participation of the cytoskeleton in responses of cells to their environment. One potential intracellular target of these compounds is the transcription factor NF-B. NF-B, named for its ability to recognize a light chain immunoglobulin gene regulatory element, can participate in the regulation of numerous genes (reviewed in Baeuerle (8)). Under basal conditions, it usually exists as a heterodimer of 50-and 65-kDa subunits bound to an inhibitor protein IB in the cytoplasm. Various stimuli cause IB to dissociate from the complex allowing the heterodimer to migrate to the nucleus and activate gene expression. Because NF-B is inv...

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.

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BackgroundEndothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2‾), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs.MethodGene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2‾ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement.ResultsWe showed that the O2‾ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC.ConclusionOur findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.