Background/Aim: Indoxyl sulfate, a uremic toxin, is considered a risk factor for cardiovascular disease (CVD) in chronic kidney disease (CKD). The present study aimed to determine whether indoxyl sulfate increases the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) by reactive oxygen species (ROS)-induced activation of nuclear factor-ĸB (NF-ĸB) in vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with indoxyl sulfate. The expression of ICAM-1 and MCP-1 in HUVEC was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Phospho-NF-ĸB p65 (Ser 536), an active form of the NF-ĸB subunit, was determined by Western blotting. Results: Indoxyl sulfate significantly increased the mRNA expression of ICAM-1 and MCP-1 in HUVEC in a time- and concentration-dependent manner. Inhibitors of NF-ĸB (ammonium pyrrolidinedithiocarbamate and isohelenin) and an antioxidant (N-acetyl-L-cysteine) suppressed the indoxyl sulfate-induced expression of ICAM-1 and MCP-1 in HUVEC. Indoxyl sulfate increased phospho- NF-ĸB p65 in HUVEC, and N-acetyl-L-cysteine suppressed it. Conclusions: Indoxyl sulfate upregulates the expression of ICAM-1 and MCP-1 by ROS-induced activation of NF-ĸB in vascular endothelial cells. Thus, indoxyl sulfate may play an important role in the development of CVD in CKD by increasing the endothelial expression of ICAM-1 and MCP-1.
Background/Aim: Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Oxidative stress and nitric oxide (NO) deficiency play an important role in vascular endothelial cell dysfunction in chronic kidney disease. To determine if the uremic toxin indoxyl sulfate (IS) induces oxidative stress and inhibits NO production and cell viability in human umbilical vein endothelial cells (HUVEC). Methods: The production of reactive oxygen species (ROS), superoxide, NO and peroxynitrite was measured using a fluorescence microplate reader. The expression of NADPH oxidases (Nox4, Nox2) was analyzed by quantitative reverse transcription-polymerase chain reaction. Cell viability was examined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Results: IS induced ROS generation in HUVEC. An inhibitor of NADPH oxidase showed an inhibitory effect on IS-induced ROS production. However, the inhibitors of xanthine oxidase, mitochondrial electron transport and NO synthase did not show any significant effect on IS-induced ROS production. Antioxidants such as vitamin E, N-acetyl-L-cysteine and vitamin C inhibited IS-induced ROS production. IS induced the expression of Nox4 mRNA and the production of superoxide and peroxynitrite in HUVEC. IS inhibited NO production in HUVEC. IS inhibited cell viability, and antioxidants preserve the inhibitory effect of IS on cell viability. Conclusions: IS inhibits NO production and cell viability by inducing ROS through induction of Nox4 in HUVEC.
Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where it has been shown to promote tumor cell invasion upon phosphorylation. One mechanism by which EGFR promotes tumor progression is by activating signal cascades that lead to loss of E-cadherin, a transmembrane glycoprotein of the cell-cell adherence junctions; however mediators of these signaling cascades are not fully understood. One such mediator, RhoC, is activated upon a number of external stimuli, such as epidermal growth factor (EGF), but its role as a mediator of EGF-stimulated migration and invasion has not been elucidated in HNSCC. In the present study, we investigate the role of RhoC as a mediator of EGF-stimulated migration and invasion in HNSCC. We show that upon EGF stimulation, EGFR and RhoC were strongly activated in HNSCC. This resulted in activation of the phosphatidylinositol 3-Kinase Akt pathway (PI3K-Akt), phosphorylation of GSK-3β at the Ser9 residue, and subsequent down regulation of E-cadherin cell surface expression resulting in increased tumor cell invasion. Knockdown of RhoC restored E-cadherin expression and inhibited EGF-stimulated migration and invasion. This is the first report in HNSCC demonstrating the role RhoC plays in mediating EGF-stimulated migration and invasion by down-regulating the PI3K-Akt pathway and E-cadherin expression. RhoC may serve as a treatment target for HNSCC.
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