Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells

Abstract

Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 µg/mL or 2 µg/mL) (0.2 µM or 0.8 µM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action.