Celastrol Inhibits Lipopolysaccharide-Induced Angiogenesis by Suppressing TLR4-Triggered Nuclear Factor-Kappa B Activation

Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine <i>Tripterygium wilfordii</i> Hook F. In this study, we investigated the effect of celastrol on lipopolysaccharide (LPS)-activated LP-1 human multiple myeloma cell-induced angiogenesis, and identified its molecular mechanism of action. Migration of human umbilical vein endothelial cells (HUVECs) was tested using a wound-healing assay. HUVEC invasion was assayed using a Transwell chamber. Cell surface expression of Toll-like receptor 4 (TLR4) was analyzed by flow cytometry. Angiogenic factor vascular endothelial growth factor (VEGF) level was quantified by LUMINEX and protein expression was analyzed by Western blot. Translocation of nuclear factor-kappa B (NF-&#954;B) was observed by fluorescence microscopy. Celastrol inhibited LPS-stimulated LP-1 human multiple myeloma-induced HUVEC migration and invasion in a concentration-dependent manner. Wound diameters increased by 72.9, 165.4 and 246.2% at 0.025, 0.05 and 0.1 µ<smlcap>M</smlcap>, respectively, compared to LPS alone. A 45-74% inhibition of LPS-dependent cell invasion was achieved in the presence of 0.025-0.1 µ<smlcap>M</smlcap> celastrol. Celastrol significantly downregulated LPS-induced TLR4 expression and inhibited LPS-induced VEGF secretion in LP-1 cells. VEGF levels decreased by 64.8, 84.4 and 92.9% after coexposure to celastrol at 0.025, 0.05 and 0.1 µ<smlcap>M</smlcap>, respectively, compared to LPS alone. Celastrol also inhibited the I&#954;B kinase (IKK)/NF-&#954;B pathway induced by LPS. Protein levels of NF-&#954;B p65, IKKa and I&#954;B-a decreased in a dose-dependent manner after coexposure to celastrol. Celastrol also blocked nuclear translocation of the p65 subunit. These results suggest that celastrol inhibits LPS-induced angiogenesis by suppressing TLR4-triggered NF-&#954;B activation.