Inhibition of angiogenic key features in vitro: the alkaloid narciclasine blocks proliferation, migration, and tube formation of primary human endothelial cells

Plants of the Amaryllidaceae family have been used in traditional medicine for the treatment of cancer for a long time. Narciclasine, an isocarbostyril alkaloid from Narcissus and Haemanthus species, was reported to exhibit pro-apoptotic effects selectively for cancer cells. Although narciclasine is an interesting compound for cancer therapy, its effects on endothelial cells in the context of tumor angiogenesis have been neglected so far.

We aimed to elucidate the action of narciclasine on in vitro key features of angiogenesis, i.e. on the proliferation, migration, and tube formation of cultured endothelial cells (ECs). Treatment of ECs with narciclasine up to a concentration of 300 nM for 24 or 48h did not affect the metabolic activity, while cell proliferation was concentration-dependently reduced with an IC50 value of 61 nM. The compound lowered the number of endothelial cells in the S- as well as G2/M-phase and increased the cell number in the G1/G0-phase of the cell cycle (flow cytometric analysis). Interestingly, narciclasine (300 nM) did neither inhibit the activation of Erk1/2 and Akt (Western blot analysis), nor interfere with endothelial nitric oxide (NO) production (DAF-2 and arginine/citrulline conversion assay). Moreover, narciclasine (300 nM) reduced the undirected migration of ECs (scratch assay) by 54% and the chemotactic migration (Boyden chamber assay) by 77%. Microscopical analysis of the cytoskeleton revealed that narciclasine increased the formation of F-actin stress fibers. Most importantly, the alkaloid inhibited the formation of endothelial tube-like structures on Matrigel, since it reduced the number of junctions and tubules as well as the total length of tubules.

Taken together, we could demonstrate that the isocarbostyril alkaloid narciclasine effectively decreases proliferation, migration, and tube formation of human endothelial cells and, therefore, will be further evaluated for its anti-angiogenic properties.