Effects of Macrolides on Angiogenesis Induced by Human Lung Cancer A549 Cells.

Abstract

Objective: We investigated the effects of two macrolides on the induction of angiogenesis by human bronchiolo-alveolar carcinoma A 549 cells in vitro. Methods: We chose clarithromycin (CAM) and midecamycin (MDM) as 14-and 16-membered ring macrolide antibiotics, respectively. By using human umbilical vein endothelial cells (HUVEC), we performed a wounding cell migration assay, proliferation assay and tube formation assay with MATRIGEL® to investigate the effects of these macrolides on angiogenesis in both a direct manner, and an indirect manner mediated by A549 cells. We next measured the concentrations of angiogenic factors such as basic fibroblast growth factor (basic-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) present in culture supernatant from macrolide-treated A 549 cells by enzyme-linked immunosorbent assay (ELISA) and also measured the concentrations of urokinase plasminogen activator (u-PA) in culture supernatant from macrolide-treated HUVEC and macrolide-treated A 549 cells by ELISA. Results: The culture supernatant from A 549 cells promoted each step of HUVEC angiogenesis; migration, proliferation, and tube formation. The culture supernatant from A 549 cells treated with CAM and MDM at 0.1 and 1.0μ g/ml which correspond to their concentrations in serum at clinical administration inhibited migration of HUVEC. However, the inhibitory effect of MDM was weak. The culture supernatant from A 549 cells treated with CAM, but not with MDM, inhibited proliferation and tube formation of HUVEC. Both macrolides showed no direct effect on HUVEC angiogenesis. The concentrations of basic-FGF in culture supernatant from A 549 cells were below the assay sensitivity limit. A549 cells produced interleukin-8 (IL-8) as well as VEGF, and both macrolides inhibited the production of these factors. The culture supernatant from MDM-treated A 549 cells did not affect proliferation or tube formation of HUVEC. Both macrolides suppressed A549 cells from secreting u-PA, an endothelial cell migration factor, at 0.1 and 1.0μg/ml, but promoted them at 10μg/ml. This biphasic manner of the effect of macrolides on u-PA production was similar to that on HUVEC migration. Conclusion: The present results indicate that macrolides, especially a 14-membered ring macrolide, are a potential inhibitor of angiogenesis promoted by lung cancer cells. Their sites of action may be on cancer cells but not endothelial cells. However, the precise mechanism of the inhibitory effect of macrolides remains to be determined.