Angiogenic effect of interleukin-8 in breast cancer and its association with estrogen receptor

Abstract

To explore the angiogenic effect of interleukin-8 (IL-8) in breast cancer and its association with estrogen receptor (ER). The supernatants of culture liquid of breast cancer cells of different lines with high expression of Il-8 (MDA-MB-231 and MDA-MB-157), moderate expression of Il-8 (SKBr-3), or low expression of Il-8 (T47D and ZR75-1) were collected. These different conditioned media and human umbilical cord vein endothelial cells (HUVECs) were used in cell migration test to calculate the number of migrating HUVECs. Neutralizing antibody of IL-8 was added into above supernatants to observe the change in HUVECs migration. Mouse-tail collagen was added into the 12-well plate and then solidified, HUVECs were added thereon, and different supernatants were used as culture fluid so as to observe the angiogenesis of HUVECs on the collagen. HUVECs were cultured in the supernatants of culture liquid of different breast cells and CyQUANT dye was added at different time points so as to observe the proliferation of HUVECs by CCD imaging system. The supernatants of the culture fluid of the MDA-MB-231, SKBr-3, or T47D cells with or without fibroblast growth factor (FGF)-2 were injected subcutaneously into mice. Five days later the mice were killed to strip the skin to observe the angiogenesis microscopically. Supernatant chip test was made to measure the concentrations of IL-8 in different supernatants. Estrogen receptor (ER)-alpha and pN1481 Luc containing IL-8 promoter or p Luc0 not containing IL-8 promoter were transfected into different human breast cancer cells. Dual-luciferase assay was performed to study the regulation of IL-8 level by ER. The numbers of migrating HUVECs cultured in the supernatants of MDA-MB-231 cells, SKB-Br-3 cells, and T47D cells were 7800 +/- 368, 6510 +/- 419, and 3470 +/- 297 respectively (P < 0.05). After the addition of IL-8 neutralizing antibody, the number of migrating HUVECs cultured in the supernatant of MDA-MB-231 cells was 4700 +/- 233 and 4900 +/- 328 respectively, both significantly lower than those before the addition (both P < 0.05). In comparison with those cultured in the supernatants of the breast cancer cells expressing lower level of IL-8, the HUVECs cultured in the supernatants of the breast cancer cells expressing higher level of IL-8 tended to form more microangioid structure and proliferate more rapidly (P < 0.05). The skin of the mice injected with the supernatants of the breast cancer cells expressing higher level of IL-8 showed more blood vessel formation. Transient transfection test showed that the IL-8 level of the MDA-MB-231 cells transfected with ER was decreased by 8.8 folds compared with that of the ER negative MDA-MB-231 cells. Dual-luciferase assay showed that the activity of IL-8 promoter was significantly down-regulated by ERalpha (r = 0.856, P < 0.05). IL-8 is the key factor involved in angiogenesis of human breast cancer cell. The IL-8 level in human breast cancer cells is negatively correlated with ER status. Exogenous ER may down-regulate the expression of IL-8 in breast cancer cell.