Figure 4

The mechanism of ET-1-induced expression of CXCR4 in 6-10B nasopharyngeal carcinoma cells. A. The ET-1-induced expression of CXCR4 in 6-10B nasopharyngeal carcinoma cells is mainly mediated through ETAR. Initially, 6-10B cells were serum-starved for 24 hours and pretreated with a selective ETAR antagonist, BQ123, for 2 hours. The cells were then either left unstimulated or stimulated with 10 nM ET-1 for 24 hours. The CXCR4 and GAPDH levels of whole-cell extracts were determined by western blotting. B-E. The ET-1-induced stimulation of CXCR4 expression was activated via the PI3K/AKT/mTOR or MAPK1/ERK1/2 signaling pathway in 6-10B nasopharyngeal carcinoma cells. B. Initially, 6-10B cells were serum-starved and treated with 10 nM ET-1 for various times, as indicated. Equal volumes of whole-cell lysate were analyzed by western blotting for AKT (phosphorylated on serine 473; P-AKT), total AKT, ERK1/2 (phosphorylated at threonine 202 and tyrosine 204; P-ERK1/2), total ERK1/2, and anti-GAPDH (loading control). C. Initially, 6–10 B cells were serum-starved and pretreated with LY-294002 (50 μM), wortmannin (10 μM), or rapamycin (50 μM) for 2 hours and then incubated in the presence of ET-1 (10 nM) for 24 hours. The CXCR4 levels were then analyzed by western blotting. D. The dose-dependent effects of LY-294002 on CXCR4 expression were evaluated in the presence of ET-1 (10 nM) for 24 hours. E. Initially, 6–10 B cells were serum-starved and pretreated with U0126 (10 μM), PD-98059 (50 μM), or SB203580 (30 μM) for 2 hours and then incubated in the presence of ET-1 (10 nM) for 24 hours. The CXCR4 levels were then analyzed by western blotting. ET-1 stimulation activated the AKT and MAPK signaling pathways, whereas blocking the PI3K/AKT or MAPK signal prevented ET-1-induced CXCR4 expression. Representative results from one of three independent experiments are shown.