The effects of cetuximab alone and in combination with endostatin on vascular endothelial growth factor and interleukin-8 expression in human lung adenocarcinoma cells

Abstract

Background: Angiogenesis, the growth of new blood vessels, plays an important role in tumor growth and metastasis. Both cetuximab and endostatin have been found to reduce the expression of endothelial-stimulating growth factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8. However, the effects of cetuximab alone or in combination with endostatin on human lung adenocarcinoma cell growth remain unclear. Objective: The aim of this study was to evaluate the cellular and molecular effects of cetuximab alone and in combination with endostatin on human lung adenocarcinoma cell lines HI 299, SPC-A1, and H460 in vitro. Methods The epidermal growth factor receptor (EGFR) status of a panel of human lung adenocarcinoma cell lines was characterized using Western blot analysis. We used a modified tetrazolium salt assay to evaluate the growth-inhibitory effects of cetuximab and endostatin alone and in combination on the cell lines. We also determined the effects of these 2 drugs on VEGF and IL-8 expression using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Cells were treated for 4 days with cetuximab 12.5 μ/mL, endostatin 25 μ/mL, or cetuximab 12.5 μg/mL + endostatin 25 μg/mL. Untreated cells cultured for 4 days served as controls. Results: EGFR expression in the H1299 cells was higher than in the SPC-A1 and H460 cells. Varying concentrations of cetuximab alone were associated with a significant growth-inhibitory effect on all 3 cell lines in a dose-dependent manner after 4 days of exposure compared with controls (all, P < 0.05). Compared with controls, varying concentrations of endostatin alone were not associated with significant inhibition of cell growth in any of the 3 cell lines. The inhibitory ratio of cetuximab + endostatin at varying concentrations was significantly greater than that of cetuximab alone (all, P < 0.05). On ELISA, either drug alone was associated with significant reductions in secreted VEGF and IL-8 in the HI 299, SPC-A1, and H460 cell lines (all, P < 0.05), with the exception of IL-8 concentration in the H460 cells. Mean (SD) VEGF expression with combination treatment in the H1299 and SPC-A1 cell lines (687 [21] and 629 [23] pg/mL, respectively) was significantly lower than with cetuxi-mab alone (878 [31] and 708 [20] pg/mL; both, P < 0.001); in the H460 cell line, combination treatment was not associated with a significant further reduction in VEGF expression. IL-8 concentrations with cetuximab in the H1299, SPC-A1, and H460 cell lines were 628 (20), 484 (29), and 532 (28) pg/mL, respectively, while the IL-8 concentrations with the combination treatment were 516 (20), 480 (18), and 467 (30) pg/mL. An enhanced effect of endostatin on IL-8 was observed in the H1299 and H460 cell lines ( P < 0.001 and P = 0.018, respectively); however, no enhanced effect in the SPC-A1 line was observed. Similar results for VEGF and IL-8 expression were found using Western blot analysis. Conclusions: The results from this in vitro study suggest that cetuximab treatment might both inhibit human lung adenocarcinoma cell line growth and reduce the expression of VEGF and IL-8, which are the biomarkers of angiogenesis. Endostatin was not associated with inhibition of human lung adenocarcinoma cell line growth directly. Findings with the combination of cetuximab + endostatin suggest that endostatin might enhance the antiangiogenic and antitumor activity of cetuximab through an apparent effect on VEGF expression and, to a lesser degree, on IL-8 expression.