Vascular endothelial growth factor promotes the expression of cyclooxygenase 2 and matrix metalloproteinases in Lewis lung carcinoma cells

Abstract

Vascular endothelial growth factor (VEGF) plays a critical role in tumor progression, angiogenesis and metastasis. Cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)2, MMP9 and wild-type (WT) p53 has been found to regulate the production of VEGF. Whether VEGF regulates the production of COX-2, MMP2, MMP9 and WTp53, however, has yet to be determined. This study examined the influence of the overexpression or knockdown of VEGF on the protein levels of COX-2, MMP2, MMP9 and WTp53 as well as cell growth and cell cycle progression in Lewis lung carcinoma (LLC) cells. LLC cells were transfected with pIRES2-VEGF-GFP in the VEGF-overexpressing group (LLC-VEGF), pIRES2-GFP in the mock group (LLC-GFP) or pSUPER-VEGF-GFP in the VEGF knockdown group (LLC-RNAi). Protein levels were detected by western blot analysis. LLC cell growth exhibited no marked change in the LLC-VEGF group, but was significantly retarded in the LLC-RNAi group. Further examination revealed that more cells entered the S stage in the LLC-VEGF group than in the control (or mock) group (45.3 vs. 29.1%, P<0.05), and that cell growth was retarded in the LLC-RNAi group. Moreover, COX-2 and MMP2 and MMP9 proteins were significantly increased in the LLC-VEGF group (approximately 1.84-, 1.89- and 1.83-fold, respectively, vs. control, P<0.05), but significantly decreased in the LLC-RNAi group, whereas the expression of WTp53 exhibited the opposite pattern of change. VEGF expression was positively correlated with COX-2, MMP2 and MMP9 expression (r=0.984, r=0.978, r=0.969, respectively, P<0.01) and negatively correlated with WTp53 (r=-0.833, p<0.01). The activities of MMP2 and MMP9 were increased in the LLC-VEGF group. In conclusion, VEGF overexpression may promote the expression of COX-2 and MMPs, but inhibits WTp53 production in LLC cells; VEGF underexpression may have an inverse effect. These changes are closely correlated with the infiltration and metastasis of lung cancer.

Figure 1

VEGF concentration and its effect on the expression of MMP2, MMP9, COX-2 and WTp53. (A) Equal amount of protein extracts from whole-cell lysates were subjected to western blotting with β-acting serving as an internal control. The results are representative of at least three independent experiments. (B) Bar graphs of VEGF, MMP2, MMP9, COX-2 and WTp53 protein ratios of the four groups. ^*p<0.05, compared with the LLC group; ^#p<0.05, compared with the LLC-VEGF group.

Figure 2

VEGF has no direct effect on the growth of Lewis lung cancer cells in vitro. Cells (1×10⁴ cells/ml) were seeded into a 96-well plate, and the number of cells was counted directly every day. The results showed that there was no significant difference in cell growth between the LLC-VEGF or LLC-GFP and LLC groups, but the growth rate was significantly repressed on the 4th and 5th day in the LLC-RNAi group compared with the control (P<0.05).

Figure 3

The effect of VEGF on the cell cycle distribution. DNA status was determined by flow cytometry in the cells of the (A) LLC, (B) LLC-VEGF, (C) LLC-RNAi and (D) LLC-GFP groups. (E) Cell cycle distributions of the four groups. ^*P<0.05; LLC-VEGF or LLC-RNAi group vs. LLC group.

Figure 4

VEGF promotes MMP2 and MMP9 activities in Lewis lung cancer cells. Gelatin zymography was used to detected the MMP activities. The activities of MMP2 and MMP9 were significantly higher in the LLC-VEGF group than in the other groups, but decreased in the LLC-RNAi group.