Table 1.
Effects of AF3485 on PGE2 release and growth in non tumor cells.
Figure 1.
AF3485 inhibits human recombinant mPGES-1 activity in vitro and selectively inhibits IL-1β-induced PGE2 production in A549 cells.
Structure of AF3485 (A). Effect of AF3485 (B) on hmPGES-1 expressed in bacterial membrane. A549 cells pre-treated with IL-1β (10 ng/ml, 18 h) then treated with AF3485 (C) (0.01–100 µM, 30 min). PGE2 and PGF2α levels measured by ELISA. Data reported as % inhibition of PGE2 (□) or PGF2α levels (▿).
Table 2.
Effects of AF3485 on leukotriene biosynthesis.
Figure 2.
AF3485 inhibits IL-1β-induced PGE2 production in A431 cells.
(A) COX-1, -2 and mPGES-1 mRNA expression in A431 cells in presence/absence of IL-1β (10 ng/ml, 18 h). Data reported as fold increase vs. unstimulated control. **P<0.01 vs Control. (B) Western blot of COX-1, -2, and mPGES-1 expression in A431 cells in presence/absence of IL-1β (10 ng/ml, 18 h). (C) PGE2 levels in unstimulated A431 cells treated with AF3485 (0–100 µM, 30 min). PGE2 levels measured by ELISA (24 h from plating.) Data reported as ng/ml. *P<0.05; **P<0.01; ***P<0.001 vs Control. (D) PGE2 levels in unstimulated or IL-1β (10 ng/ml, 24 h) pre-treated A431 cells, and treated with AF3485 (10 µM, 30 min). The data are reported as ng/ml. **P<00.1 vs Cont; ##P<0.01 vs Cont; §§P<0.01 vs IL-1β (10 ng/ml).
Figure 3.
The mPGES-1 inhibitor AF3485 reduces A431 tumor growth in xenograft nude mice.
(A) Tumor volume measured in athymic mice inoculated with A431 cells and treated with vehicle (Ctr, 0.5% MTC), AF3485 (20, 1, or 0.1 mg/kg/mouse), or AG1478 (400 µg/mouse). (B) EGFR phosphorylation in xenograft tumor tissues reported as optical density (OD = ratio between phospho-tyr and EGFR expression). *P<0.05 vs Cont. (C) Representative images of the proliferative Ki67 index in tumor sections.
Figure 4.
IL-1β-induced PGE2 transactivates EGFR in tumor cells.
(A) EGFR phosphorylation in A431 pretreated with IL-1β (10 ng/ml, 18 h), then treated with AF3485 (0.1–100 µM, 30 min). (B) EGFR phosphorylation in A431 in the conditions described in panel A, in presence/absence of PGE2 (1 µM). (C) EGFR phosphorylation in A431 silenced for mPGES-1, then treated with IL-1β (10 ng/ml, 18 h). (D) mPGES-1 expression in A431 silenced with siRNA for mPGES-1 or control siRNA. Gels (Western blot) are representative of three experiments with similar results.
Figure 5.
AF3485 inhibits tumor growth and angiogenesis.
Representative images of histological analysis of CD31 in tumor sections from (a) control, (b) AF3485-treated mice. Images taken at 40X. Quantification of microvessel density in tumors. **P<0.01 vs Cont.
Figure 6.
The mPGES-1 inhibitor AF3485 reduces VEGF and FGF-2 expression in tumors and in A431 cells.
(A) VEGF and FGF-2 levels in tumors specimens and (B) in A431 cells treated with or without IL-1β (10 ng/ml, 18 h) in presence/absence of AF3485 (10 µM). *P<0.05, **P<0.01. ADU = arbitrary density unit ± standard deviation. (C) Pseudocapillary formation of EC exposed for 16 h to conditioned media from A431 treated with 0.1% FBS (panel a: control), IL-1β (panel b: 10 ng/ml, 18 h), AF3485 (panel c: 10 µM, 24 h), IL-1β + AF3485 (panel d: 24 h) *P<0.01 vs. cont, #p<0.01 vs. IL-1β. (D) VEGF and FGF-2 expression in A431 cells co-cultured with NIH-3T3 for 24 h.