Effects of Two-by-Two Combination Therapy with Valproic Acid, Lithium Chloride, and Celecoxib on the Angiogenesis of the Chicken Chorioallantoic Membrane

Abstract

Background: The synergistic effects of valproic acid (VPA), lithium (Li), and celecoxib (CX) have been shown in combination therapy against the proliferation and metastasis of numerous cancers. Angiogenesis plays a critical role in the pathogenesis of tumor growth and metastasis. The aim of the present study was to evaluate the antiangiogenic effects of VPA, lithium chloride (LiCl), and CX, alone or in 2-by-2 combinations, using the chicken chorioallantoic membrane (CAM) assay.

Methods: Fertilized chicken eggs were randomly divided into 10 groups: control, VPA (1.8 and 3.6 µmol/CAM), Li (0.15 and 0.60 µmol/CAM), CX (0.02 and 0.08 µmol/CAM), VPA+Li, VPA+CX, and CX+Li (n=10 per group). A window was made on the eggshells and the CAMs were exposed to a filter disk containing VPA, LiCl, and CX, alone or in 2-by-2 combinations. The control CAMs were treated with distilled water (vehicle). Three days after the treatment, the number of vessel branch points was counted in each CAM. The data were analyzed using SPSS, version 15.One-way ANOVA, followed by the Tukey tests, was used to compare the groups. A P<0.05 was considered a statistically significant difference between the groups.

Results: According to the results, all the tested drugs decreased the number of the vessel branch points in a dose-dependent manner compared to the control group (P<0.001). In addition, combinations of the drugs were more effective in decreasing angiogenesis than the use of each drug alone.

Conclusion: These findings suggest that 2-by-2 combinations of VPA, CX, and LiCl can be considered an effective antiangiogenesis therapeutic modality.

Keywords: Celecoxib; Combined modality therapy; Lithium chloride; Valproic acid; Angiogenesis inhibitors.

Figure1

Micrograph (A) shows the distribution of blood vessels in a vehicle-treated chicken chorioallantoic membrane (CAM) in the control group and when analyzed using Wimasis Image Analysis software (B) to quantify the vessel branch points. Lines marked in red are vessels and points marked in white are vessel branch points (arrows).

Figure2

Micrographs show vessel branch points in the control vehicle-treated chicken chorioallantoic membrane (CAM) (A) and analyzed micrograph (A´), CAM treated with 1.8 µmol of valproic acid (VPA) (B) and analyzed micrograph (B´), and CAM treated with 3.6 µmol of VPA (C) and analyzed micrograph (C´). The number of the vessel branch points was counted in each group and analyzed using one-way ANOVA, followed by the Tukey post hoc test (D). The data are presented as mean±SD of the number of the vessel branch points in each group (n=10).

Figure3

Micrographs show vessel branch points in the control vehicle-treated chicken chorioallantoic membrane (CAM)(A) and analyzed micrograph (A´), CAM treated with 0.15 µmol of lithium chloride (LiCl) (B) and analyzed micrograph (B´), and CAM treated with 0.60 µmol of LiCl (C) and analyzed micrograph (C´). The number of the vessel branch points was counted in each group and analyzed using one-way ANOVA, followed by the Tukey post hoc test (D). The data are presented as mean±SD of the number of the vessel branch points in each group (n=10).

Figure4

Micrographs show vessel branch points in the control vehicle-treated chicken chorioallantoic membrane (CAM) (A) and analyzed micrograph (A´), CAM treated with 0.02 µmol of celecoxib (CX)(B) and analyzed micrograph (B´), and CAM treated with 0.08 µmol of CX (C) and analyzed micrograph (C´). The number of the vessel branch points was counted in each group and analyzed using one-way ANOVA, followed by the Tukey post hoc test (D). The data are presented as mean±SD of the number of the vessel branch points in each group (n=10).

Figure5

Micrographs show vessel branch points in the Li (0.60 µmol)+VPA (3.6 µmol)-treated chicken chorioallantoic membrane (CAM)(A) and analyzed micrograph (A´), CAM treated with Li (0.60 µmol)+CX (0.08 µmol) (B) and analyzed micrograph (B´), and CAM treated with VPA (3.6 µmol)+CX(0.08 µmol) (C) and analyzed micrograph (C´). The number of the vessel branch points was counted in each group and analyzed using one-way ANOVA, followed by the Tukey post hoc test (D). The data are presented as mean±SD of the number of the vessel branch points in each group (n=10).