A Study of the Effect of Drug Combinations on Angiogenesis

Journal of Taibah University Medical Sciences. 2023 Mar 8; 18 (5): 1011-1017.

Authors: Qadir A, Samad DA, Asif M, Ali MM, Zain S.

Angiogenesis is defined as the process used to form new blood vessels from pre-existing vasculature. Vandetanib is a multi-kinase inhibitor that inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2, the epidermal growth factor receptor, proto-oncogene RET, and MMPs. Studies have also reported that celecoxib possesses an anti-cancer effect and is also capable of inhibiting angiogenesis by targeting VEGF and MMPs.

• Vandetanib and celecoxib stock solutions were serially diluted in a culture medium to obtain final concentrations of vandetanib (1, 2, and 4 μM) and celecoxib (1.5, 3, and 4.5 μg/mL).
• Isolation and primary culture of human umbilical vein endothelial cells (HUVECs). HUVECs were isolated from umbilical cords using methods described previously. The cells were cultured in EBM-2 media supplemented with 100 U/mL penicillin/streptomycin and 15% FBS.
• An MTT assay (methylthiazolyldiphenyl tetrazolium bromide) was performed to investigate the effect of drugs on the growth of HUVECs. HUVEC apoptosis was examined by flow cytometry using an annexin apoptosis detection kit.
• The process of angiogenesis was demonstrated using a tube formation assay. After the incubation, the effect on the formation of endo-tubes was inspected using a phase contrast microscope, and images were recorded at 10× magnification.
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• HUVECs exhibited typical cobblestone morphology under phase contrast microscopy. The purity of HUVECs was determined by flow cytometry using positive and negative markers. HUVECs were positive for CD144 and negative for CD14.
• When HUVECs were treated with vandetanib, celecoxib, or a combination of both drugs, cell viability decreased with drug concentration, and the rates of apoptosis were 13.1%, 9%, and 23.7%.

Fig. 1 Left: Effect of drugs on HUVEC tube formation; Right: Effect of drugs on angiogenic genes.

• Vandetanib and celecoxib are both effective in inhibiting tube formation, and their combination showed more pronounced inhibition than either drug alone. Analysis showed that vandetanib and celecoxib both reduced the expression levels of tumor angiogenic genes (MMP-2 and MMP-9) and that their combination resulted in a greater extent of reduction in expression levels.

This study demonstrated that the combination of vandetanib and celecoxib exerted a significantly greater inhibitory effect on in vitro angiogenesis than vandetanib alone. The results suggest that the combination of these drugs might be an effective therapeutic option for treating tumors.

Cell Line Testing and Assays

Cell Angiogenesis Assays

3D Angiogenesis Assay

Reference

1. Qadir A, et al. (2023). "Investigating the effect of vandetanib and celecoxib combination on angiogenesis." J Taibah Univ Med Sci. 18 (5), 1011-1017.

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