Introduction

• Angiogenesis is the formation of new blood cells that sprout from pre-existing ones. • Essential for a tumor to grow larger then 1-2 mm, which is easily removed with surgery. • Two key proteins Vascular Endothelial Growth Factor (VEGF) and Basic Fibroblast Growth Factor (bFGF) are key to the pathway of angiogenesis because of their ability to induce blood vessel formation.

Figure 1. Angiogenesis in direction of a tumor

•Thalidomide has anti-angiogenic and anti-inflammatory capabilities. •Originally used to treat morning sickness but caused birth defects in unborn fetuses.• Anti-angiogentic compounds as treatment for a tumor have advantages such as a decrease in tumor size, they are less toxic than traditional anti-cancer agents, and do no need to breach the blood-brain barrier.

Objective

To screen thalidomide analogs for anti-angiogenic activity for subsequent use in mechanistic studies.

Abstract

Angiogenesis is the growth of new blood vessels from pre-existing ones. This is a key process in the growth of cancerous tumors. Thalidomide has been discovered to have the ability to inhibit angiogenesis. Using a Chorioallantoic Membrane Assay (CAM) protocol as a model, chicken eggs were used to stimulate the angiogenesis process. Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF), key proteins in the angiogenesis pathway, were used to stimulate it. First, a system was established to test the anti-angiogenic ability of thalidomide. This was then followed by a screening of different analogs of thalidomide using the same system. The follow up study was to determine if any of the analogs were good candidates for further mechanistic study. Analogs are blinded screens to minimize bias. This research can be used to further study the possibility of thalidomide and anti-angiogenic compounds as a treatment for cancer.

Methods

White Longhorn Chicken Eggs incubated 10 days

Treated with PBS and VEGF and bFGF and incubated 3 more days to stimulate Angiogenesis

Treated with Thalidomide or other anti-angiogenic compound

Treated portion of the chorioallantoic membrane (CAM) was cut off and fixed

in a 5mL 4% paraformaldehyde in 12.5 uL triton X-100

Figure 2. CAM Assay of a developing chicken embryo.

CAMs were imaged and the number of blood vessels were counted and an average of blood

vessels for each CAM sample was recorded

Figure 3a. Embryos treated on a day 10 with 25 ul of VEGF/bFGF solution on micro disks on the CAM. 3b. Certain CAMs treated 3 days later with 25 ul of thalidomide . CAMs isolated 2 days later, fixed in 4% paraformaldehyde, then imaged using a dissecting microscope.

Results

Figure 4a.Effects of thalidomide on VEGF/FGF stimulated angiogenesis on a CAM assay. Control is untreated CAM. Imaged CAMs were magnified, a grid of 8 x 4 squares was transposed over the image and the blood vessels in each square were counted. Average was taken of 7 squares best representing the entire image.

Figure 4b. Effects of thalidomide on VEGF/FGF stimulated angiogenesis on CAM assay. Control is untreated, SU 416 control treated only with SU 416, SU 416 treated with VEGF/FGF and SU 416. Results were obtained same as above.

Conclusions

1.The CAM assay is a good in vivo system to screen anti-angiogenic potential of compounds since thalidomide produced expected results (Figure 4a.).2.Analogs of thalidomide have different anti-angiogenic potential (Figure 4b). We looked for a 50% reduction in angiogenesis.3.Based on the data, WL722 and XZ424 showed a 50% or more reduction and will be followed up with a mechanistic study.

Works Cited

Acknowledgements

A very special thank you to Dr. Kaltreider for all his help and assistance to me during this process. Thank you to Dr. Ihnat and his lab in Oklahoma for all the assistance. Also, thank you to all the YCP biology faculty for their assistance in the absence of Dr. K.

Tumor

1. Bauditz, J., Schachschal, G., Wedel, S., Lochs, H. 2004. Thalidomide for treatment of severe intestinal bleeding. GUT Online. 53:609-612. Available from: GUT Online.

2. D’Amato, R., Loughnan, E. F., Folkman, J. 1994. Thalidomide is an inhibitor of Angiogenesis. Proceedings of the National Academy of Sciences. 91: 4082-4085. Available from: High- Wire Press.

3. Eleutherakis-Papaiakovou, V., Bamias, A., Dimopoulos, M. A. 2004. Thalidomide in Cancer Medicine. Annals of Oncology. 15:1151-1160. Available from: High-Wire Press.

4. Danser, PA; Thorpe, JE; Shenoy, SS; Warnke, LW; Dandajena, TC; Ihnat, MA. Arsenic at the Drinking Water MCL of 10 PPB Triggers the Angiogenic “Switch” in Skin Tumors. University of Oklahoma.

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Figure 4b.

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Figure 4a.