Anti-angiogenic effect of high doses of ascorbic acid

Nina A Mikirova, Thomas E Ichim, Neil H Riordan, Nina A Mikirova, Thomas E Ichim, Neil H Riordan

Abstract

Pharmaceutical doses of ascorbic acid (AA, vitamin C, or its salts) have been reported to exert anticancer activity in vitro and in vivo. One proposed mechanism involves direct cytotoxicity mediated by accumulation of ascorbic acid radicals and hydrogen peroxide in the extracellular environment of tumor cells. However, therapeutic effects have been reported at concentrations insufficient to induce direct tumor cell death. We hypothesized that AA may exert anti-angiogenic effects. To test this, we expanded endothelial progenitor cells (EPCs) from peripheral blood and assessed, whether or not high dose AA would inhibit EPC ability to migrate, change energy metabolism, and tube formation ability. We also evaluated the effects of high dose AA on angiogenic activities of HUVECs (human umbilical vein endothelial cells) and HUAECs (human umbilical arterial endothelial cells). According to our data, concentrations of AA higher than 100 mg/dl suppressed capillary-like tube formation on Matrigel for all cells tested and the effect was more pronounced for progenitor cells in comparison with mature cells. Co-culture of differentiated endothelial cells with progenitor cells showed that there was incorporation of EPCs in vessels formed by HUVECs and HUAECs. Cell migration was assessed using an in vitro wound healing model. The results of these experiments showed an inverse correlation between AA concentrations relative to both cell migration and gap filling capacity. Suppression of NO (nitric oxide) generation appeared to be one of the mechanisms by which AA mediated angiostatic effects. This study supports further investigation into non-cytotoxic antitumor activities of AA.

Figures

Figure 1
Figure 1
Effect of high doses of ascorbic acid on capillary tube formation by endothelial progenitor cells. Formation of capillary tube structure by EPCs in control well (a) and in well treated by 3 mg/ml of ascorbic acid (b).
Figure 2
Figure 2
Effect of high doses of ascorbic acid on capillary tube formation by mature endothelial cells. Capillary tube formation by HUVECs in control well without addition of ascorbate (a) and in well treated by 3 mg/ml of ascorbic acid (b).
Figure 3
Figure 3
Ascorbic acid attenuates tube formation in HUVECs, HUAECs and EPCs. Average data for three cell lines treated by different concentrations of AA during 3–6 hrs. Number of intact loops in wells treated by ascorbic acid was normalized on the number of intact loops in control wells.
Figure 4
Figure 4
Effect of high doses of ascorbic acid on endothelial cell migration. Wound was created by sterile plastic scraper and width of gap was measured after 5 hrs and 8 hrs. The ability of cell migration was calculated as the ratio of the gaps after five and eight hours of the cells' treatment by different concentrations of AA to the initial width of the gap.
Figure 5
Figure 5
Effect of NOS inhibitor L-NAME on capillary formation by endothelial cells. Comparison of the capillary tube structure for endothelial cells treated by 2 mM of nitric oxide synthase inhibitor (b) with control well (a).
Figure 6
Figure 6
Nitric oxide inhibitor attenuates formation of capillary network on Matrigel by endothelial cells. Dependence of the number of closed loops formed by HUVECs on the concentration of NO inhibitor.

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