Down regulation of u-PA by a nutrient mixture in hemangioma (EOMA) cells by inducing caspase-dependent apoptosis

Abstract

Hemangiomas are the most common congenital vascular and benign tumor in infants and children. Most hemangiomas do not cause major symptoms to require intervention, however, the larger hemangiomas have tendency to bleed and may require surgical removal. Experimental studies have demonstrated the role of urokinase plasminogen activator (u-PA), especially cell surface u-PA, as an initiator of extra-cellular matrix proteolysis and associated tumor cell invasion. Aim: To examine, whether the antitumor effects of a specific nutrient mixture are due to induction of apoptosis by inhibition of u-PA. Materials and Methods: A nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract which has showed anticancer activity against a number of cancer cell lines was used as an experimental composition. EOMA cells were grown in appropriate media with antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with nutrition mixture at 10, 100, 1000 µg/ml in triplicate. Analysis of u-PA activity was carried out by fibrin zymography. Morphological changes and caspase activation associated with apoptosis induction was checked by H&E staining and Live Green caspase assay, respectively. Apoptosis inducing anticancer drug camptothecin (10 µM) was used as positive control. Results: The nutrition mixture exhibited dose response toxicity with maximum toxicity 55% (p < 0.001) at 1000 µg/ml. EOMA cells expressed u-PA, which was inhibited by nutrition mixture in a dose-dependent manner. The caspase analysis revealed a dose dependent increase in apoptosis of EOMA hemangioma cells, with an increasing apoptosis observed at 100 μg/ml, and maximum at 1000 μg/ml. Cells treated with nutrition mixture showed significantly more apoptotic changes than the control or camptothecin-treated cells. Conclusion: These results suggest that NM may induce apoptosis of hemangioma cells in vitro thus warranting further investigation. Key Words: nutrient mixture, urokinase plasminogen activators, hemangioma, EOMA, caspase, apoptosis.