Akt1-Dependent Pathways Involved in the Regulation of Tumor Conditioned Medium -Induced Endothelial Cell Migration and Survival

Purpose: Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary- like structures in angiogenesis models in vitro. However, the molecular events triggered by CM are not fully understood. Here, we aimed to examine the effects of CM from human lung carcinoma A549 on cultures of primary human umbilical veins endothelial cells (HUVECs). Methods: After treatment of HUVECs with the CM, cell migration was assessed by wound-healing assay, cell viability was evaluated by XTT assay, and apoptosis and cell death of HUVECs was analyzed by flow cytometry. Akt activation was assessed by Western blotting. To dissect the direct role of Akt, small interfering RNA (siRNA) against Akt1 was used. Results: The CM significantly stimulated cell migration and promoted cell viability. The CM also significantly inhibited low seruminduced apoptosis and cell death of HUVECs. Moreover, addition of CM activated the PI3K-Akt pathway: the level of p-Akt was increased by CM, and both the increase in p-Akt and the increase in migration and survival were blocked by an inhibitor of PI3K (wortmaninn) or by special knock-down Akt1. Conclusion: The results indicated that the angiogenic effects of A549-CM are largely mediated through activation of the PI3K-Akt in endothelial cells, and that the Akt1 is crucial in such a process, which may provide a therapeutic strategy of decreasing tumor angiogenesis.