Involvement of Akt1/protein kinase Balpha in tumor conditioned medium-induced endothelial cell migration and survival in vitro

Endothelial cell migration and survival might be called “major angiogenic responses”. 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 tumor CM are not fully understood. Here, we examined the effects of the CM from human lung carcinoma cell lines A549 and SPC-A-1 on cultures of primary human umbilical veins endothelial cells (HUVECs). 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. Phosphorylation of Akt was assessed by Western blotting. To dissect the direct role of Akt, small interfering RNA (siRNA) against Akt1 was used. Both A549 and SPC-A-1 CM significantly stimulated cell migration. However, only A549 CM promoted cell viability and inhibited low serum-induced apoptosis and cell death of HUVECs, but SPC-A-1-CM showed no effects on survival of HUVECs. Meanwhile, A549 CM was found to be able to induce much more phosphorylation of Akt compared to SPC-A-1 CM treated group. The inhibitor of PI3K (wortmaninn) or Akt1 siRNA blocked A549 CM-induced migration and survival of HUVECs. These 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 this process, which may provide a therapeutic target for decreasing tumor angiogenesis.