Stable silencing of dll4 gene suppresses the growth and metastasis of esophagus cancer cells by attenuating Akt phosphorylation

-Like ligand 4 (DLL4) has recently been reported to be involved in the process of cancer angiogenesis and considered to play a vital role in vascular endothelial growth factor (VEGF) signaling, while the role of DLL4 in cancer metastasis and growth has not been systematically studied. In the present study, the esophagus cancer cell line Eca109 was infected in vitro with a lentiviral vector loaded with dll4-shRNA to obtain a stable cell line of DLL4 expression which was downregulated through puromycin screening. The migration and invasion ability of the Eca109 (dll4-shRNA) cells were evaluated by scratch and Transwell assays, respectively. The underlying signaling pathway was further explored by western blotting. Subsequently, to explore the role of dll4 in the development of esophagus cancer cells in vivo, a xenograft model was established by intraperitoneal injection with Eca109 dll4(-shRNA) cells containing luciferase activity in nude mice. Then, small animal imaging system was used to evaluate the volume and metastatic potential of the tumors. Additionally, the overall survival rate of the nude mice was also recorded. Following infection with lentivirus, the expression of DLL4 in the Eca109 cells could be stably silenced through screening with puromycin, which was confirmed by western blotting. The scratch and Transwell assays demonstrated that downregulated DLL4 significantly diminished the aggressive invasion and migration properties of the Eca109 cells. The underlying mechanisms may be attributed to the inactivation of the PI3K/Akt/E-cadherin pathway by western blotting. Finally, the results from the in vivo study indicated that the tumor growth rate in the Eca109 dll4(-shRNA) group, as displayed by the tumor volume and the weak staining of the proliferating cell nuclear antigen (PCNA), was significantly slower than the control group, and the metastasis ability of the Eca109 dll4(-shRNA) cells was also dramatically abolished in vivo. It was also observed that downregulated DLL4 led to the formation of less pulmonary nodules in mice lungs and to a prolonged survival rate of nude mice. In summary, this study revealed that DLL4 has pathophysiological roles on the progression of esophagus cancer cells, including migration, invasion and apoptosis, which indicated that DLL4 may be considered as a potent therapeutic target for the treatment of malignant esophageal cancer.