Effect of Akt on the function of esophageal squamous cancer cell Eca109 and the expression of vasculogenic mimicry-related genes

Abstract

To investigate the inhibitory effect of siRNA targeting Akt on the biological behavior of esophagus squamous cell carcinoma cell line in vitro and to explore the relationship between Akt and vasculogenic mimicry (VM)-related genes in esophageal squamous cell carcinoma. The plasmid-harboring small interfering RNA targeting Akt was introduced into Eca109 cells by liposome-mediated transfection. The proliferation of Eca109 cell was determined by colony formation assay. The cellular migration was evaluated by Transwell migration assay. And three dimensional cell culture was employed to observe and count the number of capillary structure for each cell group. Flow cytometry (FCM) was used to detect the apoptotic rate of Eca109, Eca109/Neo and Eca109/siRNA Akt cells under normoxia exposure. The apoptotic rate was assessed by Annexin V/7-AAD double labeling. And the expressions of Akt and matrix metalloproteinase-2 (MMP-2) protein were detected by Western blotting. The results of Western blotting showed that the expression of Akt in stably transfected group were significantly lower than empty carrier and untransfected groups (0.03 ± 0.01 vs 1.49 ± 0.39 and 1.47 ± 0.41, both P < 0.05). Transwell migration assay showed that fewer Eca109/8 cells could move through the artificial basement membrane as compared with untransfected and empty carrier groups (48 ± 9 vs 128 ± 10 and 122 ± 11, both P < 0.05). Clone formation number of stably transfected group was significantly lower than empty carrier and untransfected groups (63 ± 7 vs 148 ± 11 and 163 ± 15, both P < 0.05). Annexin V/7-AAD double standard method demonstrated that the apoptotic rate of stably transfected group was much more than those of untransfected and empty carrier groups (12.2% ± 1.6% vs 4.8% ± 0.8% and 4.2% ± 0.8%, both P < 0.05). Eca109 and Eca109/Neo cells were capable of forming the in vitro structures of VM. And the number of tube-shaped structure in stably transfected group was markedly less than those of untransfected and empty carrier groups (14.0 ± 1.2 vs 30.0 ± 1.2 and 27.7 ± 1.5, both P < 0.05). MMP-2 protein expression in stably transfected group was less than those of untransfected and empty carrier groups (both P < 0.05). The PI(3)K/Akt pathway is involved in the regulation of VM formation in esophageal squamous cell carcinoma through the action of MMP-2. Blockade of this pathway may provide a new therapeutic approach to human esophagus squamous cell carcinoma.