Adenovirus-mediated Pigment Epithelium-derived Factor Inhibited Human Esophageal Carcinoma Cells And Human Umbilical Vein Endothelial Cells In Vitro

Objectives: Pigment epithelium-derived factor(PEDF) is pluripotent inboth anti-tumor effects and antiangiogenesis. In this study, we investigated thebiological activities of PEDF in both esophageal carcinoma cell lines-109(ECa109)and human umbilical vein endothelial cells(HUVECs) in vitro, andhope to explore a new approach of gene therapy for esophageal cancer and topromote the translational medical research on PEDF as well.Methods: The adenoviruses encoding PEDF (Ad-PEDF) were amplifiedin Human Embryonic Kidney293T cells, the lysate of the cells was collected,and the titers of viruses in the lysate were measured according to the TCID50principle. The293T cells and ECa109cells and HUVECs were infected withthe amplified Ad-PEDF, and the expression of PEDF in both cell lines wasdetermined by RT-PCR and Western-Blot. The Transwell tests were carriedout to investigate the retardation of invasion and migration of ECa109cellsand HUVECs by PEDF. The tests of capillary-like tube formation byHUVECs were acted to assess the inhibitive effects of PEDF on HUVECs.The proliferation and viability tests via CCK8kit were executed to evaluatethe inhibition activities of PEDF on the growth of both ECa109cells andHUVECs. Cell apoptosis assays by Flow Cytometer(FCM) were conducted toevaluate anti-tumor effects of PEDF on ECa109cells.Results: Ad-PEDF recombinants were amplified largely in293Tcells. A target stripe of1.25kbp was observed by Agarose Gel Electrophoresis, and the expression of human PEDF proteins in bothECa109cells and HUVECs infected with Ad-PEDF were comfirmed byWestern blotting. Transwell tests demonstrated that Ad-PEDF group couldsignificantly suppress the invasion and migration of ECa109cells and themigration of HUVECs compared with the Ad-GFP group and the blank group(P<0.01). The neoangiogenisis of HUVECs were significantly restrained byAd-PEDF compared with Ad-GFP group, and the inhibitive effects becamemore prominent with the increased titer of the virus (P<0.05). Theproliferation of ECa109cells and HUVECs were curbed by Ad-PEDF and theproliferation inhibition activity of PEDF on HUVECs was noted (P<0.01).The ECa109cells were infected by Ad-PEDF and Ad-GFP of different titers,the apoptosis rates of ECa109induced by Ad-PEDF or Ad-GFP showed nosignificant difference when the virus titer is1.5×106PFU/ml. But when thevirus titer is over1.5×106PFU/ml, the apoptosis rates of ECa109induced byAd-PEDF were higher than that induced by Ad-GFP, again in a trend of titerdependence (P<0.01). The apoptosis rates of ECa109induced by Ad-GFP ofdifferent titers showed no significant difference.Conclusions: Our data demonstrated that PEDF could suppress theinvasion and migration of ECa109cells, but the incremented ECa109cellapoptosis induced by PEDF was unobvious. The proliferation, migration andcapillary-like tube formation of HUVECs were apparently inhibited by PEDF.Based on the results above we infer that the anti-tumor effects of PEDF inesophageal carcinoma were probably implemented by inhibiting endothelialcells from proliferating and migrating. As the anti-angiogenic and anti-tumouractivities of PEDF display a strong potential in applying PEDF treatment intumor, the development of effectiveness, safety and efficient delivery systems for the use of PEDF in cancer treatment will required more work.