Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 23 2 2024 04 07 220 230 Zohreh Mehmandoostli Department of Biology, Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran Mahmood Dehghani Ashkezari Department of Biology, Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran Seyed Morteza Seifati Department of Biology, Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran Vida Sadeghi Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran AND Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Reza Falak Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran AND Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Gholam Ali Kardar Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran AND Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 2023 05 14 2024 01 02 During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-β and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and β-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of β-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-β in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/3834 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/3834/2036