The Evaluation of the N-cadherin Promoter’s ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines

Zohreh Mehmandoostli; Mahmood  Dehghani Ashkezari; Seyed Morteza Seifati; Vida Sadeghi; Reza Falak; Gholam Ali Kardar

doi:10.18502/ijaai.v23i2.15327

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
Reza Falak Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran
Gholam Ali Kardar Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ijaai.v23i2.15327

Keywords: Cancer; Diphtheria toxin; Epithelial-mesenchymal transition; N-cadherin; Promoter

Abstract

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.