Targeting MCF-7 Cell Line by Listeriolysin O Pore Forming Toxin Fusion with AHNP Targeted Peptide

Fotoohi-Ardakani, Gholamreza; Kheirollahi, Majid; Zarei Jaliani, Hossein; Noorian, Mohadese; Ansariniyia, Hossein

Targeting MCF-7 Cell Line by Listeriolysin O Pore Forming Toxin Fusion with AHNP Targeted Peptide

Authors

¹ Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease and Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan; Department of Medical Genetics, School of Medicine, Protein Engineering Laboratory, Shahidsadoughi University of Medical Sciences, Yazd, Iran

² Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease and Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

³ Department of Medical Genetics, School of Medicine, Protein Engineering Laboratory, Shahidsadoughi University of Medical Sciences, Yazd, Iran

⁴ Department of Medical Genetics, Shahid Sadoughi University of Medical Science, Yazd, Iran

⁵ Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Science, Yazd, Iran

Abstract

Background: Tumor-targeting peptides are attracting subjects in cancer therapy. These peptides, which are widely studied, deliver therapeutic agents to the specific sites of tumors. In this study, we produced a new form of recombinant listeriolysin O (LLO) with genetically fused Anti-HER2/neu peptide (AHNP) sequence adding to its C-terminal end. The aim of the study was to engineer this pore-forming toxin to make it much more specific to tumor cells. Materials and Method and Results: Two forms of the toxin (with and without peptide) were subcloned into a bacterial expression plasmid. Subcloning was performed using a polymerase chain reaction (PCR) product as a megaprimer in a quick-change PCR to introduce the whole insert gene into the expression plasmid. After expression of two recombinant forms of LLO in BL21 DE3 cells, purification was performed using Ni-NTA affinity column. MDA-MB-231 and MCF-7 cell lines (as negative and positive controls, respectively) were treated with both LLO toxins to evaluate their cytotoxicity and specificity. The IC₅₀ of LLO on MDA-MB-231 and MCF-7 cells was 21 and 5 ng/ml, respectively. In addition, IC₅₀ for the fusion AHNP-LLO toxin was 140 and 60 ng/ml, respectively. It was found that the cytotoxicity of the new engineered AHNP-LLO toxin has decreased by about 9x compared to the wild-type toxin and the specificity of the AHNP-LLO toxin has been also reduced. Conclusions: Results show that the C-terminal of the LLO should not be modified and it seems that N-terminal of the toxin should be preferred for engineering and adding peptide modules.

Keywords

References

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