Targeting the EGFR in cancer cells by fusion protein consisting of arazyme and third loop of TGF-alpha: an in silico study.

The anticancer effects of arazyme, a bacterial metalloprotease, have been revealed in previous studies. Because of the overexpression of epidermal growth factor receptor (EGFR) in tumor cells, targeting this receptor is one of the approaches to cancer therapy. In the present study, we designed fusion protein by using a non-mitogenic binding sequence of TGFα, arazyme, and a suitable linker. The I-TASSER and Robetta web servers were employed to predict the territory structures of the Arazyme-linker-TGFαL3, and TGFαL3-linker-Arazyme. Then, models were validated by using PROCHECK, ERRAT, ProSA, and MolProbity web servers. After docking to EGFR, Arazyme-linker-TGFαL3 showed a higher binding affinity and was selected to be optimized through 100 ns Molecular dynamic (MD) simulation. Next, the stability of ligand-bound receptor was examined utilizing MD simulation for 100 ns. Furthermore, the binding free energy calculation and free energy decomposition were carried out employing MM-PBSA and MM-GBSA methods, respectively. The root mean square deviation and fluctuation (RMSD, RMSF), the radius of gyration, H-bond, and binding free energy analysis revealed the stability of the complex during simulation time. Finally, linear and conformational epitopes of B cells, as well as MHC class I and MHC class II were predicted by using different web servers. Meanwhile, the potential B cell and T cell epitopes were identified throughout arazyme protein sequence. Collectively, this study suggests a novel chimera protein candidate prevent cancer cells potentially by inducing an immune response and inhibiting cell proliferation. Communicated by Ramaswamy H. Sarma.

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells.

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.