Cytotoxic Evaluation of Trastuzumab in HER2-Positive MDA-MB-453 Breast Cancer Cells

Vollständiger Abstract

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This study aims to provide an integrated computational and experimental approach in the development of recombinant trastuzumab with specificity towards the HER2-positive breast cancer cell surface antigen. Using the molecular docking approach with the HADDOCK protocol, the interaction between the antibody and the antigen is shown to be stable through the computation of the docking scores and the low intermolecular RMSD. In addition, the electrostatic and van der Waals components of the interaction are significant. Using the information obtained in the molecular docking studies, a mammalian expression construct of the antibody is generated and is shown to be successfully propagated in Escherichia coli. Using the transformed cells, the recombinant antibody is shown to be successfully produced in ExpiCHO-S™ cells with sustained cell viability. Using affinity chromatography and size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis, the antibody is shown to be highly homogeneous with minimal aggregation and is further confirmed through the analysis of the structure of the antibody using SDSPAGE in reducing and non-reducing conditions. The functional evaluation by MTT assay showed a dosedependent decrease in cell viability in MDA-MB-453 cells, with an IC₅₀ value of ~700 nM. Scratch wound healing assays showed significant inhibition in cell migration whereas AO/EtBr staining and DNA fragmentation assays confirmed apoptosis-mediated cell death. Overall, these results validate the synthesis of high-purity biologically active rTrastuzumab and its potent cytotoxic, anti-proliferative and anti-migratory activities, making it relevant in targeted breast cancer therapy.