Different Concentrations of Produced Recombinant TNF-α Protein Caused Different Cellular Viability in Breast Cancer Cell Lines

Abstract

Background: Breast cancer is a prevalent malignancy among women globally. The implementation of early detection methods and improved treatment strategies have shown a considerable decrease in the financial burden associated with breast cancer mortality. One of the inflammatory cytokines is tumor necrosis factor (TNF), produced during chronic inflammation, apoptosis, tumor proliferation, and angiogenesis. The main objectives of this study were to produce recombinant TNF-α protein and assess its impact on the MCF-7 and MDA-MB-468 cancer cell lines. Methods: The present study employed the SHuffle® T7 express strain of E. coli to synthesize recombinant TNF-α protein, primarily due to its advantageous characteristics of cost-effectiveness and high productivity. The molecular weight of the protein was determined using SDS-PAGE and Coomassie blue staining techniques, and its presence was subsequently verified through Western blot analysis. Subsequently, the cells were treated with the produced recombinant protein, and the cellular viability was assessed using the MTT assay. Then, the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) technique was employed to quantify the expression level of the Caspase-3 gene, known as an apoptotic factor, and the interleukin-1 (IL-1) gene, recognized as a pro-inflammatory factor, in both cell lines. Results: Lower quantities of recombinant TNF-α protein were shown to cause cellular cytotoxicity, according to the findings of the MTT test, while cellular proliferation was observed at increased levels of recombinant TNF-α protein. The results of the qRT-PCR test exhibited a lack of identifiable patterns in both cell lines. Conclusion: The findings indicate that cellular viability is significantly decreased at a minimal concentration of 5-10 ng/mL of TNF-α, whereas it is noticeably elevated at concentrations higher than 10 ng/mL. Furthermore, gene expression analysis revealed a significant reduction in IL-1 expression in both cell lines after administering a high dose of TNF-α.