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Several studies have been conducted to find suitable combinations of drugs to increase the efficacy of chemotherapy and reduce the resistance of tumor cells to treatment. Lipopolysaccharide (LPS), as a ligand for Toll-like receptor 4 (TLR-4), can modify immune responses in different cancers. Although multiple studies have been performed in this area, the effect of LPS on tumor cells remains controversial. In the present study, the cytotoxic effects of 5-fluorouracil (5-FU), with or without LPS, were evaluated in human breast cancer cell line (MCF-7) on apoptosis and gene expression in downstream signaling pathways. MCF-7 was obtained from the Pasteur Institute of Iran. The effects of LPS and 5-FU on cytotoxicity, apoptosis, and gene expression in NF-κB, ERK, and AKT signaling pathways were evaluated by MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and qRTPCR, respectively. Our findings showed that LPS alone did not significantly affect cytotoxicity or apoptosis, compared to the control cells (untreated cells), while combined with 5-FU, it caused a significant increase in the apoptosis of cancer cells and decreased cell viability. It was also concluded that LPS in combination with 5-FU increased TLR-4 expression and downregulated gene expression in NF-κB, ERK, and AKT pathways (p=0.001). Although the role of LPS in tumor inhibition or progression remains controversial, our findings suggest that LPS can be considered a novel complementary approach intranslational oncology research of breast cancer therapy. [ABSTRACT FROM AUTHOR]