Combination of 5-fluorouracil and Lipopolysaccharide Synergistically Induces Cytotoxicity and Apoptosis in MCF-7 Human Breast Cancer Cells

  • Negin Nokhandani Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran AND Department of Immunology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
  • Mahdieh Naghavi Alhosseini Department of Immunology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
  • Ali Memarian Department of Immunology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
  • Homa Davoodi Mail Cancer Research Center, Golestan University of Medical Sciences, Gorgan, Iran
Breast cancer‏, Cytotoxicity, Lipopolysaccharides, Toll-like receptor 4, 5-Fluorouracil


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 qRT-PCR, 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 down-regulated 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.


1. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, et al. Global cancer observatory: cancer today. IARC Publ. 2018.
2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.
3. Hansen R, Quebbeman E, Beatty P, Ritch P, Anderson T, Jenkins D, et al. Continuous 5-fluorouracil infusion in refractory carcinoma of the breast. Breast Cancer Res Treat. 1987;10(2):145-9.
4. Tobias J. Current role of chemotherapy in head and neck cancer. Drugs. 1992;43(3):333-45.
5. Iacopetta B, Kawakami K, Watanabe T. Predicting clinical outcome of 5-fluorouracil-based chemotherapy for colon cancer patients: is the CpG island methylator phenotype the 5-fluorouracilresponsive subgroup? Int J Clin Oncol. 2008;13(6):498-503.
6. Bernhard H, Jäger-Arand E, Bernhard G, Heike M, Klein O, Riemann J, et al. Treatment of advanced pancreatic cancer with 5-fluorouracil, folinic acid and interferon alpha-2A: results of a phase II trial. Br J Cancer. 1995;71(1):102.
7. Marquette C, Nabell L. Chemotherapy-resistant metastatic breast cancer. Curr Treat Options Oncol. 2012;13(2):263-75.
8. Ahmed A, Redmond HP, Wang JH. Links between Toll-like receptor 4 and breast cancer. Oncoimmunology. 2013;2(2):e22945.
9. Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388(6640):394.
10. Tomalka JA, De Jesus TJ, Ramakrishnan P. Sam68 is a regulator of Toll-like receptor signaling. Cell Mol Immunol. 2017;14(1):107.
11. Merrell MA, Ilvesaro JM, Lehtonen N, Sorsa T, Gehrs B, Rosenthal E, et al. Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity. Mol Cancer Res. 2006;4(7):437-47.
12. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer. Nat Rev Cancer. 2009;9(1):57-63.
13. Tsan MF, Gao B. Endogenous ligands of Toll‐like receptors. J Leukoc Biol. 2004;76(3):514-9.
14. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140(6):883-99.
15. Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol. 2006;117(5):979-87.
16. Beutler B, Du X, Poltorak A. Identification of Toll-like receptor 4 (Tlr4) as the sole conduit for LPS signal transduction: genetic and evolutionary studies. J Endotoxin Res. 2001;7(4):277-80.
17. Rakoff-Nahoum S, Medzhitov R. Toll-like receptors and cancer. Nat Rev Cancer. 2009;9(1):57.
18. Murad S. Toll-like receptor 4 in inflammation and angiogenesis: a double-edged sword. Front Immunol. 2014;5:313.
19. Lee MS, Kim Y-J. Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu Rev Biochem. 2007;76:447-80.
20. Garay RP, Viens P, Bauer J, Normier G, Bardou M, Jeannin J-F, et al. Cancer relapse under chemotherapy: why TLR2/4 receptor agonists can help. Eur J Pharmacol. 2007;563(1-3):1-17.
21. Okamoto H, Shoin S, Koshimura S, Shimizu R. Studies on the anticancer and streptolysin S-forming abilities of hemolytic streptococci. Jpn J Microbiol. 1967;11(4):323-36.
22. Otto F, Schmid P, Mackensen A, Wehr U, Seiz A, Braun M, et al. Phase II trial of intravenous endotoxin in patients with colorectal and non-small cell lung cancer. Eur J Cancer. 1996;32(10):1712-8.
23. Harmey JH, Bucana CD, Lu W, Byrne AM, McDonnell S, Lynch C, et al. Lipopolysaccharide‐induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion. Int J Cancer. 2002;101(5):415-22.
24. Jagavelu K, Routray C, Shergill U, O'Hara SP, Faubion W, Shah VH. Endothelial cell toll‐like receptor 4 regulates fibrosis‐associated angiogenesis in the liver. Hepatol. 2010;52(2):590-601.
25. Liu X, Liang J, Li G. Lipopolysaccharide promotes adhesion and invasion of hepatoma cell lines HepG2 and HepG2. 2.15. Mol Biol Rep. 2010;37(5):2235-9.
26. Davoodi H, Hashemi SR, Seow HF. 5-Fluorouracil induce the expression of TLR4 on HCT116 colorectal cancer cell line expressing different variants of TLR4. Iran J Pharm Res. 2013;12(2):453.
27. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.
28. Gessner PK. Isobolographic analysis of interactions: an update on applications and utility. Toxicol. 1995;105(2-3):161-79.
29. Chou T-C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27-55.
30. Chou T-C, Talalay P. Analysis of combined drug effects: a new look at a very old problem. Trends Pharmacol Sci. 1983;4:450-4.
31. Edwardson DW, Boudreau J, Mapletoft J, Lanner C, Kovala AT, Parissenti AM. Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance. PloS one. 2017;12(9):e0183662.
32. Chicoine MR, Zahner M, Won EK, Kalra RR, Kitamura T, Perry A, et al. The in vivo antitumoral effects of lipopolysaccharide against glioblastoma multiforme are mediated in part by Toll-like receptor 4. Neurosurg. 2007;60(2):372-81.
33. Chung YH, Kim D. Enhanced TLR4 expression on colon cancer cells after chemotherapy promotes cell survival and epithelial–mesenchymal transition through phosphorylation of GSK3β. Anticancer Res. 2016;36(7):3383-94.
34. Yang H, Wang B, Wang T, Xu L, He C, Wen H, et al. Toll-like receptor 4 prompts human breast cancer cells invasiveness via lipopolysaccharide stimulation and is overexpressed in patients with lymph node metastasis. PloS one. 2014;9(10):e109980.
35. Mehmeti M, Allaoui R, Bergenfelz C, Saal LH, Ethier SP, Johansson ME, et al. Expression of functional toll like receptor 4 in estrogen receptor/progesterone receptor-negative breast cancer. Breast Cancer Res. 2015;17(1):130.
36. Yang H, Zhou H, Feng P, Zhou X, Wen H, Xie X, et al. Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion. J Exp Clin Cancer Res. 2010;29(1):92.
37. Hsu RY, Chan CH, Spicer JD, Rousseau MC, Giannias B, Rousseau S, et al. LPS-induced TLR4 signaling in human colorectal cancer cells increases β1 integrin-mediated cell adhesion and liver metastasis. Cancer Res. 2011;71(5):1989-98.
38. Li J, Yin J, Shen W, Gao R, Liu Y, Chen Y, et al. TLR4 Promotes Breast Cancer Metastasis via Akt/GSK3β/β‐Catenin Pathway upon LPS Stimulation. Anat Rec. 2017;300(7):1219-29.
39. Tang X-Y, Wang H, Zhu Y-Q, Wei B. Expression and functional research of TLR4 in human colon carcinoma. Am J Med Sci. 2010;339(4):319-26.
40. Kang MH, Oh SC, Lee HJ, Kang HN, Kim JL, Kim JS, et al. Metastatic function of BMP-2 in gastric cancer cells: the role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression. Exp Cell Res. 2011;317(12):1746-62.
41. Han D, Wu G, Chang C, Zhu F, Xiao Y, Li Q, et al. Disulfiram inhibits TGF-β-induced epithelial-mesenchymal transition and stem-like features in breast cancer via ERK/NF-κB/Snail pathway. Oncotarget. 2015;6(38):40907.
42. Zhao Y, Sharfman NM, Jaber VR, Lukiw WJ. Down-regulation of essential synaptic components by GI-tract microbiome-derived lipopolysaccharide (LPS) in LPS-treated human neuronal-glial (HNG) cells in primary culture; relevance to Alzheimer's disease (AD). Front Cell Neurosci. 2019;13:314.
How to Cite
Nokhandani N, Naghavi Alhosseini M, Memarian A, Davoodi H. Combination of 5-fluorouracil and Lipopolysaccharide Synergistically Induces Cytotoxicity and Apoptosis in MCF-7 Human Breast Cancer Cells. Iran J Allergy Asthma Immunol. 19(4):424-434.
Original Article(s)