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Amphotericin B abolishes the cytotoxic effect of Toll-like receptor 9 agonist CpG Oligonucleotides in breast cancer cell line MCF-7

Suresh Madheswaran, Venil N Sumantran, Sudhakar Natarajan.




Abstract

Background: Toll-like receptors (TLRs) are key sensors of microbial components and triggers signals responsible for the activation of innate immune responses. Unmethylated cytosine-guanosine oligodeoxynucleotides called CpG ODNs act as a ligand for TLR9.

Aims and Objectives: The present study was conducted to determine whether the presence of antimycotic amphotericin B (AmB) used in the culture medium has any profound effect on MCF-7 cells treated with CpG.

Materials and Methods: MCF-7, a breast adenocarcinoma cell line, was cultured and maintained in the media with the absence of AmB (Group 1) and presence of AmB at 0.25 μg/ml (Group 2). The morphology of MCF-7 cells grown in these two groups of media was assessed, and the cytotoxicity of CpG treated in both the groups was evaluated by MTT assay.

Results: Visualizing the monolayer of MCF-7 cells grown in two groups of media revealed identical morphology, growth pattern, and confluence. MCF-7 cells in Group 1 media treated with the CpG ODN at 1, 2, and 3 μM for 24 h showed a dose-dependent cytotoxicity in 13%, 18%, and 22% of the cells, respectively, whereas treatment with CpG ODN at 1, 2, and 3 μM for 48 h demonstrated a better cytotoxicity of 16%, 27%, and 37% of cells, respectively. On the other hand, MCF-7 cells in Group 2 media treated with the CpG ODN at 1, 2, and 3 μM for 24 h and 48 h showed poor cytotoxic effect.

Conclusion: While comparing the effect of CpG ODN in Group 1 (AmB−) versus Group 2 (AmB+) for 24- and 48-h duration in MCF-7 cells evidenced that the response of CpG ODN was significantly different between the two groups (P < 0.01). The results strongly evidenced that the presence of AmB in culture media during the maintenance of cell line shown to decrease the cytotoxic effects of CpG ODN. Hence, the present study suggests to avoid using AmB in cell culture, particularly while conducting in vitro studies related to TLR signaling.

Key words: Cell Culture; Toll-like Receptor; Cytosine-guanosine; Antimycotic Agent; Amphotericin B; Cytotoxic Activity






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