Genistein enhances TLR3-mediated apoptosis and immune signaling in breast cancer cells

Breast cancer is one of the most common malignant tumors globally and the second leading cause of cancer-related death in women. Toll-like receptors (TLR) constitute a family of transmembrane receptors playing a crucial role in innate immunity. TLR3 is a type of TLR that is activated following Poly (I:C) double-stranded RNA binding. TLR3 activation leads to tumor suppression, and TLR3 directly causes apoptotic effects in cancer cells. Genistein (GEN), a phytoestrogen found in soy, inhibits cellular proliferation, induces apoptosis, and arrests the cell cycle. Therefore, it is important to determine the roles of immunotherapeutic agents targeting TLR3 in cancer treatment. The study aimed to determine the anti-inflammatory effect of GEN on breast cancer cells for the first time. The anti-inflammatory effects of GEN on the TLR3 signaling pathway were evaluated using Annexin V and cell cycle analysis, immunofluorescence assay, acridine orange staining, Western blotting, and ELISA cytokine release level in MCF-7 (hormone-dependent) and MDA-MB-231 (triple negative) breast cancer cells. The GEN alone treatment increased apoptosis, cell cycle arrest, apoptotic cell morphology, and the expression of TLR3, IRF3, AP-1, and p-NF-kB proteins. Additionally, higher levels of INF-β and TNF-α in both cells compared to treatment with Poly I:C alone were detected. These effects were more pronounced in MCF-7 cells than in MDA-MB-231 cells. Stimulation of the TLR3 signaling pathway was enhanced in the presence of GEN, leading to increased apoptosis.