Investigation of the anti-tumor effect of chrysin on breast cancer cells through immunomodulatory pathways

Chrysin (5,7-dihydroxyflavone) is a flavonoid naturally found in plants and bee products, recognized for its significant biological activities, particularly its anti-cancer properties. These beneficial effects are partly attributed to its ability to activate immune cells. The primary aim of this study is to investigate how chrysin modulates the activities of natural killer (NK-92) cells, Jurkat T cells, and RAW 264.7 macrophages, and their anti-tumoral potentials against two different breast cancer cell lines, MCF-7 and MDA-MB-231. The second objective is to assess the effects of chrysin on the expression of programmed death-ligand 1 (PD-L1), a protein involved in immune evasion, as well as the expression of survival proteins p-akt and p-mTOR in breast cancer cells. Firstly, the anti-proliferative effects of chrysin on breast cancer and normal breast cell lines, its reactive oxygen species (ROS) production activity, and apoptotic effects were measured using MTT assay, DCF-DA fluorescent probe with immunofluorescence (IF) staining, flow cytometry, and acridine orange/ethidium bromide (AO/EB) double staining, as well as Annexin V/PI staining for IF staining and flow cytometry analyses. Proliferation of NK-92, Jurkat T cells, and RAW 264.7 macrophages was analyzed by WST-1 assay. The activity of NK-92 cells was evaluated through CD56+ surface protein expression analysis via flow cytometry in the presence of IL-2, and IFN-γ production was measured spectrophotometrically by enzyme-linked immunosorbent assay (ELISA). Changes in the activity of PHA-M-induced Jurkat T cells were determined by ELISA. Activation of the macrophage cell line was analyzed through measurements of TNF-α and nitric oxide (NO) levels using ELISA and Griess reagent. Chrysin-treated NK-92 cells were directly co-cultured with breast cancer cells; cancer cell viability was measured by MTT assay, and the amounts of cytokines and granzyme-B in supernatants were measured using ELISA, while NO levels were measured using Griess reagent. Chrysin-exposed breast cancer cells were co-cultured directly with PHA-M-stimulated Jurkat T cells; cytokine levels in the supernatants were determined by ELISA. Macrophage cells treated with chrysin were co-cultured with breast cancer cell supernatants; the apoptosis level in breast cancer cells was demonstrated using AO/EB double staining and Alexa-fluor-488-coupled caspase-3 measurement by IF method. PD-L1 protein levels in EGF-induced breast cancer cells were analyzed by IF staining and western blotting, while the expression of p-Akt and p-mTOR proteins in breast cancer cells was evaluated by western blotting. Chrysin exhibited cytotoxic and apoptotic activity in MCF-7 and MDA-MB-231 cells by increasing intracellular ROS production. Moreover, it significantly enhanced the cytotoxicity of NK-92 and T cells against both breast cancer cell lines, with the most prominent effect observed in MCF-7 cells (20%). This cytotoxic effect was further increased in the presence of IL-2 and PHA-M but decreased in EGF-treated breast cancer cells. The anti-tumoral response of NK-92 cells against breast cancer cell lines was associated with increased production of granzyme-B, TNF-α, and NO. Similarly, the activation of Jurkat T cells against breast cancer cell lines was characterized by increased production of granzyme-B, IL-2, and IFN-γ. The anti-cancer response of macrophage cells was related to increased apoptosis in breast cancer cells. Furthermore, chrysin-stimulated NK-92 cells were characterized by increased IFN-γ production and CD56 expression, Jurkat T cells by increased IL-2 production, and RAW 264.7 macrophages by increased NO production. In two different breast cancer cell lines, the PD-L1 and p-Akt protein levels elevated by EGF were inhibited by chrysin, while no significant increase in p-mTOR protein expression was observed. The findings suggest that chrysin significantly contributes to the activation and functional enhancement of NK-92, T, and macrophage cells against two different breast cancer cell lines. It also supports the potential use of chrysin as both a therapeutic and chemopreventive agent in cancer treatment by reducing PD-L1and p-Akt levels, which play a role in cancer progression. Keywords: Chrysin, NK cell, T cell, Macrophage, Breast Cancer, Immune Checkpoint Proteins