Mechanistic Insights into the Anti-Proliferative Action of Gut Microbial Metabolites against Breast Adenocarcinoma Cells

Abstract

The gut microbiota undergoes metabolic processes to produce by-products (gut metabolites), which play a vital role in the overall maintenance of health and prevention of disease within the body. However, the use of gut metabolites as anticancer agents and their molecular mechanisms of action are largely unknown. Therefore, this study evaluated the anti-proliferative effects of three key gut microbial metabolites-sodium butyrate, inosine, and nisin, against MCF7 and MDA-MB-231 breast adenocarcinoma cell lines. To determine the potential mechanistic action of these gut metabolites, flow cytometric assessments of apoptotic potential, reactive oxygen species (ROS) production measurements and proteomics analyses were performed. Sodium butyrate exhibited promising cytotoxicity, with IC₅₀ values of 5.23 mM and 5.06 mM against MCF7 and MDA-MB-231 cells, respectively. All three metabolites were found to induce apoptotic cell death and inhibit the production of ROS in both cell lines. Nisin and inosine indicated a potential activation of cell cycle processes. Sodium butyrate indicated the possible initiation of signal transduction processes and cellular responses to stimuli. Further investigations are necessary to ascertain the effective therapeutic dose of these metabolites, and future research on patient-derived tumour spheroids will provide insights into the potential use of these gut metabolites in cancer therapy.

Keywords: breast cancer; gut microbial metabolites; gut microbiome; inosine; nisin; postbiotics; sodium butyrate.

Figure 1

Reactive oxygen species (ROS) inhibitory percentage for nisin, inosine, and sodium butyrate, at 3 and 2 mg/mL, against the MCF7 and MDAMB231 cells after 4 h of treatment (n = 3). * Indicates p-value: 0.01 < p-value < 0.05. ** Indicates p-value ≤ 0.01. For the MCF7 cells, inosine exhibited significantly more ROS inhibition than did sodium butyrate at 3 mg/mL (p < 0.05). For the MDA-MB-231 cells, inosine, at both 3 and 2 mg/mL showed significantly more ROS inhibition (p < 0.05) compared to sodium butyrate at 3 mg/mL.

Figure 2

Flow cytometric assessment of the apoptotic profiles of the MCF7 (a,b) and MDAMB231 (c,d) breast cancer cells after 24 h of treatment with nisin, inosine, and sodium butyrate (n = 3). * indicates 0.01 < p-value < 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001; **** indicates p ≤ 0.0001 compared to the negative control. (a) All three metabolites showed significantly lower live cells and higher apoptotic (early- and late-stage) cells compared to the negative control in the MCF7 cell line. Notably, inosine exhibited the greatest activity among the three metabolites with significantly lower live cells (p < 0.0001) and higher apoptotic (early- and late-stage) cells (p < 0.0001) compared to the negative control. (b) Represented are the density plots of each drug treatment that is most representative of the average data from the flow cytometric analyses, with Q3-1 = necrotic cells, Q3-2 = late-stage apoptotic cells, Q3-3 = live cells, and Q3-4 = early-stage apoptotic cells. (c) All three metabolites showed significantly lower live cells and higher apoptotic (early- and late-stage) cells compared to the negative control in the MDAMB231 cell line. Sodium butyrate exhibited significantly lower live cells (p < 0.05) and greater apoptotic (early- and late-stage) cells (p < 0.01) compared to nisin, inosine and the negative control. (d) The density plots of each drug treatment shown here are the most representative of the average data from the flow cytometric analyses, with Q3-1 = necrotic cells, Q3-2 = late-stage apoptotic cells, Q3-3 = live cells, and Q3-4 = early-stage apoptotic cells.

Figure 3

The STRING network of associated expressed genes in the MCF7 cells following sodium butyrate treatment for 24 h, with the connecting lines depicting significant interactions between multiple genes. The thicker lines indicate a more significant association, whereas the thinner lines are indicative of a less significant association.

Figure 4

The STRING network of associated expressed genes in the MCF7 cells following 24 h of inosine treatment, with the connecting lines depicting significant interactions between multiple genes. The thicker lines indicate a more significant association, whereas the thinner lines are indicative of a less significant association.

Figure 5

The STRING network of associated expressed genes in the MCF7 cells following 24 h of nisin treatment, with the connecting lines depicting significant interactions between multiple genes. The thicker lines indicate a more significant association, whereas the thinner lines are indicative of a less significant association.