The anti-proliferative activity and molecular mechanisms of action of gut microbial metabolites against human breast adenocarcinoma cell lines in vitro

Abstract

The complex and diverse interplay between gut microbiota and cancer has been an evolving area in oncology research. The gut microbiota undergoes metabolic processes to produce metabolites as by-products which play a vital role in the overall maintenance of health and homeostasis within the body, as well as in the prevention of infections and diseases. However, there is limited evidence supporting the use of gut microbial metabolites as anticancer agents and their molecular mechanisms of action have been largely unknown. Therefore, this study aimed to identify the anticancer potential of three key gut microbial metabolites- sodium butyrate, inosine, and nisin, against two breast adenocarcinoma cell lines, and determine the underlying molecular pathways using cellular, molecular and proteomics studies. In assessing the anti-proliferative effects using the Alamar Blue assay, sodium butyrate exhibited a promising level of cytotoxicity with IC50 values of 576 μg/mL (5.23 mM) and 557 μg/mL (5.06 mM) against the MCF7 and the MDA-MB-231 cells, respectively. Inosine displayed an IC50 value of 2743 μg/mL (10.23 mM) against the MCF7 cells and weak activity against the MDA- MB-231 cells. Nisin presented with marginal activity against both breast cancer cell lines, with a maximum inhibitory effect of 24.46% for the MDA-MB-231 cells, and 14.34% for the MCF7 cells at the highest tested concentration of 3000 μg/mL (894.44 μM). However, all three metabolites were found to induce apoptotic cell death in both cell lines in the flow cytometric analyses compared to the negative control. Furthermore, these metabolites inhibited the production of reactive oxygen species (ROS) in both the MCF7 and MDA-MB-231 cell lines. To understand the differential expression of key proteins in the MCF7 cells after treatment with the three metabolites, proteomics studies were performed using label-free bottom-up quantification via nano-ultra high-performance liquid chromatography coupled with quadruple time of flight mass spectrometry. Both nisin and inosine triggered the activation of cell cycle processes. Cell cycle arrest is a therapeutic trait of many anticancer drugs. Sodium butyrate primarily initiated signal transduction processes and cellular responses to stimuli within the MCF7 cells, including regulation of HSF1-mediated heat shock response and inducing estrogen-dependent gene expression. Sodium butyrate followed by inosine was found to be the most effective gut metabolite in inhibiting cell proliferation and ROS production and initiating apoptotic cell death in the present study. As such, the findings of the present study warrant further investigation to ascertain the effective therapeutic dose of these gut metabolites and their mechanisms of action in vivo. Future research on different breast cancer cell lines and patient-derived tumour spheroids will provide insights into the potential use of these gut metabolites in cancer therapy.

Date of Thesis submission	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Deep Bhuyan (Supervisor)