Combined Effects of Withaferin A and Sodium Butyrate on NF-κB Signaling and Epigenetic Regulation in Breast Cancer...

Background/Objectives: There is a clear need for more options to control the progression of breast cancer and prevent the occurrence of breast cancer in minority populations that have a higher rate of mortality due to triple-negative breast cancer (TNBC) subtypes. Prevalent nutraceuticals such as Ashwagandha (also known as the Indian Winter Cherry) have anti-inflammatory and apoptotic capabilities, as well as the ability to inhibit cancer growth. The purpose of this study is to analyze the novel combination of withaferin A (derived from the Indian Winter Cherry and known to have histone deacetylase inhibition capabilities) and sodium butyrate (a short-chain fatty acid produced from the gut microbiome and known to have DNA methyltransferase inhibition capabilities) treatment on breast cancer-derived cell lines. There is a scientific gap of possible causality of decreasing breast cancer progression when treated with sodium butyrate and withaferin A. Methods: Two in vitro cell viability assays were utilized consisting of [MTT (4,5 Dimethylthiazol-2-yl)] and the neutral red assay to analyze the impact of treatment of compounds alone and in combination on breast cancer cells for 72 h. The Highest Single Agent (HSA) combination analysis was utilized to derive combination indexes for our breast cancer cell types. Protein and gene expression was investigated for Class 1 histone deacetylases, de novo DNA methyltransferase, the p65 subunit of NF-κB, and NFκB1. Lastly, DNA methyltransferase enzymatic activity was analyzed via the Epigentek DNMT Activity/Inhibition ELISA Easy Kit. Results: Through the cell viability assay [MTT (4,5 Dimethylthiazol-2-yl)], MCF-7, MDA-MB-231, and MDA-MB-157 cells were found to have a decrease in cell viability due to combinatorial treatment with withaferin A and sodium butyrate. Western blot results depicted a decrease in protein expression levels for DNA methyltransferases due to the administration of 2.5 mM sodium butyrate and 0.2 µM withaferin A alone and in combination for breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-157. Additionally, the combination of these two components have successfully inhibited the progression of the NFκB1 gene within analysis through the quantitative polymerase chain reaction (qPCR). Conclusions: The novel combination of withaferin A and sodium butyrate have markedly reduced the progression of breast cancer-derived cell lines for cell viability, epigenetic DNMT gene expression, as well as inhibiting NFκB1 signaling on the gene expression level.