Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

Transcriptomic and Epigenomic Changes in Human Leukocytes Upon 8 Weeks Supplementation with Monomeric and Oligomeric Flavanols

International audienceConsumption of flavanol-rich foods is associated with a reduced risk of cardiovascular diseases, which was linked to improvements in endothelial function. A recent randomized, double-blind, placebo controlled clinical trial unveiled pleiotropic health benefits in the vasculature of healthy male smokers upon 8 weeks supplementation with daily 200 mg flavanols. In order to unravel the flavanols’ underlying molecular mechanisms, we investigated the transcriptomic and epigenomic changes in leukocytes. Methods: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). Results: Flavanol consumption significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that flavanols exposure of monocytes attenuates their adhesion to TNF-α- timulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that flavanols decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes’ DNA methylation was observed. As a consequence, on group level, changes due to flavanols supplementation could not be found. Conclusion: Altogether, flavanols may elicit protective effects in the vasculature by decreasing inflammatory and cell adhesion pathways at the transcriptional level. Moreover, smoking history may be a confounding factor in epigenetic profiling studies of leukocytes from subjects involved in a flavanol-rich diet intervention