Pharmacological Levels of Withaferin A (Withania somnifera) Trigger Clinically Relevant Anticancer Effects Specific to Triple Negative Breast Cancer Cells

• DOI: 10.1371/journal.pone.0087850
• Publication Date: 2014-02-03T22:07:24Z
• Authors: Katarzyna Szarc v...; Ken Op de Beeck; Dariusz Ratman; An Wouters; Ilse M. Beck; Ken Declerck; Karen Heyninck; Erik Fransen; Marc Bracke; Karolien De Bosscher; Filip Lardon; Guy Van Camp; Wim Vanden Berghe
• Keywords: EXPRESSION; 0301 basic medicine; METASTASIS SUPPRESSOR; TUMOR-METASTASIS; Science; INVASION; Blotting, Western; Apoptosis; Triple Negative Breast Neoplasms; KAPPA-B; Real-Time Polymerase Chain Reaction; 03 medical and health sciences; Medicine and Health Sciences; Biomarkers, Tumor; Tumor Cells, Cultured; Humans; RNA, Messenger; PLASMINOGEN-ACTIVATOR; Withanolides; Cell Proliferation; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Gene Expression Profiling; Q; Cell Cycle; METHYLATION; R; EPITHELIAL-MESENCHYMAL TRANSITION; 3. Good health; HISTONE DEMETHYLASES; GROWTH; Medicine; Female; Human medicine; Engineering sciences. Technology; Research Article; Phytotherapy

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.