Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway

Abstract

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms

“…A recent study by Heyninck et al suggested that WA interacts with Keap1 in endothelial cells [67]. While a direct biochemical interaction is possible, our results suggest that this WA-Keap1 interaction is not a determinant of Nrf2 induction in a functional context.…”

Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products

“…Previous studies have uncovered several targets of withaferin A, including C328 on vimentin as well as several cysteines on Keap1 (Bargagna-Mohan et al, 2007; Heyninck et al, 2016). In our study, we identified C328 on vimentin as a site of IAyne labeling, but this site was not a target of withaferin A, as evidenced by a light to heavy ratio of 1.0 from in vitro treatment of 231MFP breast cancer cell proteomes with withaferin A (10 μM) (Table S1) as well as a lack of competition observed between withaferin A and IAyne labeling of pure human vimentin by gel-based ABPP (Fig.…”

“…We further optimized this hit to generate JNS 1-40, a lead covalent ligand with nanomolar potency that selectively targets C377 of PPP2R1A in vitro , in situ , and in vivo to recapitulate the effects observed with withaferin A: activating PP2A, inactivating AKT signaling, and impairing breast cancer pathogenicity. Previous studies have identified various additional targets of withaferin A, including vimentin, KEAP1, Hsp90, and IKKβ (Bargagna-Mohan et al, 2007; Heyninck et al, 2014, 2016; Yu et al, 2010). Withaferin A has also been shown to affect important processes such as proteostasis (Tao et al, 2015).…”

Withaferin A attenuates bleomycin‐induced scleroderma by targeting FoxO3a and NF‐κβ signaling: Connecting fibrosis and inflammation

“…Withaferin A (WFA) is a highly oxygenated withanolide and steroidal lactone which is one of the active and abundant constituents of Withania somnifera (Ashwagandha) used for centuries in the Indian Ayurvedic medicine practice . WFA is known to have numerous health benefits such as immune modulation and anti‐inflammatory effects, demonstrated in clinical trials .…”