## 3 Keep1-Nrf2-ARE Signaling System
### Withaferrin A (WFA)  
Withaferrin A (**4**) was isolated from traditional Indian medicinal herb _Withania somifera_ roots and has been studied extensively for its anti-inflammatory, and cardioprotective effects [124]. It has been found to be effective in ameliorating a wide variety of cancers including breast, cervical, ovarian, prostate, oral, glioblastoma, and pancreatic cancer [125; 126]. The chemical structure of WFA contains \(\alpha\),\(\beta\)-unsaturated carbonyl moieties [127]. Therefore, it might work as a Michael acceptor and a thiol modifier; in particular, WFA might have an interesting role in the modification of Keap1-cysteine thiol residues. This modification might help Nrf2 to disassociate from Keap1, thus allowing it to be activated. The Nrf2-induced oxidative stress response pathway was implicated in the anticancer action of WFA [128]. According to this model, under oxidative stress, WFA bound with Keap1, freeing Nrf2 to be translocated into the nucleus. Thus, the enhanced Nrf2 bound with AREs in order to upregulate its downstream antioxidant enzymes. Robust induction of Nrf2 by WFA was recently reported in the treatment of human breast cancer cells [129]. This study concluded that Nrf2 helped in the mediation of apoptosis and autophagy in several breast cancer cells. Induction by WFA of Nrf2 and its associated detoxifying enzymes was also reported to produce an effective cytoprotective response in both in vitro and in vivo models [130]. This withaferrin A-mediated Nrf2 and ARE induction followed a unique Keap1-independent phosphatase and tension homolog (PTEN)/PI3K/AKT signaling pathway. In this case, the Nrf2-dependent cytoprotective genes were significantly induced even under Keap1-deprived conditions. WFA upregulated the mRNA expression of Nrf2 and its downstream antioxidant genes HO-1, glutathione-disulfide reductase (GSR), and NQO1 in Ca9-22 oral cancer cells [131]. In addition, it facilitated mild phosphorylation of the MAPK family proteins including ERK1/2, JNK, and p38. The same study reported a migration inhibitory role of WFA against oral cancer, where it abolished matrix metalloproteinase (MMP)-2 and MMP-9 activities. In J82 bladder cancer cells, WFA responded remarkably to oxidative stress by upregulating the expression of Nrf2 and its antioxidant enzymes CAT, SOD1, TXN, GSR, NQO1, and HO-1 [132]. Moreover, WFA mediated DNA strand breaks and oxidative DNA damage to the bladder cancer cells. _Withania somifera_ containing WFA demonstrated antioxidant and anti-inflammatory activity in BV-2 microglial cells, wherein upregulated Nrf2/HO-1 and downregulated NF-kB expression played the key role [133]. The apoptosis-inducing role of WFA has also been reported, in which ERK, JNK, and p38 MAPK signaling pathways are involved, resulting in the phosphorylation of Nrf2 and enhancement of HO-1 levels [134]. WFA suppressed necrotic hepatocyte injury by enhancing Nrf2, GCLC and NQO1, and abrogating IL-6, TNF-\(\alpha\) and IL-1\(\beta\) expression [135]. In the same study, mitochondrial Bax translocation, JNK expression and nitrotyrosine generation were reduced in WFA-treated mice. A review on the anticancer action of WFA reported that WFA upregulated Nrf2 protein expression, thereby enhancing the expressions of HO-1, SOD, CAT, Gpx and NQO1 [127]. The accumulated evidence supporting the chronic inflammation ameliorating role of WFA was gathered in a recent review [136]. Here, reduced NF-\(\kappa\)B and increased Nrf2 expression were implicated in the anti-inflammatory role of WFA. Additionally, signaling pathways like AKT/mTOR, MAPK, and JAK/STAT were involved in exerting this effect. In a recent study, inhibition of NF-\(\kappa\)B expression was implicated in the anti- hepatocellular carcinoma effect mediated by WFA [137]. The anti-angiogenic pathway of WFA against lung cancer involved the inhibition of transforming growth factor \(\beta\)1 (TGF-\(\beta\)1), TNF-\(\alpha\), and nuclear translocation and phosphorylation of NF-\(\kappa\)B [138].