Broad-spectrum antitumor properties of Withaferin A: a proteomic perspective

Abstract

The multifunctional antitumor properties of Withaferin A (WA), the manifold studied bioactive compound of the plant Withania somnifera, have been well established in many different in vitro and in vivo cancer models. This undoubtedly has led to a much better insight in the underlying mechanisms of WAs broad antitumor activity range, but also raises additional challenging questions on how all these antitumor properties could be explained on a molecular level. Therefore, a lot of effort was made to characterize the cellular WA target proteins, since these binding events will lead and initiate the observed downstream effects. Based on a proteomic perspective, this review provides novel insights in the molecular chain of events by which WA potentially exercises its antitumor activities. We illustrate that WA triggers multiple cellular stress pathways such as the NRF2-mediated oxidative stress response, the heat shock response and protein translation events and at the same time inhibits these cellular protection mechanisms, driving stressed cancer cells towards a fatal state of collapse. If cancer cells manage to restore homeostasis and survive, a stress-independent WA antitumor response comes into play. These include the known inhibition of cytoskeleton proteins, NFκB pathway inhibition and cell cycle inhibition, among others. This review therefore provides a comprehensive overview which integrates the numerous WA-protein binding partners to formulate a general WA antitumor mechanism.

Fig. 1. Overview of the dual action antitumor mechanism of WA: inducing anti-stress pathways and at the same time inhibiting them. (I) WA treatment results in electrophilic stress and binding of WA to Keap1 results in (II) Nrf-2 activation and translocation. (III) Activation of Nrf-2 downstream anti-oxidative pathways that are prone to (IV) WA–protein binding and inhibition. As result WA induces and inhibits the anti-oxidative pathways simultaneously. (V) Low, basal, ROS levels further accumulate due to inhibition of anti-oxidative pathways. (VI) ROS accumulation triggers Keap1 and (VII) further activates Nrf-2. The resulting imbalanced cytoprotection/ROS levels will eventually determine cellular fate: live or die.

Fig. 2. Under quiescent conditions, binding of Kelch-like ECH-associated protein 1 (Keap1) with nuclear factor erythroid-derived 2-like 2 (Nrf2) facilitates the sustained ubiquitination and proteasome degradation of Nrf2. Upon oxidative stress, Keap1 becomes inactivated through binding of its cysteine thiols with electrophilic groups. This releases Keap1 from Nrf2 which allows Nrf2 translocation to the nucleus. There it forms heterodimers with small Maf proteins containing a leucine zipper (bZIP) domain and binds with the ARE of target genes such as NAD(P)H dehydrogenase [quinone] 1 (NQO1) and heme-oxygenase 1 (HMOX1) which initiate stress homeostasis mechanisms.

Fig. 3. Overview of the autophagy pathway and its main components. Within each step of the formation of the autolysosome, WA targets the 5 proteins (gene names HDAC6, PLAA, WIPI2, SNAPIN and TUBB), suggesting serious disruption of the autophagy process with subsequent cellular impotency to deal with the WA-induced cellular stress. Marked in red are the WA-target proteins (gene names).

Fig. 4. Heat shock factor-1 transcription factor (HSF1): a stress-inducible and DNA-binding transcription factor.

Fig. 5. Overview of ERAD. Newly synthesized proteins are guided towards the Golgi structures in case of properly folding. Misfolded proteins are recognized by glycan processing and marked for proteasome degradation. Marked in red are the WA-target proteins (gene names).

Fig. 6. Overview of the proposed WA antitumor mechanism based on stress dependent (left) and – independent (right) effects. All proteins are represented by their gene names. Black = WA target; red = upregulated protein expression upon WA treatment; green = downregulated protein expression upon WA treatment. RP: regulatory particle. CP: core particle.

Fig. 7. Schematic overview of the coupling between redox state of the metabolism and the cell cycle.