Apigenin and Luteolin Regulate Autophagy by Targeting NRH-Quinone Oxidoreductase 2 in Liver Cells

Abstract

Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

The Unusual Cosubstrate Specificity of NQO2: Conservation Throughout the Amniotes and Implications for Cellular Function

“…It has previously been observed that reduction of the flavin cosubstrate and binding of chloroquine resulted in a change in conformation or dynamics, which could affect interactions of NQO2 with other molecules ( Leung and Shilton, 2013 ). A recent manuscript implicating NQO2 in regulation of autophagy concluded that the mechanism may not require catalytic activity, but instead depend on signaling through ligand binding and changes in protein-protein interactions ( Janda et al, 2021 ). Another possibility is that NQO2 signals through the production of reactive oxygen species, which have been detected in vitro ( Reybier et al, 2011 ) and in cells ( Cassagnes et al, 2015 ) during the reduction of quinones by NQO2 using a suitable cosubstrate such as NRH or BNAH.…”

Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: a potential novel target in Parkinson’s disease.

“…Thus, S29434 by acting as a protective agent on astrocytes, might delay neuronal damages in PD and other pathological conditions concerning OS and autophagy impairment mediated by NQO2, suggesting that NQO2 inhibitors might have a therapeutic potential for the treatment of cerebral injuries involving oxidative neurodegeneration. The vast literature suggesting a protective effect of several natural polyphenols in neurodegeneration strongly supports this idea, since NQO2 is a well-documented target of these compounds [39,64]. Future studies should validate this hypothesis.…”

“…Overall, due to the reported speci city of S29434 towards NQO2 [38], these and our previous data strongly suggest that NQO2 is an important modulator of autophagy. The question whether it is due to the enzymatic activity of NQO2 or to the formation of NQO2 complexes with protein partners hypothetically regulated by S29434 and other inhibitors, as suggested previously [39], is not yet clear.…”

“…NQO2 enzymatic activity assays were performed as described previously [39] with the following modi cations. The cellular NQO2 activity was measured in U373 cell lysates.…”

“…PQ has been shown to inhibit basal autophagy, while the treatment with S29434 stimulated autophagy in astrocytes [30]. Recent data indicate that the function of this enzyme is not limited to generation of OS and inhibition of autophagy, but NQO2 plays a role in avone-induced autophagy acting as a receptor of pro-autophagic ligands including avonoids and S29434 [39].…”