Artesunate-induced mitophagy alters cellular redox status

Abstract

Artesunate (ART) is a prominent anti-malarial with significant anti-cancer properties. Our previous studies showed that ART enhances lysosomal function and ferritin degradation, which was necessary for its anti-cancer properties. ART targeting to mitochondria also significantly improved its efficacy, but the effect of ART on mitophagy, an important cellular pathway that facilitates the removal of damaged mitochondria, remains unknown. Here, we first observed that ART mainly localizes in the mitochondria and its probe labeling revealed that it binds to a large number of mitochondrial proteins and causes mitochondrial fission. Second, we found that ART treatment leads to autophagy induction and the decrease of mitochondrial proteins. When autophagy is inhibited, the decrease of mitochondrial proteins could be reversed, indicating that the degradation of mitochondrial proteins is through mitophagy. Third, our results showed that ART treatment stabilizes the full-length form of PTEN induced putative kinase 1 (PINK1) on the mitochondria and activates the PINK1-dependent pathway. This in turn leads to the recruitment of Parkin, sequestosome 1 (SQSTM1), ubiquitin and microtubule-associated proteins 1A/1B light chain 3 (LC3) to the mitochondria and culminates in mitophagy. When PINK1 is knocked down, ART-induced mitophagy is markedly suppressed. Finally, we investigated the effect of mitophagy by ART on mitochondrial functions and found that knockdown of PINK1 alters the cellular redox status in ART-treated cells, which is accompanied with a significant decrease in glutathione (GSH) and increase in mitochondrial reactive oxidative species (mROS) and cellular lactate levels. Additionally, knockdown of PINK1 leads to a significant increase of mitochondrial depolarization and more cell apoptosis by ART, suggesting that mitophagy protects from ART-induced cell death. Taken together, our findings reveal the molecular mechanism that ART induces cytoprotective mitophagy through the PINK1-dependent pathway, suggesting that mitophagy inhibition could enhance the anti-cancer activity of ART.

Targeting autophagy enhances the anticancer effect of artemisinin and its derivatives

“…Using an ART probe with an alkyne moiety, we confirmed the direct interaction of ART with PINK1 and parkin. Our results also showed that ART stabilizes PINK1, and the resultant accumulation of mitochondrial PINK1 enhances the phosphorylation of ubiquitin to activate Parkin . These damaged mitochondria are selectively removed by mitophagy …”

“…In the process of mitophagy, these ubiquitinated mitochondrial proteins are recognized by autophagy receptors such as SQSTM1/p62 or other molecules that interact with LC3 to form autophagosomes . We identified the involvement of the PINK1‐parkin pathway in ART‐induced mitophagy (Figure ) . Using an ART probe with an alkyne moiety, we confirmed the direct interaction of ART with PINK1 and parkin.…”

“…In our study, we demonstrated that ART induces mitophagy, as indicated by the reduction in mitochondrial proteins, the induction of autophagosome formation and their subsequent fusion with both mitochondria and lysosomes. We first confirmed that ART stays mainly in the mitochondria using a red fluorescence‐tagged ART probe.…”

“…We first confirmed that ART stays mainly in the mitochondria using a red fluorescence‐tagged ART probe. Furthermore, we identified a large list of mitochondrial membrane proteins or enzymes covalently bound to artemisinin by using an alkyne‐tagged artemisinin probe, suggesting that ART alters mitochondrial functions. Most importantly, we observed that, in ART‐treated cells, there is a clear overlap between mitochondrial membrane proteins and autophagosomes, indicating that damaged mitochondrial proteins participate in the formation of autophagosomes.…”

“…Artesunate activates the PINK1‐parkin pathway and induces mitophagy . ART, artesunate; PINK1, PTEN‐induced kinase 1…”