Hydrogen peroxide (H₂O₂) is an oxidizing agent that induces cellular damage at inappropriate concentrations and gives rise to an arrest during cell cycle progression, causing cell death. Recent evidence indicates that H₂O₂ also acts as a promoter for cell cycle progression by oxidizing specific thiol proteins. The intracellular concentration of H₂O₂ is regulated tightly, enabling its use as a cellular signaling molecule while minimizing its potential to cause cellular damage. Peroxiredoxins (Prxs) have peroxidase activity toward H₂O₂, organic hydroperoxides, and peroxynitrite for protecting cells from oxidative stress. They are suggested to work as signaling mediators, allowing the local accumulation of H₂O₂ by inactivating their peroxidase activity uniquely compared with other antioxidant proteins such as catalase and glutathione peroxidase. Given that Prxs are highly sensitive to oxidation by H₂O₂, they act as sensors and transducers of H₂O₂ signaling via transferring their oxidation state to effector proteins. The concentrations of intracellular H₂O₂ increase as the cell cycle progresses from G₁ to mitosis. Here, we summarize the roles of Prxs with regard to the regulation of cell cycle-dependent kinase activity and anaphase-promoting complex/cyclosome in terms of changes in H₂O₂ levels. Protection of the cell from unwanted progression of the cell cycle is suggested to be a role of Prx. We discuss the possible roles of Prxs to control H₂O₂ levels.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.

The serine/threonine kinase AKT is a major effector during phosphatidylinositol 3-kinase (PI3K)-driven cell signal transduction in response to extracellular stimuli. AKT activation mechanisms have been extensively studied; however, the mechanism underlying target of rapamycin complex 2 (mTORC2) phosphorylation of AKT at Ser473 in the cellular endomembrane system remains to be elucidated. Here, we demonstrate that endocytosis is required for AKT activation through phosphorylation at Ser473 via mTORC2 using platelet-derived growth factor-stimulated U87MG glioma cells. mTORC2 components are localized to early endosomes during growth factor activation, and the association of mTORC2 with early endosomes is responsible for the local activation of AKT, which is critical for specific signal transduction through glycogen synthase kinase-3 beta and forkhead box O1/O3 phosphorylation. Furthermore, endosomal phosphoinositide, represented by PtdIns(3,4)P₂, provides a binding platform for mTORC2 to phosphorylate AKT Ser473 in endosomes through mammalian Sty1/Spc1-interacting protein (mSIN), a pleckstrin homology domain-containing protein, and is dispensable for AKT phosphorylation at Thr308. This PtdIns(3,4)P₂-mediated endosomal AKT activation provides a means to integrate PI3K activated by diverse stimuli to mTORC2 assembly. These early endosomal events induced by endocytosis, together with the previously identified AKT activation by PtdIns(3,4,5)P₃, contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.