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.

Receptor-mediated endocytosis (RME) is a commonly recognized receptor internalization process of receptor degradation or recycling. However, recent studies have supported that RME is closely related to signal propagation and amplification from the plasma membrane to the cytosol. Few studies have elucidated the role of H₂O₂, a mild oxidant among reactive oxygen species (ROS) in RME and second messenger of signal propagation. In the present study, we investigated the regulatory function of H₂O₂ in early endosomes during signaling throughout receptor-mediated endocytosis. In mammalian cells with a physiological amount of H₂O₂ generated during epidermal growth factor (EGF) activation, fluorescence imaging showed that the levels of two activating phosphorylations on Ser⁴⁷³ and Thr³⁰⁸ of Akt were transiently increased in the plasma membrane, but the predominant p-Akt on Ser⁴⁷³ appeared in early endosomes. To examine the role of endosomal H₂O₂ molecules as signaling mediators of Akt activation in endosomes, we modulated endosomal H₂O₂ through the ectopic expression of an endosomal-targeting catalase (Cat-Endo). The forced removal of endosomal H₂O₂ inhibited the Akt phosphorylation on Ser⁴⁷³ but not on Thr³⁰⁸. The levels of mSIN and rictor, two components of mTORC2 that work as a kinase in Akt phosphorylation on Ser⁴⁷³, were also selectively diminished in the early endosomes of Cat-Endo-expressing cells. We also observed a decrease in the endosomal level of the adaptor protein containing the PH domain, the PTB domain, and the Leucine zipper motif 1 (APPL1) protein, which is an effector of Rab5 and key player in the assembly of signaling complexes regulating the Akt pathway in Cat-Endo-expressing cells compared with those in normal cells. Therefore, the H₂O₂-dependent recruitment of the APPL1 adaptor protein into endosomes was required for full Akt activation. We proposed that endosomal H₂O₂ is a promoter of Akt signaling.