ROS generation and JNK activation contribute to 4-methoxy-TEMPO-induced cytotoxicity, autophagy, and DNA damage in HepG2 cells

Abstract

4-Methoxy-TEMPO, a derivative of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is a stable nitroxide radical and is generally used in organic and pharmaceutical syntheses for the oxidation of alcohols. Previously, we reported the involvement of reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) in TEMPO-induced apoptosis in mouse L5178Y cells. In this study, we investigated 4-methoxy-TEMPO induced toxicity in human HepG2 hepatoma cells and its underlying mechanisms. Treatments with 4-methoxy-TEMPO (0.5–5 mM for 2–6 h) caused oxidative stress as demonstrated by increased intensity of the ROS indicator H2DCF-DA, decreased levels of glutathione. 4-Methoxy-TEMPO treatment also induced DNA damage as characterized by increased levels of DNA tail intensity in the Comet assay, increased phosphorylation of related proteins including γ-H2A.X, p-Chk1, and p-Chk2, and activation of MAPK signaling pathways. In addition, 4-methoxy-TEMPO also induced autophagy as demonstrated by the conversion of LC3B-I to II, decreased level of p62, and the appearance of GFP-LC3B punctae. To investigate the crosstalk between different signaling pathways, pretreatment of HepG2 with N-acetylcysteine, an ROS scavenger, attenuated 4-methoxy-TEMPO-induced DNA damage, suppressed JNK activation, and diminished autophagy induction. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased DNA damage levels induced by 4-methoxy-TEMPO. These results suggest that multiple mechanisms including ROS generation, DNA damage, and MAPK activation contribute to 4-methoxy-TEMPO-induced toxicity.

Antitumor activity of Raddeanin A is mediated by Jun amino‐terminal kinase activation and signal transducer and activator of transcription 3 inhibition in human osteosarcoma

“…JNK are extremely important in the process of apoptotic cell death as one subgroup of the MAPK family. JNK and ERK, as stress‐activated protein kinases, are activated by various stress stimuli, such as bioactive compounds, to induce apoptosis . Consistent with these results, we observed that JNK activation and ERK1/2 activation were involved in RA‐induced apoptosis in a drug concentration‐ and time‐dependent method.…”

“…As a heterogeneous group of diatomic oxygen molecules from free and nonfree radical species, ROS trigger apoptosis by causing various cellular stresses and regulate several apoptotic effectors, such as caspases, Bcl‐2, and cytochrome c . Broadly speaking, ROS are capable of activating p38 MAPK, JNK, ERK, and Akt pathways, depending on the context …”

“…8,9 As a heterogeneous group of diatomic oxygen molecules from free and nonfree radical species, ROS trigger apoptosis by causing various cellular stresses and regulate several apoptotic effectors, such as caspases, Bcl-2, and cytochrome c. 8 Broadly speaking, ROS are capable of activating p38 MAPK, JNK, ERK, and Akt pathways, depending on the context. 8,[10][11][12] Jun amino-terminal kinase, a stress-activated protein kinase of the MAPK family, regulates a variety of cellular events, including autophagy and apoptosis, which are complex events depending on cell type, nature of the death stimulus and activity of other signaling pathways. 13 A major target of the JNK signaling pathway is the activator protein-1 (AP-1) transcription factor, which is activated in part by the phosphorylation of c-Jun and related molecules.…”

Functional Role of p53 in the Regulation of Chemical-Induced Oxidative Stress

“…The activation of MAPKs, especially the stress-activated kinase, JNK, is common under conditions of oxidative stress. In most cases, MAPK activation occurs downstream of ROS generation [81,82]; however, activated MAPKs can also positively regulate ROS generation in certain conditions [44,83], (Huo et al 2016).…”

The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets

“…4-Methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl, shortened to MetT, is a stable cyclic nitroxide derivative based on TEMPO, with a substitution at position 4 for a methoxy group [15]. The complete characterisation of the functional difference of this substitution to other nitroxides is lacking in the literature, but MetT appears to have similar activity to other cyclic nitroxides [16].…”