Tissue transglutaminase 2 expression is epigenetically regulated in human lung cancer cells and prevents reactive oxygen species-induced apoptosis

PURPOSE: Tissue transglutaminase 2 (TG2) is a stress-regulated protein and associated with cancer cell survival. However, the effects of TG2 expression in human non-small-cell lung cancer (NSCLC) cells on reactive oxygen species (ROS) production and redox homeostasis have not been fully elucidated. MATERIALS AND METHODS: We investigated the TG2 expression and activity in A549, H1299, H1355, and H460 lung cancer cells by Western blots and quantitative polymerase chain reaction assay. The enzyme-linked immunosorbent assay was used for transglutaminase activity. The epigenetic expression was characterized with histone deacetylase inhibitor trichostatin A and DNA methyltransferase inhibitor 5-Aza treatment. TG2 expression was inhibited by siRNA transfection and the intracellular calcium was measured by Flow-3AM assay, apoptosis was analyzed by Annexin V/propidium iodide assay, and intracellular ROS was detected by fluorescence-activated cell sorting analysis. The ROS scavenger N-acetyl-L-cysteine (NAC) was applied to reduce TG2-knockdown-induced oxidative stress. RESULTS: Only A549 cells expressing high levels of TG2 correlated with high TG2 activity. The expression of TG2 can be regulated by epigenetic regulation in A549, H1299, and H1355 cells. The data also show that TG2 reduction induces apoptosis in A549 and H1299 cells. Furthermore, increased intracellular ROS and calcium levels were both detected in TG2-reduced cells. Moreover, endoplasmic reticulum stress inhibitor (salubrinal) and antioxidant NAC were able to reduce ROS and calcium levels to recover cell viability. Interestingly, the extrinsic and intrinsic apoptosis pathways were activated with a p53 independence upon TG2 reduction. TG2 reduction not only attenuated AKT activation but also reduced superoxide dismutase 2 (SOD2) expression. Exogenous NAC partially recovered SOD2 expression, indicating that mitochondrial-mediated apoptosis accounts for a part of but not all of the TG2-reduction-related death. CONCLUSION: TG2 plays a protection role in NSCLC cell lines. Regardless of the endogenous level of TG2 and p53 status, reduction of TG2 may result in oxidative stress that induces apop-tosis. Therefore, target TG2 expression represents a logical strategy for NSCLC management.