Mitochondria are well known for their important roles in oxidative phosphorylation, amino acid metabolism, fatty acid oxidation and ion homeostasis. Although the effects of mitochondrial dysfunction on tumorigenesis in various cancer cells have been reported, the correlation between mitochondrial dysfunction and epithelial‑to‑mesenchymal transition (EMT) in lung cancer development and metastasis has not been well elucidated. In the present study, the effects of mitochondrial dysfunction on EMT and migration in lung cancer cells were investigated using inhibitors of mitochondrial respiration, oligomycin A and antimycin A. Oligomycin A and antimycin A induced distinct mesenchymal‑like morphological features in H23, H1793 and A549 lung cancer cells. In addition, they decreased the expression levels of the epithelial marker protein E‑cadherin, but increased the expression levels of the mesenchymal marker proteins Vimentin, Snail and Slug. The results of immunofluorescence staining indicated that oligomycin A and antimycin A downregulated cortical E‑cadherin expression and upregulated the expression of Vimentin. In addition, oligomycin A and antimycin A increased the migration and invasion of A549 lung cancer cells, and promoted the expression levels of phosphorylated (p)‑protein kinase B (AKT) and p‑AMP‑activated protein kinase (AMPK). Notably, the production of reactive oxygen species by oligomycin A and antimycin A did not affect the expression of EMT protein markers. Conversely, treatment with the AKT inhibitor wortmannin and the AMPK inhibitor Compound C upregulated E‑cadherin and downregulated Vimentin expression. These results suggested that oligomycin A and antimycin A may induce migration and invasion of lung cancer cells by inducing EMT via the upregulation of p‑AKT and p‑AMPK expression.

A prior study identified that 4-O-methylascochlorin (MAC), a methylated derivative of ascochlorin (ASC) from the fungus Ascochyta viciae, activates autophagy in leukemia cells by suppressing c-Myc phosphorylation. However, the effects of MAC on autophagy in other cancer cells remain unknown. In the present study, we demonstrated that MAC activated autophagy in human glioblastoma. MAC increased expression of autophagy-related proteins, such as LC3-II and Beclin-1. Moreover, MAC stimulated AMP-activated protein kinase (AMPK) phosphorylation and suppressed phosphorylation of the mTOR, p70S6K, and 4EBP1. The well-known AMPK activator metformin increased LC3-II levels, which were augmented by MAC cotreatment. AMPK knockdown decreased LC3-II levels and inhibited MAC activation of autophagy. Furthermore, MAC suppression of c-Myc expression activated autophagy. Treatment with the c-MYC inhibitor, 10058-FA, induced autophagy, as did c-Myc small interfering RNA knockdown. These effects were augmented by MAC cotreatment. Taken together, these findings indicated that MAC induces autophagy in human glioblastoma by activating AMPK signaling and inhibiting c-Myc protein expression in human glioblastoma.

Background and Aims Activation of G protein-coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R. However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2 expression and phosphorylation, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R. Tripartite motif-containing protein 28 (TRIM28) was identified as a substrate of ERK1 as well as a vasopressin-responsive protein via bioinformatic tools. We further evaluated whether TRIM28 plays a role in vasopressin-mediated regulation of AQP2 in the kidney CD. Method To identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on high-throughput profiles of transcriptome and proteome (http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); and 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate protein (TRIM28) was examined by immunohistochemistry. The role of TRIM28 in vasopressin-mediated AQP2 regulation was examined by quantitative real-time PCR (qRT-PCR) and semiquantitative immunoblotting after RNA interference of TRIM28 in mpkCCDc11 cells. Results Immunoblotting of mpkCCDc11 cells revealed that both p-ERK1/2 and pS261-AQP2 expression was decreased in response to dDAVP (10-9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase-substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed TRIM28, as the substrate of ERK1, has the only one phosphorylation site which was down-regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin-protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP-induced AQP2 mRNA and protein up-regulation was significantly attenuated in mpkCCDc11 cells with siRNA-mediated knockdown of TRIM28. Conclusion We identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.