No abstract

No abstract

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

SUMMARY: Cu, Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H202 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H202 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H202, lipid peroxidation was gradually increased in a time-dependent manner.The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H202 concentrations.Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and I-I202 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H20~ system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.

SUMMARYThe free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wildtype enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.Key words: copper,zin-superoxide dismutase, mutant, hydroxyl radical, copper. 1191All rights of reproduction in any form reserved, Vol. 46, No. 6, 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALAndersen et al. [7] reported a Cu,Zn-SOD exon 4 mutation (D90A) with novel features in Swedish FALS patients and the disease has only been observed in patients homozygous for this mutation, whereas heterozygotes remained unaffected. Furthermore, there was essentially no reduction in erythrocyte Cu,Zn-SOD dismutation activity. Thus, [7] suggested that this Cu,Zn-SOD mutation caused ALS by a gain-of-function rather than by the loss of dismutation activity.In view of these considerations, the present work represents an investigation of the free radical-generating functions of the D90A mutant and the oxidative damage to DNA, as a first attempt to establish the extent of any correlation between the gain-of-function and the pathology of FALS. MATERIALS AND METHODS Expression and purification of wild-type and mutated human Cu, Zn-SOD