Growth inhibitory factor (GIF), a brain-specific member of the metallothionein family (MT-III), has been characterized as a inhibitory substance for neurotrophic factors in Alzheimer's disease brains. However, the function of GIF, other than the inhibition of neurotrophic factors, remains unknown. We demonstrate here that exogenous GIF prevents neurite extension of cortical neurons in the early period of differentiation and the death of differentiated neurons caused by high oxygen exposure. Down-regulation of GIF in cortical neurons with antisense S-oligonucleotides promoted neuronal death under high oxygen conditions. ESR spin-trapping studies demonstrated that GIF at 2-6 M scavenged hydroxyl radicals generated by a Fenton-type reaction or the photolysis of hydrogen peroxide much more effectively than the same concentration of metallothionein I؉II. GIF did not scavenge either superoxide produced by the xanthine/xanthine oxidase reaction or NO generated from 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene. Moreover, GIF at 40 -80 M inhibited tyrosine nitration by peroxynitrite as efficiently as metallothionein I؉II at the same concentration. These results indicate that GIF prevents neurite extension of neurons in the early period of differentiation and supports the survival of differentiated neurons by scavenging hydroxyl radicals.

Pancreatic cancer, composed of heterogeneous cancer cells, alters epithelial to mesenchymal features during growth and metastasis. In this study, we aimed to characterize pancreatic ductal adenocarcinoma (PDAC) cells showing epithelial or mesenchymal features in 3D culture. In 3D culture, PK-1 cells had high E-cadherin and low vimentin expression and exhibited a round-like appearance encircled by flat cells. PANC-1 cells had high vimentin and low E-cadherin expression and formed grape-like spheres. PK-1 cells had secretary granules and many microvilli, desmosomes, and adherens junctions, while PANC-1 cells had few microvilli, adherens junction, and no desmosomes. Cytokeratin 7, trypsin, CA19-9, and E-cadherin were highly expressed in PK-1 cells but not in PANC-1 cells. Ki-67 was diffusely expressed in PANC-1 spheres but was restricted to the peripheral flat cells of PK-1 spheres. PANC-1 and PK-1 cells were positive for transforming growth factor (TGF) β receptor II and phosphorylated smad2/3, but PK-1 cells were smad4 negative. Taken together, 3D culture enhanced morphofunctional differences of PDAC cells showing epithelial or mesenchymal characteristics, and epithelial phenotype maintenance may be due to the ineffectiveness of the TGF-β pathway. Clarification of heterogeneity using 3D culture may be useful for development of individualized diagnostic and therapeutic methods in patients with PDAC. Due to the development of early detection methods and new treatments including surgery, chemotherapy, radiotherapy, and immunotherapy for cancers, the average survival rates of major cancers are over 50% 1. However, the five-year survival rate for patients with pancreatic cancer is only 8% 2. Pancreatic ductal adenocarcinoma (PDAC) is a major histological pancreatic cancer subtype. While surgical treatment offers the only possible cure for PDAC, 80% of patients with PDAC are inoperable at diagnosis. Even after surgery, the five-year survival rate is 15-20%, due to the high PDAC metastatic rate and local recurrence 3. Currently, chemotherapies or chemoradiotherapies are able to reduce tumor size and improve the prognosis, but these treatments do not fully cure the patients. Morbidity and mortality of PDAC are high in aged people and the worldwide progressive aging of society suggests a rapid increase in pancreatic cancer related deaths in the near future 4. Recent studies have shown that cancer stem cells (CSCs) contribute to the heterogeneity of various cancers. The "stem cell theory" of cancer implies that CSCs are responsible for tumor initiation, growth, and metastasis.

Genetic, transcriptional, and morphological differences have been reported in pancreatic ductal adenocarcinoma (PDAC) cases. We recently found that epithelial or mesenchymal features were enhanced in three-dimensional (3D) cultures compared to two-dimensional (2D) cultures. In this study, we examined the differences in the morphological and functional characteristics of eight PDAC cell lines in 2D and 3D cultures. Most PDAC cells showed similar pleomorphic morphologies in 2D culture. Under 3D culture, PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres encircled with flat lining cells, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative. In 3D culture, gemcitabine was more effective for the spheres formed by PDAC cells with epithelial features, while abraxane was more effective on those with mesenchymal features. The expression levels of drug transporters were highest PDAC cells with high vimentin expression levels. These findings indicate that PDAC cells possess various levels of epithelial and mesenchymal characteristics. The 3D-culture method is useful for investigating the diversity of PDAC cell lines and may play important roles in the development of personalized early diagnostic methods and anticancer drugs for PDAC.

Longitudinal age changes in the exhalation rates of ethane and pentane, which are used as the indices of in vivo lipid peroxidation, from rats under ad libitum feeding and food restriction were measured. The exhalation rate of ethane or pentane from old, ad libitum-fed rats is higher than that of the corresponding hydrocarbon from young, ad libitum-fed rats, as reported previously. There is no difference between the exhalation rates of each hydrocarbon from young, ad libitum-fed and food-restricted rats. However, the exhalation rate of pentane from old, food-restricted rats is significantly lower than that from old, ad libitum-fed rats, while the exhalation rate of pentane from old, food-restricted rats tends to be lower than that from old, ad libitum-fed rats. In addition, the exhalation rate of pentane from old, food-restricted rats is equivalent to that from young, ad libitum-fed rats. These results are consistent with the views that in vivo lipid peroxidation in rats is increased during aging, and that the age-dependent increase in in vivo lipid peroxidation is suppressed by food restriction.

The effects of aging and food restriction on the activities and mRNA levels of antioxidant enzymes in rat livers were examined. Rats were fed ad libitum every day (AL) or ad libitum on every other weekday (FR). At 30 months of age, the catalase and glutathione peroxidase activities were lower, whereas the thiobarbituric acid (TBA) value, an index of lipid peroxidation of the AL rats, was higher than that at younger ages. At 33 months of age, copper/zinc superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase activities increased, and the TBA value of the FR rats remained unchanged as compared with those at younger ages. Until old age, food restriction gave rather decreasing effects on antioxidant enzyme activities. Furthermore, antioxidant enzyme activities and the TBA values of the FR rats were higher at the end of a fasting period than those at the end of a feeding period.