Resveratrol, a SIRT1 activator, attenuates aging-associated alterations in skeletal muscle and heart in mice.

Aging is associated with impairment of multiple organs, including skeletal muscle and heart. In this study, we investigated whether resveratrol, an activator of an NAD+-dependent protein deacetylase Sirtuin-1 (SIRT1), attenuates age-related sarcopenia and cardiomyocyte hypertrophy in mice. Treatment of mice with resveratrol (0.4 g/kg diet) from 28 weeks of age for 32 weeks prevented aging-associated shortening of rotarod riding time. In the tibialis anterior (TA) muscle, histogram analysis showed that the atrophic muscle was increased in 60-week-old (wo) mice compared with 20-wo mice, which was attenuated by resveratrol. In the heart, resveratrol attenuated an aging-associated increase in the cardiomyocyte diameter. Acetylated proteins were increased and autophagic activity was reduced in the TA muscle of 60-wo mice compared with those of 20-wo mice. Resveratrol treatment reduced levels of acetylated proteins and restored autophagic activity in the TA muscle. Aging-related reduction in myocardial autophagy was also suppressed by resveratrol. Skeletal muscle-specific SIRT1 knockout mice showed increases in acetylated proteins and atrophic muscle fibers and reduced autophagic activity in the TA muscle. These results suggest that activation of SIRT1 by treatment with resveratrol suppresses sarcopenia and cardiomyocyte hypertrophy by restoration of autophagy in mice.

Activation of SIRT1 promotes membrane resealing via cortactin.

Muscular dystrophies are inherited myopathic disorders characterized by progressive muscle weakness. Recently, several gene therapies have been developed; however, the treatment options are still limited. Resveratrol, an activator of SIRT1, ameliorates muscular function in muscular dystrophy patients and dystrophin-deficient mdx mice, although its mechanism is still not fully elucidated. Here, we investigated the effects of resveratrol on membrane resealing. We found that resveratrol promoted membrane repair in C2C12 cells via the activation of SIRT1. To elucidate the mechanism by which resveratrol promotes membrane resealing, we focused on the reorganization of the cytoskeleton, which occurs in the early phase of membrane repair. Treatment with resveratrol promoted actin accumulation at the injured site. We also examined the role of cortactin in membrane resealing. Cortactin accumulated at the injury site, and cortactin knockdown suppressed membrane resealing and reorganization of the cytoskeleton. Additionally, SIRT1 deacetylated cortactin and promoted the interaction between cortactin and F-actin, thus possibly enhancing the accumulation of cortactin at the injury site. Finally, we performed a membrane repair assay using single fiber myotubes from control and resveratrol-fed mice, where the oral treatment with resveratrol promoted membrane repair ex vivo. These findings suggest that resveratrol promotes membrane repair via the SIRT1/cortactin axis.

Downregulation of IGFBP5 contributes to replicative senescence via ERK2 activation in mouse embryonic fibroblasts.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) are secretory proteins that regulate IGF signaling. In this study, we investigated the role of IGFBP5 in replicative senescence in embryonic mouse fibroblasts (MEFs). During passages according to the 3T3 method, MEFs underwent senescence after the 5th passage (P5) based on cell growth arrest, an increase in the number of cells positive for senescence-associated ß-galactosidase (SA-ß-GAL) staining, and upregulation of p16 and p19. In P8 MEFs, IGFBP5 mRNA level was markedly reduced compared with that in P2 MEFs. Downregulation of IGFBP5 via siRNA in P2 MEFs increased the number of SA-ß-GAL-positive cells, upregulated p16 and p19, and inhibited cell growth. Incubation of MEFs with IGFBP5 during serial passage increased the cumulative population doubling and decreased SA-ß-GAL positivity compared with those in vehicle-treated cells. IGFBP5 knockdown in P2 MEFs increased phosphorylation levels of ERK1 and ERK2. Silencing of ERK2, but not that of ERK1, blocked the increase in the number of SA-ß-GAL-positive cells in IGFBP5-knockdown cells. The reduction in the cell number and upregulation of p16 and p21 in IGFBP5-knockdown cells were attenuated by ERK2 knockdown. Our results suggest that downregulation of IGFBP5 during serial passage contributes to replicative senescence via ERK2 in MEFs.

Different Antioxidative and Antiapoptotic Effects of Piceatannol and Resveratrol.

Resveratrol affords protection against reactive oxygen species (ROS)-related diseases via activation of SIRT1, an NAD+-dependent deacetylase. However, the low bioavailability of resveratrol limits its therapeutic applications. Since piceatannol is a hydroxyl analog of resveratrol with higher bioavailability, it could be an alternative to resveratrol. In this study, we compared the cytotoxicity, antioxidative activity, and mechanisms of cytoprotection of piceatannol with those of resveratrol. In C2C12 cells incubated with piceatannol, electrospray ionization mass spectrometry analysis showed that piceatannol was present in the intracellular fraction. A high concentration (50 µM) of piceatannol, but not resveratrol, induced mitochondrial depolarization and apoptosis. However, piceatannol at 10 µM inhibited the increase in mitochondrial ROS level induced by antimycin A, and this ROS reduction was greater than that by resveratrol. The reduction in hydrogen peroxide-induced ROS by piceatannol was also greater than that by resveratrol or vitamin C. Piceatannol reduced antimycin A-induced apoptosis more than did resveratrol. SIRT1 knockdown abolished the antiapoptotic activity of resveratrol, whereas it blocked only half of the antiapoptotic activity of piceatannol. Piceatannol, but not resveratrol, induced heme oxygenase-1 (HO1) expression, which was blocked by knockdown of the transcription factor NRF2, but not by SIRT1 knockdown. HO1 knockdown partially blocked the reduction of ROS by piceatannol. Furthermore, the antiapoptotic action of piceatannol was abolished by HO1 knockdown. Our results suggest that the therapeutic dose of piceatannol protects cells against mitochondrial ROS more than does resveratrol via SIRT1- and NRF2/HO1-dependent mechanisms. The activation of NRF2/HO1 could be an advantage of piceatannol compared with resveratrol for cytoprotection. SIGNIFICANCE STATEMENT: This study showed that piceatannol and resveratrol were different in cytotoxicity, oxidant-scavenging activities, and mechanisms of cytoprotection. Protection by piceatannol against apoptosis induced by reactive oxygen species was superior to that by resveratrol. In addition to the sirtuin 1-dependent pathway, piceatannol exerted nuclear factor erythroid 2-related factor 2/heme oxygenase-1-mediated antioxidative and antiapoptotic effects, which could be an advantage of piceatannol compared with resveratrol.

Spatiotemporal metabolic dynamics of the photosensitizer talaporfin sodium in carcinoma and sarcoma.

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co-localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin- and caveolae-dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K-Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin- and caveolae-dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K-Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.

Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8.

BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria.

It is reported that some, but not all, bacteria in human faeces are coated with secretory immunoglobulin A (S-IgA). We evaluated the proportion of S-IgA-coated bacteria to total intestinal bacteria (S-IgA coating ratio) in the gastrointestinal tract of two different strains of mice supplied by two different suppliers. The S-IgA coating ratio was significantly different in each gastrointestinal segment and between mouse suppliers. The amount of non-bacteria-bound IgA (free IgA) in each gastrointestinal segment indicated that this difference in the S-IgA coating ratio might not be due to the amount of secreted IgA. Furthermore, immunoblotting analysis revealed that only a small amount of IgA (<5% to free-IgA) was used for the coating. This indicates that, although sufficient S-IgA was secreted to coat the entire intestinal population of bacteria, only some part of the bacteria were coated with S-IgA. This study suggests that the amount of luminal S-IgA may not determine the S-IgA coating ratio, and that the amount of IgA coating intestinal commensal bacteria is very small.

Postnatal changes in the expression of genes for cryptdins 1-6 and the role of luminal bacteria in cryptdin gene expression in mouse small intestine.

Although there have been many fascinating studies on cryptdins, the information for each cryptdin isoform was not completely provided. In this study, the postnatal changes in the gene expression of cryptdin 1-6 were evaluated, and the patterns of change were compared between conventional and germ-free mice. Two patterns of postnatal change were observed: gene expression of cryptdins 1, 3 and 6 increased gradually, and that of cryptdins 2 and 5 increased rapidly. Gene expression of cryptdin 4 increased gradually in the ileum but rapidly in the jejunum. Conventional mice showed significantly higher gene expression for all isoforms than germ-free mice. Interestingly, the difference in the gene expression for cryptdin 2, 4 and 5 between the jejunum and ileum seemed to be increased by the presence of the luminal bacteria. The results indicate that cryptdin isoforms develop differently depending on the isoform type, and that the gene expression of all cryptdin isoforms was affected by the presence of the luminal bacteria.