Acceleration of Protein Degradation by 20S Proteasome-Binding Peptides Generated by In Vitro Artificial Evolution.

Although the 20S core particle (CP) of the proteasome is an important component of the 26S holoenzyme, the stand-alone 20S CP acts directly on intrinsically disordered and oxidized/damaged proteins to degrade them in a ubiquitin-independent manner. It has been postulated that some structural features of substrate proteins are recognized by the 20S CP to promote substrate uptake, but the mechanism of substrate recognition has not been fully elucidated. In this study, we screened peptides that bind to the 20S CP from a random eight-residue pool of amino acid sequences using complementary DNA display an in vitro molecular evolution technique. The identified 20S CP-binding amino acid sequence was chemically synthesized and its effects on the 20S CP were investigated. The 20S CP-binding peptide stimulated the proteolytic activity of the inactive form of 20S CP. The peptide bound directly to one of the α-subunits, opening a gate for substrate entry on the α-ring. Furthermore, the attachment of this peptide sequence to α-synuclein enhanced its degradation by the 20S CP in vitro. In addition to these results, docking simulations indicated that this peptide binds to the top surface of the α-ring. These peptides could function as a key to control the opening of the α-ring gate.

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real-time bioluminescence imaging.

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

Decreased levels of PDI and P5 in oligodendrocytes in Alzheimer's disease.

Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S-nitrosylated PDI and S-nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.

Discovery of protein disulfide isomerase P5 inhibitors that reduce the secretion of MICA from cancer cells.

In order to regulate the activity of P5, which is a member of the protein disulfide isomerase family, we screened a chemical compound library for P5-specific inhibitors, and identified two candidate compounds (anacardic acid and NSC74859). Interestingly, anacardic acid inhibited the reductase activity of P5, but did not inhibit the activity of protein disulfide isomerase (PDI), thiol-disulfide oxidoreductase ERp57, or thioredoxin. NSC74859 inhibited all these enzymes. When we examined the effects of these compounds on the secretion of soluble major histocompatibility complex class-I-related gene A (MICA) from cancer cells, anacardic acid was found to decrease secretion. In addition, anacardic acid was found to reduce the concentration of glutathione up-regulated by the anticancer drug 17-demethoxygeldanamycin in cancer cells. These results suggest that anacardic acid can both inhibit P5 reductase activity and decrease the secretion of soluble MICA from cancer cells. It might be a novel and potent anticancer treatment by targeting P5 on the surface of cancer cells.

Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide.

BACKGROUND: Heat shock protein (Hsp) 90 and Hsp70 are indispensable for cell survival under conditions of stress. They bind to client proteins to assist in protein stabilization, translocation of polypeptides across the cell membrane, and recovery of proteins from aggregates in the cell. Therefore, these proteins have recently emerged as important targets in the treatment of cancer. We previously reported that the newly designed Antp-TPR hybrid peptide targeting Hsp90 induced cytotoxic activity to cancer cells both in vitro and in vivo. METHODS: To further improve the cytotoxic activity of Antp-TPR toward cancer cells, we investigated the effect of a Hsp70-targeted peptide, which was made cell-permeable by adding the polyarginine with a linker sequence, on the cytotoxic activity of Antp-TPR in breast cancer cell lines. RESULTS: It was revealed that Antp-TPR in the presence of a Hsp70-targeted peptide induced effective cytotoxic activity toward breast cancer cells through the descrease of Hsp90 client proteins such as p53, Akt, and cRaf. Moreover, the combined treatment with these peptides did not induce the up-regulation of Hsp70 protein, as determined by western blotting, a promoter assay using a luminometer, and single-cell level imaging with the LV200 system, although a small-molecule inhibitor of Hsp90, 17-allylamino-demethoxygeldanamycin (17-AAG), did induce the up-regulation of this protein. We also found that treatment with Antp-TPR, Hsp70-targeted peptide, or a combination of the two did not induce an increase in the glutathione concentrations in the cancer cells. CONCLUSION: These findings suggest that targeting both Hsp90 and Hsp70 with Antp-TPR and Hsp70-targeted peptide is an attractive approach for selective cancer cell killing that might provide potent and selective therapeutic options for the treatment of cancer.

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay.

Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions.

Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O(2) concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in therapeutic applications in the clinic, especially when cells are cultured under hypoxic conditions.

Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells.

BACKGROUND: Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo. RESULTS: In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition. CONCLUSION: These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer.

Targeted deletion of ecto-5'-nucleotidase results in retention of inosine monophosphate content in postmortem muscle of medaka (Oryzias latipes).

Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5'-nucleotidase genes, designated as ecto-5'-nucleotidase-a (nt5ea) and ecto-5'-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP-degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed considerable higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents than that in the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cell lines, and in vivo analysis revealed that this EGFR-lytic peptide displayed significant antitumor activity in a xenograft model of human breast cancer which was resistant to tyrosine kinase inhibitor drugs. As an attempt to improve the selective anticancer activity of EGFR-lytic peptide, we modified the EGFR-binding peptide through introducing the mutation of amino acid according to biophysical analysis by biomolecular interaction and circular dichroism (CD) spectra. When cytotoxic activity of EGFR-lytic or EGFR(2R)-lytic hybrid peptides was investigated in various human cancer and normal cell lines, it was demonstrated that EGFR(2R)-lytic, in which second histidine (H) of EGFR-binding peptide was replaced to arginine (R) had 1.2-1.9-fold higher cytotoxic activity than that of original EGFR-lytic peptide. In vivo analysis also revealed that this modified peptide displayed significant antitumor activity at as low as 1 mg/kg dosage. These results suggest that mutated arginine on EGFR-lytic peptide produces higher binding ability to EGFR on cancer cells, and thereby the improved anticancer activity.

Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.

Designed hybrid TPR peptide targeting Hsp90 as a novel anticancer agent.

BACKGROUND: Despite an ever-improving understanding of the molecular biology of cancer, the treatment of most cancers has not changed dramatically in the past three decades and drugs that do not discriminate between tumor cells and normal tissues remain the mainstays of anticancer therapy. Since Hsp90 is typically involved in cell proliferation and survival, this is thought to play a key role in cancer, and Hsp90 has attracted considerable interest in recent years as a potential therapeutic target. METHODS: We focused on the interaction of Hsp90 with its cofactor protein p60/Hop, and engineered a cell-permeable peptidomimetic, termed "hybrid Antp-TPR peptide", modeled on the binding interface between the molecular chaperone Hsp90 and the TPR2A domain of Hop. RESULTS: It was demonstrated that this designed hybrid Antp-TPR peptide inhibited the interaction of Hsp90 with the TPR2A domain, inducing cell death of breast, pancreatic, renal, lung, prostate, and gastric cancer cell lines in vitro. In contrast, Antp-TPR peptide did not affect the viability of normal cells. Moreover, analysis in vivo revealed that Antp-TPR peptide displayed a significant antitumor activity in a xenograft model of human pancreatic cancer in mice. CONCLUSION: These results indicate that Antp-TPR peptide would provide a potent and selective anticancer therapy to cancer patients.

A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.

BACKGROUND: Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. METHODS: In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. RESULTS: The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC(50) values as low as 4.0-9.3 µM. Normal cells were less sensitive to this molecule, with IC(50) values > 50 µM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47D cancer cells just in 10 min, to effectively kill these cells and induce approximately 80% apoptotic cell death but not in normal cells. The intravenous administration of TfR-lytic peptide in the athymic mice model significantly inhibited tumor progression. CONCLUSIONS: TfR-lytic peptide might provide a potent and selective anticancer therapy for patients.

Protein disulfide isomerase-immunopositive inclusions in patients with amyotrophic lateral sclerosis.

The major pathological hallmarks of amyotrophic lateral sclerosis (ALS) are neuronal cytoplasmic inclusions (NCIs) and swollen neurites. Superoxide dismutase (SOD)-1-immunopositive NCIs are observed in patients with familial ALS (FALS), and TAR DNA-binding protein 43kDa (TDP-43)-immunopositive NCIs are found in patients with sporadic ALS (SALS). Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum. Post mortem spinal cord specimens from five patients with SALS and one with FALS (I113T), and five normal controls were utilized in this immunohistochemical study. We found PDI-immunopositive swollen neurites and NCIs in the patients with ALS. Furthermore, PDI was colocalized with TDP-43 and SOD1 in NCIs. The accumulation of misfolding proteins may disturb axon transport and make swollen neurites. As the motor neuron is the longest cell in the nervous system, the motor system may selectively be disturbed. In conclusion, we assume that PDI is S-nitrosylated in the affected neurons, which inhibits its enzymatic activity and thus allows protein misfolding to occur in ALS.

Characterization of antilytic peptide antibody: application for the detection of lytic-based hybrid peptide in serum samples.

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)-lytic peptide, made by chemical conjugation of targeted binding peptide and cell-killing, lytic-peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR-lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic-peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic-based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA-MB-231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15-30 min of i.v. administration of EGFR-lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic-based peptides to investigate their in vivo stability and pharmacokinetics.

Protein disulfide isomerase immunopositive glial cytoplasmic inclusions in patients with multiple system atrophy.

BACKGROUND: Glial cytoplasmic inclusions (GCIs) are the pathological hallmarks of multiple system atrophy (MSA) and α-synuclein is abnormally deposited in GCIs. Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum (ER). Nitric-oxide-induced (NO-induced) S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response in neurodegenerative diseases. MATERIALS AND METHODS: Postmortem brain specimens from five patients with MSA and five normal control brains were utilized in this immunohistochemical study. RESULTS: We found GCIs positive for anti-PDI antibody in the brain of patients with MSA. In addition, we observed colocalization of α-synuclein and leucine-rich repeat kinase 2 (LRRK2) with PDI in GCIs. As LRRK2 immunoreactivity is associated with one of the earliest oligodendrocytic abnormalities in MSA, colocalization of LRRK2 and PDI in GCIs may be a link to the ER stress of glial cells in the early stages of MSA. CONCLUSIONS: In MSA, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur.

Oral administration of ferulic acid or ethyl ferulate attenuates retinal damage in sodium iodate-induced retinal degeneration mice.

Epidemiological studies indicate that the daily intake of antioxidants from a traditional Asian diet reduces the risk of developing age-related macular degeneration. Many of the phytochemicals that are abundant in whole grains exhibit a wide variety of biological activity such as antioxidant, anti-inflammatory, and neuroprotective effects. Ferulic acid (FA) is a phenolic acid found in vegetables and grains that has therapeutic potential for diabetes mellitus, Alzheimer's disease, and other diseases. We investigated the retinal protective effect of FA in a sodium iodate (NaIO3)-induced model of retinal degeneration. In a human retinal pigment epithelial cell line, FA attenuated H2O2-induced injury and lipopolysaccharide- or 7-ketocholesterol-induced inflammation. In mice, the oral administration of FA or its analog, ethyl ferulate, attenuated the morphological and functional features of NaIO3-induced retinal degeneration according to optical coherence tomography and electroretinography. Our results demonstrate that the oral administration of FA provides protective effects to the retina, suggesting that the intake of FA as a daily supplement or daily healthy diet containing rich vegetables and whole grains may prevent age-related macular degeneration.

Use of telomelysin (OBP-301) in mouse xenografts of human head and neck cancer.

We previously reported that telomerase-specific replication-component adenovirous, Telomelysin (OBP-301) has cytotoxic activity to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro, and investigated the association between cytotoxic activity and adenoviral receptor expression. In this study, we evaluated the most appropriate way to administer telomelysin (OBP-301) in the treatment of squamous cell carcinoma of the head and neck (SCCHN), and assessed the effect of OBP-301 in large subcutaneous KCCT873 human SCCHN tumors in immunodeficient mice. We also compared antitumor responses following three intratumoral (i.t.) injections of OBP-301 given daily, every 2 days or weekly. To investigate the mechanism of the antitumor effect, we evaluated cellular infiltration in treated tumors. OBP-301 showed remarkable antitumor activity against large KCCT873 tumors, and three treatment schedules produced similar antitumor effects. The weekly regimen also significantly reduced the growth of large tumors. Immunochemistry revealed that macrophages, but not natural killer cells, were responsible for tumor regression. A regimen of three weekly injections of OBP-301 has remarkable antitumor effects against large KCCT873 tumors. These results may provide a new platform for treating patients with localized SCCHN.