N-acetyloxfenicine strongly induces mitohormesis in mice as well as in insects.

Mitohormesis defines the increase in fitness induced by adaptive responses to mild mitochondrial stress. Here, we show that N-acetyloxfenicine (NAO) exerted higher thermotolerance than an endogenous mitohormesis inducer, N-acetyltyrosine (NAT). This activity was not observed in armyworm larvae injected with oxfenicine, suggesting the importance of N-acetylation. NAO-induced hormetic effect was triggered by transient perturbation of mitochondria, which causes a small increase in ROS production and leads to retrograde responses including enhanced expression of antioxidant enzyme genes via activation of FoxO transcription factors. Furthermore, pretreatment with NAO significantly repressed stress-induced peroxidation of lipids in mice and growth of colorectal cancer HCT116 cells that had been transplanted into nude mice. Taken together, NAO is a potent mitohormesis inducer that is similar to NAT in terms of structure and functions.

Induction of cellular senescence in fibroblasts through ß1-integrin activation by tenascin-C-derived peptide and its protumor effect.

Tenascin-C is upregulated during inflammation and tumorigenesis, and its expression level is correlated with a poor prognosis in several malignancies. Nevertheless, the substantial role of tenascin-C in cancer progression is poorly understood. Previously, we found that a peptide derived from tenascin-C, termed TNIIIA2, acts directly on tumor cells to activate ß1-integrin and induce malignant progression. Here, we show that ß1-integrin activation by TNIIIA2 in human fibroblasts indirectly contributes to cancer progression through the induction of cellular senescence. Prolonged treatment of fibroblasts with TNIIIA2 induced cellular senescence, as characterized by the suppression of cell growth and the induction of senescence-associated-ß-galactosidase and p16INK4a expression. The production of reactive oxygen species and subsequent DNA damage were responsible for the TNIIIA2-induced senescence of fibroblasts. Interestingly, peptide FNIII14, which inactivates ß1-integrin, inhibited fibroblast senescence induced not only by TNIIIA2 but also by H2O2, suggesting that ß1-integrin activation plays a critical role in the induction of senescence in fibroblasts. Moreover, TNIIIA2-induced senescent fibroblasts secreted heparin-binding epidermal growth factor-like growth factor (HB-EGF), which caused preneoplastic epithelial HaCaT cells to acquire malignant properties, including colony-forming and focus-forming abilities. Thus, our study demonstrates that tenascin-C-derived peptide TNIIIA2 induces cellular senescence in fibroblasts through ß1-integrin activation, causing cancer progression via the secretion of humoral factors such as HB-EGF.

Systematic Design and Validation of Ion Channel Stabilization of Amphipathic α-Helical Peptides Incorporating Tryptophan Residues.

Aromatic interactions such as π-π interaction and cation-π interaction are present in membrane proteins and play important roles in both structure and function. To systematically investigate the effect of aromatic residues on the structural stability and ion permeability of peptide-formed ion channels, we designed several peptides with one or two tryptophan (Trp) residues incorporated at different positions in amphipathic α-helical peptides. Circular dichroism (CD) studies revealed the preferable position of Trp residues for self-association in these designed peptides. Systematically designed di-substituted peptides with two Trps at each helix termini demonstrated intermolecular Trp-Trp interactions caused by aggregation. In the presence of liposomes, Trp on the hydrophilic face of the peptide enhanced interaction with the lipid membrane to increase the amphipathic α-helical contents. Appropriate incorporation and positioning of Trp enabled peptides to form more stable channels and had notable effects with Trp di-substituted peptides. The ion channel forming capability of a series of these peptides showed that the cation-π interactions between Trp and Lys residues in adjacent transmembrane helices contribute to remarkable stabilization of the channel structure.

Anoikis resistance conferred by tenascin-C-derived peptide TNIIIA2 and its disruption by integrin inactivation.

Glioblastoma multiforme (GBM), the most common brain tumor in adults, has an extremely poor prognosis, which is attributed to the aggressive properties of GBM cells, such as dysregulated proliferation and disseminative migration. We recently found that peptide TNIIIA2, derived from tenascin-C (TNC), which is highly expressed in GBM, contributes to the acquisition of these aggressive properties through ß1-integrin activation. In general, cancer cells often acquire an additional malignant property that confers resistance to apoptosis due to loss of adhesion to the extracellular matrix, termed anoikis resistance. Our present results show that regulation of ß1-integrin activation also plays a key role in both the development and loss of anoikis resistance in GBM cells. Despite being derived from a GBM with an extremely poor prognosis, the human GBM cell line T98G was susceptible to anoikis but became anoikis resistant via treatment with peptide TNIIIA2, which is able to activate ß1-integrin. The TNIIIA2-conferred anoikis resistance of T98G cells was disrupted by further addition of peptide FNIII14, which has the ability to inactivate ß1-integrin. Moreover, anchorage-independent survival of GBM cells in suspension culture was abrogated by peptide FNIII14, but not by RGD and CS-1 peptides, which are antagonistic for integrins α5ß1, αvß3, and α4ß1. These results suggest that GBM cells develop anoikis resistance through activation of ß1-integrin by TNC-derived peptide TNIIIA2, which is abundantly released into the tumor microenvironment of GBM. Inactivation of ß1-integrin may provide a promising strategy to overcome the apoptosis resistance of cancer cells, including GBM.

Exposure of the cryptic de-adhesive site FNIII14 in fibronectin molecule and its binding to membrane-type eEF1A induce migration and invasion of cancer cells via ß1-integrin inactivation.

Cell migration is a highly coordinated process that involves not only integrin-mediated adhesion but also de-adhesion. We previously found that a cryptic de-adhesive site within fibronectin molecule, termed FNIII14, weakens cell adhesion to the extracellular matrix by inactivating ß1-integrins. Surprisingly, eukaryotic translation elongation factor-1A (eEF1A), an essential factor during protein biosynthesis, was identified as a membrane receptor that mediates the de-adhesive effect of FNIII14. Here, we demonstrate that FNIII14-mediated de-adhesion causes enhanced migration and invasion in two types of highly invasive/metastatic cancer cells, resulting in the initiation of metastasis. Both in vitro migration and invasion of highly invasive human melanoma cell line, Mum2B, were inhibited by a matrix metalloproteinase (MMP)-2/9 inhibitor or a function-blocking antibody against FNIII14 (anti-FNIII14 Ab), suggesting that MMP-mediated exposure of the cryptic de-adhesive site FNIII14 was responsible for Mum2B cell migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell line 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that the migration and invasion were dependent on the membrane levels of eEF1A. In vivo experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that the anti-FNIII14 Ab has a significant anti-metastatic effect. Thus, these results provide novel insights into the regulation of cancer cell migration and invasion and suggest promising targets for anti-metastasis strategies.

Promotion of cell adhesion by low-molecular-weight hydrogel by Lys based amphiphile.

Hydrogels formed by low-molecular hydrogelators have been used as anti-microbial agents and cell-attachment materials. However the biomedical application of low-molecular gelators is slowly progressing compared to the hydrogels formed by polymer hydrogelator that is applied to biomedical application such as tissue engineering and biomedical regions. To obtain a simple molecular model for potent and prospective usage of low-molecular hydrogelators, we designed a Lys-based hydrogelator which was mimic to the poly cationic poly-l-lysine that promotes cells to attach to a plastic plate nonspecifically. The gel-coating led to cause 10-fold cell attachment compared to no-coating well. Also five-time cells were attached to the well compared to the poly-l-lysine coating. From the competitive assay, these hydrogels could interact with cells through electrostatic interaction between positive charge from -NH3(+) in the hydrogelator and negative charge from substances on the cell surface such as glycosaminoglycans. This strong adhesive ability can be useful for the tissue engineering and molecular glue regions using low-molecular hydrogels in the future.

Development of potent antagonists for formyl peptide receptor 1 based on Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH.

While stimulation of formyl peptide receptors (FPRs) on the surface of human neutrophils induces several immune responses, under conditions of continuous activation of the receptor by agonists such as formyl-Met-Leu-Phe-OH (fMLP), neutrophil-dependent tissue damage ensues. Thus, FPR antagonists could be anticipated as drugs for FPR-related disease. In this study, Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH (Boc-FlFlF), one of several FPR subtype selective antagonists, was chosen and the positions at the Phe residues were optimized. We found that substitution with unnatural amino acids resulted in an improvement of two orders of magnitude. The most potent antagonist indicated FPR subtype selectivity at 1 µM. In addition to finding a potent antagonist, the structure-activity trends observed in this study should be valuable in designing a new type of FPR subtype selective antagonist.

Anti-tumor activities of Au(I) complexed with bisphosphines in HL-60 cells.

We found that Au(I) complexed with 2,3-bis(tert-butyl(methyl)phosphino) quinoxaline (10) was a potent anti-tumor agent (half-maximal growth inhibitory concentration, GI50=0.87µM) with broad anti-tumor activity. In particular, the activity of complex 10 was high in tumor cell lines derived from the colon and ovary. Treatment with complex 10 resulted in the apoptosis of HL-60 cells. The ligand for the preparation of complex 10 is commercially available implying that complex 10 might be a good drug candidate for cancer therapy.

A formyl peptide substituted with a conformationally constrained phenylalanine residue evokes a selective immune response in human neutrophils.

Formyl-Met-Leu-Phe-OH (fMLP) binds to formyl peptide receptors, FPR1 and FPR2, and evokes migration and superoxide anion production in human neutrophils. To obtain a more effective and selective ligand, fMLP analogs in which the Phe residue was substituted with four isomers of cyclopropanephenylalanine were synthesized. While Z-isomer peptides induced both migration and superoxide anion production, E-isomer peptides elicited only chemotaxis. Homologous receptor desensitization experiments revealed that E-isomer peptides bound to FPR2. Although a selective agonist of chemotaxis also binds to FPR2 without increasing intracellular calcium concentration, E-isomer peptide elevated the concentration to the same level as fMLP. Understanding of mechanisms responsible for the selectivity of the reported selective agonists and ∇Phe-substituted analogs should prove useful for revealing the relationship between receptor-ligand interactions and biological responses of human neutrophils.

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues.

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated ß1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.

Formation of ion-selective channel using cyclic tetrapeptides.

It is important for ion channel peptides to have energetic stability and ion-selectivity for development of some medicines. In the present study, our objective was to achieve formation of energetically stable and ion-selective channels in the membrane using cyclic tetrapeptides. We succeeded in formation of energetically stable and ion-selective channels using two cyclic tetrapeptides cyclo(D-Ala-Dap)(2) (Dap; l-2,3-diaminopropionic acid) and cyclo(D-Ala-Glu)(2). The results of ion channel recording suggested that the cationic cyclo(D-Ala-Dap)(2) was resulted in Cl(-) anion-selective and the anionic cyclo(D-Ala-Glu)(2) led to K(+) cation-selective ion channel formation, respectively. This ion selectivity may be attributed to the charge state of peptides. And a low-hydrophobic cyclic tetrapeptide; cyclo(D-Ala-Dap)(2) had a tendency to form stable ion channel compared to more high-hydrophobic ones; cyclo(D-Phe-Lys)(2), cyclo(D-Phe-Dap)(2) and cyclo(D-Ala-Lys)(2). Our findings will shed light on the field of ion channel peptide study, especially cyclic one.

Fluoroalkene modification of mercaptoacetamide-based histone deacetylase inhibitors.

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.

Ion-channel formation assisted by electrostatic interhelical interactions in covalently dimerized amphiphilic helical peptides.

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.

Superoxide production in human neutrophils is enhanced by treatment with transmembrane peptides derived from human formyl peptide receptor.

Formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been performed on the ligand binding site of FPR, FPR dynamic behavior such as receptor dimerization on the cell surface remains unknown. Recently, peptides derived from the transmembrane (TM) domains of GPCRs were shown to disrupt dimer formation by receptors and to result in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from FPR were synthesized, and their biological activities were evaluated with human neutrophils. Synthetic peptides did not exhibit agonistic or antagonistic activity toward superoxide anion production. However, Neutrophils treated with hFPRTM4 produced 4-fold superoxide anion compared with untreated cells when stimulated with FPR agonist fMLP. Short peptide fragments derived from the fourth TM region of FPR did not enhance superoxide anion production, which suggests that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides were inserted into the membrane. The addition of hFPRTM4 increased the intracellular calcium concentration, which meant the peptide activated some membrane protein on the cell surface. The present study suggests that the fourth TM domain of FPR has a function related to a priming effect.

Effect of alpha,alpha-dialkyl amino acids on the protease resistance of peptides.

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of alpha,alpha-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1' positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1' and P2 positions resulted in complete resistance.

Synthesis of chemotactic peptide analogs with a photoaffinity cross-linker as new probes for analysis of receptor-ligand interaction.

Formylpeptide receptors are well-characterized receptors which participate in host defense responses of neutrophils. We designed and synthesized chemotactic peptide analog with p-benzoylphenylalanine (Bpa) and biotin to probe structural and mechanistic aspects of peptide-receptor interaction. These peptides possess biological activities which were dependent upon spacer residue length of and Bpa position. The covalent photoaffinity label was detected by Streptavidine-blot, which was inhibited by the parent peptide.