Receptor affinity of neurotensin message segment immobilized on liposome

AbstractNeurotensin derivatives having a dioctadecyl group were synthesized and immobilized on DMPC liposome to construct a multivalent-ligand system. The derivatives are Ac-Glu[N(C18H37)2]-(Sar-Sar-Pro)n-Arg-Arg-Pro-Tyr-Ile-Leu-OH (D3nNT, n = 0,1,2,3), where a dioctadecyl group was connected to the N-terminal side of neurotensin 8–13 fragment directly or through a hydrophilic and flexible spacer chain of different lengths. The derivatives were spontaneously immobilized on DMPC liposome upon incubation overnight. The receptor affinity of the derivatives increased significantly upon immobilization on liposome. The maximum affinity was obtained by D9NT immobilized on DMPC liposome at the molar ratio of DMPC and D9NT of 200. This affinity is slightly better than the neurotensin 8–13 fragment, the message segment of the derivatives. The fluorescent microscopy using rhodamine-labelled liposome revealed that the multivalent-ligand system binds to specific receptors without dissociation of the derivative from DMPC liposome

Vertical orientation with a narrow distribution of helical peptides immobilized on a quartz substrate by stereocomplex formation

Second-harmonic generation (SHG) of a donor–π–acceptor (D–π–A) chromophore attached to helical peptides was used for the evaluation of the self-assembled monolayer (SAM) structure of a stereocomplex of helical peptides. A stereocomplex SAM of a left-handed helical conjugate (D17) and a right-handed helical conjugate (L17) showed an SHG intensity four times larger than a stereocomplex SAM of a left-handed helical D17 and a right-handed helical peptide without the D–π–A chromophore (LA16), which agrees well with dependence of SHG intensities on the surface densities of the D–π–A chromophore. The SHG intensities of enantiopure SAMs of D17 and L17 are, however, 47% and 27% of the stereocomplex SAM of D17 and L17, respectively. These differences can be explained only after taking a larger distribution of the tilt angle of the chromophore in the enantiopure SAMs than in the stereocomplex SAM of D17 and L17. On the basis of these analyses, it is concluded that the stereocomplex SAM of a left-handed helix and a right-handed helix constitutes a well-ordered structure, where the tilt angle of the helical peptide from the surface normal becomes small with a narrow distribution due to stereocomplex formation

Transformation of peptide nanotubes into a vesicle via fusion driven by stereo-complex formation.

Two types of peptide nanotubes, one is prepared from an amphiphilic peptide having a right-handed helix segment and the other from that having a left-handed helix segment, are shown to transform the morphology into a vesicle by membrane fusion due to stereo-complex formation between these helical segments

Construction and Piezoelectric Properties of a Single-Peptide Nanotube Composed of Cyclic β-peptides with Helical Peptides on the Side Chains

To develop nanopiezoelectronics, it is necessary to investigate the relationship between the sizes and piezoelectric properties of the material. Peptide nanotubes (PNTs) composed of cyclic β-peptides have been studied as leading candidates for nanopiezoelectric materials. The current drawback of PNTs is aggregation to form a PNT bundle structure due to strong dipole–dipole interactions between PNTs. Here, we report the construction and piezoelectric properties of single PNTs without nonspecific aggregation by side-chain modification of helical peptides. A cyclic tri-β-peptide with a helical peptide was prepared by multiple-step liquid-phase peptide synthesis and assembled into PNTs by the vapor diffusion method. These nanotubes were characterized by polarized light microscopy and Fourier transform infrared (FTIR) spectroscopy. Additionally, atomic force microscopy (AFM) topographic images showed nanotubes with a height of 4 nm, which corresponds to the diameter of a PNT on a gold-coated mica substrate, indicating that a single PNT was prepared successfully. The converted piezoelectric response of a single PNT was determined to be 1.39 ± 0.12 pm/V. This value was consistent with that of a PNT bundle, which reveals that the piezoelectricity of PNTs is induced by deformation of their cyclic skeletons and is independent of the bundled structure. This finding not only demonstrates a new molecular design strategy to construct these smallest piezoelectric biomaterials by controlling the supramolecular hierarchical structures but also provides insights into the correlation between molecular assembly morphology and size-dependent piezoelectric properties

MRP3 as a novel resistance factor for sorafenib in hepatocellular carcinoma

The mechanism of resistance of hepatocellular carcinoma (HCC) to sorafenib is unknown and no useful predictive biomarker for sorafenib treatment has been reported. Accordingly, we established sorafenib-resistant HCC cells and investigated the underlying mechanism of resistance to sorafenib. Sorafenib-resistant cell lines were established from the HCC cell line PLC/PRF5 by cultivation under continuous exposure to increasing concentration of sorafenib. The IC50 values of the 2 resistant clones PLC/PRF5-R1 and PLC-PRF5-R2 were 9.2±0.47 μM (1.8-fold) and 25±5.1 μM (4.6-fold) respectively, which were significantly higher than that of parental PLC/PRF5 cells (5.4±0.17 μM) (p<0.01 respectively), as determined by MTT assay. Western blot analysis of signal transduction-related proteins showed no significant differences in expression of AKT/pAKT, mTOR/pmTOR, or ERK/pERK between the 2 resistant clones versus parent cells, suggesting no activation of an alternative signal transduction pathway. Likewise, when expression of membrane transporter proteins was determined, there were no significant differences in expression levels of BSEP, MDR1, MRP2, BCRP, MRP4 and OCT1 between resistant clones and parent cells. However, the expression levels of MRP3 in the 2 resistant clones were significantly higher than that of parent cells. When MRP3 gene was knocked down by siRNA in PLC-PRF5-R2 cells, the sensitivity of the cells to sorafenib was restored. In the analysis of gene mutation, there was no mutation in the activation segment of Raf1 kinase in the resistant clones. Our data clearly demonstrate that the efflux transporter MRP3 plays an important role in resistance to sorafenib in HCC cells

Downregulation of microRNA-100/microRNA-125b

A majority of early colorectal cancers (CRCs) with submucosal invasion undergo surgical operation, despite a very low incidence of lymph node metastasis. Our study aimed to identify microRNAs (miRNAs) specifically responsible for lymph node metastasis in submucosal CRCs. MicroRNA microarray analysis revealed that miR-100 and miR-125b expression levels were significantly lower in CRC tissues with lymph node metastases than in those without metastases. These results were validated by quantitative real-time PCR in a larger set of clinical samples. The transfection of a miR-100 or miR-125b inhibitor into colon cancer HCT116 cells significantly increased cell invasion, migration, and MMP activity. Conversely, overexpression of miR-100 or miR-125b mimics significantly attenuated all these activities but did not affect cell growth. To identify target mRNAs, we undertook a gene expression array analysis of miR-100-silenced HCT116 cells as well as negative control cells. The Ingenuity Pathway Analysis, TargetScan software analyses, and subsequent verification of mRNA expression by real-time PCR identified mammalian target of rapamycin (mTOR) and insulin-like growth factor 1 receptor (IGF1R) as direct, and Fas and X-linked inhibitor-of-apoptosis protein (XIAP) as indirect candidate targets for miR-100 involved in lymph node metastasis. Knockdown of each gene by siRNA significantly reduced the invasiveness of HCT116 cells. These data clearly show that downregulation of miR-100 and miR-125b is closely associated with lymph node metastasis in submucosal CRC through enhancement of invasion, motility, and MMP activity. In particular, miR-100 may promote metastasis by upregulating mTOR, IGF1R, Fas, and XIAP as targets. Thus, miR-100 and miR-125b may be novel biomarkers for lymph node metastasis of early CRCs with submucosal invasion

Preparation of peptide- and protein-based molecular assemblies and their utilizations as nanocarriers for tumor imaging

Three types of nanocarriers from our group for imaging probes are reviewed here. Novel nanocarriers of “peptosome” and “lactosome” were prepared from amphiphilic polymers uniquely having a helical segment as a hydrophobic block. Apoferritin was chemically modified on the surface, and used for nanocontainer of a novel Gd-chelator. Lactosome was intensively studied and labeled with indocyanine, 18F, and 131I for tumor imaging by NIRF, PET, and SPECT, respectively. Those labeled lactosomes are shown to be effective for tumor imaging on the basis of the EPR effect and the stealth property in vivo. The superiority of lactosome over other commercially available imaging agents is experimentally confirmed, which is explained by the dense and thick hydrophilic polymer brush formed on the surface and the helix bundles at the hydrophobic core. Our final goal is to develop the next-generation lactosome, which is the nanocarrier which is usable for a diagnostic agent and a therapeutic agent showing the same in vivo disposition even upon frequent administration