Publications by authors named "Ikuhiko Nakase"

88 Publications

Size and surface modification of silica nanoparticles affect the severity of lung toxicity by modulating endosomal ROS generation in macrophages.

Part Fibre Toxicol 2021 06 17;18(1):21. Epub 2021 Jun 17.

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.

Background: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties.

Results: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 μm; 3 μm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 μm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs.

Conclusions: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.
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http://dx.doi.org/10.1186/s12989-021-00415-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210371PMC
June 2021

A "ligand-targeting" peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis.

PLoS One 2021 25;16(2):e0247045. Epub 2021 Feb 25.

Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247045PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906330PMC
February 2021

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

FEBS Open Bio 2021 03 18;11(3):753-767. Epub 2021 Feb 18.

Keio University School of Medicine, Tsukuba, Japan.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.
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http://dx.doi.org/10.1002/2211-5463.13107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931216PMC
March 2021

Direct entry of cell-penetrating peptide can be controlled by maneuvering the membrane curvature.

Sci Rep 2021 01 8;11(1):31. Epub 2021 Jan 8.

Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis. Here we show the conclusive evidence to support the mechanism of CPP's cytolysis and way to control it. The mechanism we proposed is attributed to biomembrane's physicochemical nature as lamellar liquid crystal (Lα). Cytolysis occurs as the temporal and local dynamic phase transitions from Lα to an undulated lamellar with pores called Mesh. We have shown this phase transfer of Lα composed of dioleoyl-phosphatidylcholine (DOPC) with water by adding oligo-arginine (Rx) as CPP at the equilibrium. Using giant unilamellar vesicle composed of DOPC as a single cell model, we could control the level of cytolysis of CPP (FITC-R8) by changing the curvature of the membrane through osmotic pressure modulation. The cytolysis of CPP utilizes biomembrane's inherent topological and functional flexibility corresponding to the stimuli.
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http://dx.doi.org/10.1038/s41598-020-79518-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794472PMC
January 2021

Antibody-Based Receptor Targeting Using an Fc-Binding Peptide-Dodecaborate Conjugate and Macropinocytosis Induction for Boron Neutron Capture Therapy.

ACS Omega 2020 Sep 2;5(36):22731-22738. Epub 2020 Sep 2.

Research Center of BNCT, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.

Boron neutron capture therapy (BNCT) is a radiation method used for cancer therapy. Cellular uptake of boron-10 (B) atoms induces cancer cell death by the generation of alpha particles and recoiling lithium-7 (Li) nuclei when the cells are irradiated with low-energy thermal neutrons. Current BNCT technology shows effective therapeutic benefits in refractory cancers such as brain tumors and head and neck cancers. However, improvements to cancer targeting and the cellular uptake efficacy of the boron compounds and the expansion of the diseases treatable by BNCT are highly desirable. In this research, we aimed to develop an antibody-based drug delivery method for BNCT through the use of the Z33 peptide, which shows specific recognition of and interaction with the Fc domain of human IgG, for on-demand receptor targeting. In addition, we determined with an assay that macropinocytosis induction during antibody-based drug delivery is crucial for the biological activity of BNCT.
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http://dx.doi.org/10.1021/acsomega.0c01377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495456PMC
September 2020

Key Process and Factors Controlling the Direct Translocation of Cell-Penetrating Peptide through Bio-Membrane.

Int J Mol Sci 2020 Jul 30;21(15). Epub 2020 Jul 30.

Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh, which has punctured bilayer morphologies.
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http://dx.doi.org/10.3390/ijms21155466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432884PMC
July 2020

Association of Hydrophobic Carboxyl-Terminal Dendrimers with Lymph Node-Resident Lymphocytes.

Polymers (Basel) 2020 Jun 30;12(7). Epub 2020 Jun 30.

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

Delivery systems to lymph node-resident T cells around tumor tissues are essential for cancer immunotherapy, in order to boost the immune responses. We previously reported that anionic dendrimers, such as carboxyl-, sulfonyl-, and phosphate-terminal dendrimers, were efficiently accumulated in lymph nodes via the intradermal injection. Depending on the terminal structure, their cell association properties were different, and the carboxyl-terminal dendrimers did not associate with any immune cells majorly. In this study, we investigated the delivery of carboxyl-terminal dendrimers with different hydrophobicity to lymph node-resident lymphocytes. Four types of carboxyl-terminal dendrimers-succinylated (C) and 2-carboxy-cyclohexanoylated (Chex) dendrimers with and without phenylalanine (Phe)-were synthesized and named C-den, C-Phe-den, Chex-den, and Chex-Phe-den, respectively. Chex-Phe-den was well associated with lymphocytes, but others were not. Chex-Phe-den, intradermally injected at the footpads of mice, was accumulated in the lymph node, and was highly associated with the lymphocytes, including T cells. Our results suggest that Chex-Phe-den has the potential for delivery to the lymph node-resident T cells, without any specific T cell-targeted ligands.
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http://dx.doi.org/10.3390/polym12071474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408625PMC
June 2020

Effects of Lyophilization of Arginine-rich Cell-penetrating Peptide-modified Extracellular Vesicles on Intracellular Delivery.

Anticancer Res 2019 Dec;39(12):6701-6709

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan

Background/aim: Extracellular vesicles (exosomes, EVs) (30-200 nm in diameter) are secreted by various cells in the body. Owing to the pharmaceutical advantages of EVs, an EV-based drug delivery system (DDS) for cancer therapy is expected to be the next-generation therapeutic system. However, preservation methods for functional and therapeutic EVs should be developed. Here, we developed the method of lyophilization of arginine-rich cell penetrating peptide (CPP)-modified EVs and investigated the effects of lyophilization on the characteristics of EVs.

Materials And Methods: Particle size, structure, zeta-potential, and cellular uptake efficacy of the arginine-rich CPP-modified EVs were analyzed. The model protein saporin (SAP), having anti-cancer effects, was encapsulated inside the EVs to assess the cytosolic release of EV content after cellular uptake.

Results: Lyophilization of the EVs did not affect their particle size, structure, zeta-potential, and cellular uptake efficacy; however, the biological activity of the encapsulated SAP was inhibited by lyophilization.

Conclusion: Lyophilization of EVs may affect SAP structures and/or reduce the cytosolic release efficacy of EV's content after cellular uptake and needs attention in EV-based DDSs.
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http://dx.doi.org/10.21873/anticanres.13885DOI Listing
December 2019

Intracellular target delivery of cell-penetrating peptide-conjugated dodecaborate for boron neutron capture therapy (BNCT).

Chem Commun (Camb) 2019 Nov;55(93):13955-13958

Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

In this study, we designed and synthesized organelle-targeted cell-penetrating peptide (CPP)-conjugated boron compounds to increase their cellular uptake and to control the intracellular locations for the induction of sophisticated anticancer biological activity in boron neutron capture therapy (BNCT), leading to anticancer effects with ATP reduction and apoptosis when irradiated with neutrons in an in vitro BNCT assay.
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http://dx.doi.org/10.1039/c9cc03924dDOI Listing
November 2019

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Int J Pharm 2019 Dec 11;572:118762. Epub 2019 Oct 11.

NanoSquare Research Institute, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan; Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan. Electronic address:

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899172PMC
December 2019

Optimization of the method for analyzing endocytosis of fluorescently tagged molecules: Impact of incubation in the cell culture medium and cell surface wash with glycine-hydrochloric acid buffer.

J Control Release 2019 09 21;310:127-140. Epub 2019 Aug 21.

Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe, Hyogo 650-8586, Japan.

To obtain the therapeutic effect of biological medicines, such as proteins and nucleic acids, these medicines must achieve their intracellular target, such as the cytoplasm, and pass through biological membrane barriers. Endocytosis is an attractive route for the intracellular delivery of such drugs, and various endocytosis inhibitors have been used as tools to study the involvement of endocytosis in the cell internalization of delivery carriers. However, the specificity of these inhibitors has been insufficiently studied, and our preliminary tests could not detect the expected effect of the well-known endocytosis inhibitors. Therefore, the present study aimed to optimize the experimental conditions to precisely analyze cellular internalization via endocytosis. We first found that incubation of model molecules, such as transferrin (Tf) and cholera toxin subunit B (CTB), in cell culture medium (DMEM) could efficiently induce their internalization to HeLa cells compared to that in transport buffer (HBSS). Moreover, we clarified that cell surface wash with glycine-hydrochloric acid buffer before confocal microscopy and flow cytometry strengthened the intracellular fluorescence of Tf, CTB, and dextran tagged with fluorescent probes possibly via the neutralization of endosomal pH. Even under the optimized condition, however, the specificity of endocytosis inhibitors was disputable. The present study suggested the importance of the optimization of the study design with endocytosis inhibitors in analyzing cellular internalization.
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http://dx.doi.org/10.1016/j.jconrel.2019.08.020DOI Listing
September 2019

Peptides with the multibasic cleavage site of the hemagglutinin from highly pathogenic influenza viruses act as cell-penetrating via binding to heparan sulfate and neuropilins.

Biochem Biophys Res Commun 2019 05 21;512(3):453-459. Epub 2019 Mar 21.

Vaccine Creation Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan; BIKEN Center for Innovative Vaccine Research and Development, The Research Foundation for Microbial Diseases of Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan; Laboratory of Nano-design for Innovative Drug Development, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan; Global Center for Medical Engineering and Informatics, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address:

Cell-penetrating peptides (CPPs) show promise as an attractive delivery vehicle for therapeutic molecules-including nucleic acids, peptides, proteins, and even particulates-into several cell types. It is important to identify new CPPs and select the optimal CPP for each application, because CPPs differ in their internalized efficiency and internalization mechanisms. Here, we identified new CPPs derived from the peptides with the hemagglutinin cleavage site (pHACS) of highly pathogenic influenza viruses. We compared the potential of peptides from the pHACS of four subtypes of influenza A virus (H1, H3, H5, and H7) and an influenza B virus (H1-pHACS, H3-pHACS, H5-pHACS, H7-pHACS, and B-pHACS, respectively) to serve as CPPs. H5-pHACS and H7-pHACS, but not the other peptides, bound to mouse dendritic cells and human epithelial cells and were internalized efficiently into these cells. H5-pHACS and H7-pHACS required glycosaminoglycans, especially heparan sulfate and neuropilins, to bind to the cells. In addition, we designed a mutant H7-pHACS with superior cell-binding capability by changing a single amino acid. Furthermore, when conjugated with antigen, H5-pHACS and H7-pHACS induced antigen-specific antibody responses, demonstrating the usefulness of this antigen-delivery vehicle. Our results will improve our understanding of the mechanisms of CPPs and facilitate the development of novel drug-delivery vehicles designed to improve therapeutic efficacy.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.068DOI Listing
May 2019

Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to Cytosol via Caveolae-Mediated Endocytosis.

Biomacromolecules 2019 05 1;20(5):1849-1859. Epub 2019 Apr 1.

Institute for Chemical Research , Kyoto University , Uji , Kyoto 611-0011 , Japan.

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.
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http://dx.doi.org/10.1021/acs.biomac.8b01299DOI Listing
May 2019

Rapid optical tissue clearing using poly(acrylamide-co-styrenesulfonate) hydrogels for three-dimensional imaging.

J Biomed Mater Res B Appl Biomater 2019 10 28;107(7):2297-2304. Epub 2019 Jan 28.

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.

Optical tissue clearing methods are attractive for three-dimensional imaging of intact tissues and organs. A CLARITY (clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ-hybridization-compatible tissue-hydrogel) method using polyacrylamide gels was recently developed. In the current study, we used poly(acrylamide-co-styrenesulfonate) gels as a polyelectrolyte gel for a passive CLARITY method. First, radical copolymerization of acrylamide (AAm) and sodium p-styrenesulfonate (SS) at different ratios was performed. The composition of the copolymer could be controlled by changing the monomer ratio. With increased SS content, the molecular weight decreased and the refractive index slightly increased. The poly(AAm-co-SS) hydrogels with a higher SS content were more swollen and looser. The clearing time could be reduced using the poly(AAm-co-SS) hydrogel with a higher SS content. Three-dimensional fluorescence imaging of the poly(AAm-co-SS) hydrogel-cleared tissues was successfully performed after treatment using a blue fluorescent DNA stain. Therefore, the use of the poly(AAm-co-SS) hydrogel improved the clearing process in the passive CLARITY method, and the technique is useful for fluorescence imaging of DNA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2297-2304, 2019.
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http://dx.doi.org/10.1002/jbm.b.34322DOI Listing
October 2019

Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells.

Biochem Biophys Res Commun 2018 12 13;507(1-4):297-303. Epub 2018 Nov 13.

Graduate School of Science, Osaka Prefecture University, 1-1 Gakuencho, Sakai-shi, Osaka, 599-8570, Japan.

Lung branching morphogenesis is a complex system involving many molecular interactions to filling the three dimensional spaces; however, the underlying developmental mechanisms are still not fully understood. In this paper, we have investigated the effect of epidermal growth factor (EGF) on normal human bronchial epithelial cells and their three-dimensional (3D) branching pattern formation by using in vitro experiments and mathematical simulation. The results show that EGF is essential for 3D branch pattern formation and its receptor is highly expressed at the tip of branches to generate the drive force for cells to migrate. Macropinocytosis induced by EGFR expression is firmly contributed to the nutrition uptake at the tip of branches. Our findings for effective branching formation of human lung cells contribute to further understanding molecular mechanisms of organogenesis, and the important mechanisms also possibly participate in related lung disease such as malformation.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.028DOI Listing
December 2018

Modular Redesign of a Cationic Lytic Peptide To Promote the Endosomal Escape of Biomacromolecules.

Angew Chem Int Ed Engl 2018 09 30;57(39):12771-12774. Epub 2018 Aug 30.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri-glutamate unit to the N-terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a Ni -dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live-cell imaging of acetylated histones.
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http://dx.doi.org/10.1002/anie.201807534DOI Listing
September 2018

In Situ Ligation of High- and Low-Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition.

Adv Sci (Weinh) 2017 11 28;4(11):1700147. Epub 2017 Jul 28.

Biofunctional Synthetic Chemistry Laboratory RIKEN Hirosawa Wako-shi Saitama 351-0198 Japan.

This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high- and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets.
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http://dx.doi.org/10.1002/advs.201700147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700463PMC
November 2017

Potential Roles of GLUT12 for Glucose Sensing and Cellular Migration in MCF-7 Human Breast Cancer Cells Under High Glucose Conditions.

Anticancer Res 2017 12;37(12):6715-6722

Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan

Background/aim: Recent reports have indicated that hyperglycaemia is associated with breast cancer progression. High glucose conditions corresponding to hyperglycaemia significantly promote migration of MCF-7 human breast cancer cells, however, little is known about the mechanisms of glucose sensing for the acquisition of migratory properties by MCF-7 cells. This study investigated glucose sensing and mediation, which are responsible for the high motility of MCF-7 cells.

Materials And Methods: We evaluated the migration of MCF-7 cells cultured in high glucose-containing medium and essential regulatory factors from the perspective of the glucose transport system.

Results: We demonstrated that glucose transporter 12 (GLUT12) protein level increased in MCF-7 cells and co-localized with actin organization under high glucose conditions. Moreover, GLUT12-knockdown completely abrogated high glucose-induced migration, indicating that GLUT12 functionally participates in sensing high glucose concentrations.

Conclusion: GLUT12 plays a critical role in the model of breast cancer progression through high glucose concentrations.
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http://dx.doi.org/10.21873/anticanres.12130DOI Listing
December 2017

Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions).

Toxins (Basel) 2017 10 12;9(10). Epub 2017 Oct 12.

Urological Research Institute, Division of Experimental Oncology, IRCCS San Raffaele Hospital, 20132 Milan, Italy.

Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications.
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http://dx.doi.org/10.3390/toxins9100314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666361PMC
October 2017

Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization.

Acc Chem Res 2017 10 14;50(10):2449-2456. Epub 2017 Sep 14.

Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University , Naka-ku, Sakai, Osaka 599-8570, Japan.

One of the recent hot topics in peptide-related chemical biology research is the potential of cell-penetrating peptides (CPPs). Owing to their ability to deliver exogenous molecules into cells easily and effectively, their flexible design that allows transporters to comprise various chemical structures and functions, and their potential in chemical and cell biology studies and clinical applications, CPPs have been attracting enormous interest among researchers in related fields. Consequently, publications on CPPs have increased significantly. Although there are many types of CPPs with different physicochemical properties and applications, arginine-rich CPPs, which include the human immunodeficiency virus type 1 (HIV-1) TAT peptide and oligoarginines, are among the most extensively employed and studied. Previous studies demonstrated the importance of the guanidino group in arginine, which confers flexibility in transporter design. Therefore, in addition to peptides, various transporters rich in guanidino groups, which do not necessarily share specific chemical and three-dimensional structures, have been developed. Typically, cell-penetrating transporters have 6-12 guanidino groups. Since the pK of the guanidino group in arginine is approximately 12.5, these molecules are highly basic and hydrophilic. Our group is interested in why these cationic molecules can penetrate cells. Understanding their mechanism of action should lead to the rational design of intracellular delivery systems that have high efficacy. Additionally, novel cellular uptake mechanisms may be elucidated during the course of these studies. Therefore, our group is trying to understand the basic aspects underlying the ability of these peptides to penetrate cells. Regarding the delivery of biopharmaceuticals including proteins and nucleic acids, achieving efficient and effective delivery to target organs and cells is one of the biggest challenges. Furthermore, when the target sites of these drug molecules are within cells, effective cell penetration becomes another obstacle. Cells are surrounded by a membrane that separates the inside of the cell from its outside. This barrier function is critical for keeping cellular contents inside cells, and without this, cells cannot function. Therefore, understanding the mechanism of action of CPPs is necessary to overcome these obstacles and will allow us not only to improve CPP-mediated delivery but also to create other types of intracellular delivery systems. In this Account, we summarize the current knowledge on the mechanisms of internalization of arginine-rich CPPs, from the viewpoints of both direct cell-membrane penetration (i.e., physicochemical aspects) and endocytic uptake (i.e., physiological aspects), and discuss the implications of this knowledge. We also discussed loosening of lipid packing as a factor to promote direct cell-membrane penetration.
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http://dx.doi.org/10.1021/acs.accounts.7b00221DOI Listing
October 2017

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with Mutations at a High Cell Density.

Anticancer Res 2017 09;37(9):4779-4788

NanoSquare Research Institution, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Japan

Background/aim: Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density.

Materials And Methods: We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities.

Results: The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-x and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death.

Conclusion: Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.
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http://dx.doi.org/10.21873/anticanres.11884DOI Listing
September 2017

Zinc and its transporter ZIP6 are key mediators of breast cancer cell survival under high glucose conditions.

FEBS Lett 2017 10 4;591(20):3348-3359. Epub 2017 Sep 4.

Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo, Japan.

Recent studies have shown that hyperglycaemia is related to breast cancer progression; however, the mechanisms underlying the relationship between hyperglycaemia and breast cancer cell survival remain unknown. Here, we demonstrate that as compared to physiological glucose conditions, high glucose conditions promote a significant increase in MCF-7 cell survival under hypoxia. High glucose levels inhibit apoptosis and induce epithelial-to-mesenchymal transition, resulting in increased cell viability under hypoxic conditions. Moreover, high glucose-treated cells display significant increases in intracellular Zn levels and reduction in mRNA expression of the zinc (Zn) transporter Zrt- and Irt-like protein 6 (ZIP6) in hypoxia. ZIP6 deficiency disturbs intracellular Zn homeostasis, leading to increased cell survival in hypoxia and reduced E-cadherin expression, indicating that decreased ZIP6 expression is strongly associated with resistance to hypoxia.
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http://dx.doi.org/10.1002/1873-3468.12797DOI Listing
October 2017

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide.

Nat Chem 2017 08 22;9(8):751-761. Epub 2017 May 22.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.
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http://dx.doi.org/10.1038/nchem.2779DOI Listing
August 2017

Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery.

Sci Rep 2017 05 16;7(1):1991. Epub 2017 May 16.

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.
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http://dx.doi.org/10.1038/s41598-017-02014-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434003PMC
May 2017

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

Sci Rep 2016 10 17;6:34937. Epub 2016 Oct 17.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.
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http://dx.doi.org/10.1038/srep34937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066177PMC
October 2016

Calmodulin EF-hand peptides as Ca -switchable recognition tags.

Biopolymers 2017 Jan;108(1)

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Calmodulin is a representative calcium-binding protein comprised of four Ca -binding motifs with a helix-loop-helix structure (EF-hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF-hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N- or C-terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild-type protein. Heterodimer formation was shown to be a function of the Ca concentration, suggesting that these structures may be used as Ca -switchable recognition tags. An example of an "EF-tag" system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2016.
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http://dx.doi.org/10.1002/bip.22937DOI Listing
January 2017

A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein-Protein Interactions by Epitope and Arginine Grafting.

Angew Chem Int Ed Engl 2016 08 28;55(36):10612-5. Epub 2016 Jul 28.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Osaka, 599-8531, Japan.

The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.
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http://dx.doi.org/10.1002/anie.201603230DOI Listing
August 2016

Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N-terminal helix derived from adenovirus internal protein VI as a model.

Biopolymers 2016 Nov;106(4):430-9

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

The N-terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33-55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild-type AdVpVI (33-55) (WT) and phenylalanine-substituted peptides (L40F) demonstrated that both peptides have positive membrane-curvature-inducing ability. These peptides showed higher binding affinity to 50-nm large unilamellar vesicles (LUVs) than to 200-nm LUVs. However, no enhanced perturbation by these peptides was observed for 50-nm LUVs compared to 200-nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50-nm LUVs than for 200-nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016.
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http://dx.doi.org/10.1002/bip.22797DOI Listing
November 2016

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

Bioconjug Chem 2016 Apr 5;27(4):1119-30. Epub 2016 Apr 5.

Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00082DOI Listing
April 2016

Suppressive effect of membrane-permeable peptides derived from autophosphorylation sites of the IGF-1 receptor on breast cancer cells.

Eur J Pharmacol 2015 Oct 12;765:24-33. Epub 2015 Aug 12.

Department of Anesthesiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan.

Insulin-like growth factor-1 (IGF-1) receptors play a crucial role in the biology of human cancer, making them an attractive target for anti-cancer agents. We previously designed oligopeptides containing the amino-acid sequences surrounding the autophosphorylation sites of the insulin receptor and found that two of them, namely, Ac-DIYET-NH2 and Ac-DYYRK-NH2, suppressed phosphorylation of purified insulin receptors in a non-ATP-competitive manner, whereas Ac-NIYQT-NH2 and Ac-NYYRK-NH2 suppressed in an ATP-competitive manner. Because the IGF-1 receptor is closely related to the insulin receptor, the aim of this study was to observe the effects of these peptides, which correspond to the amino-acid sequences of the autophosphorylation sites of the IGF-1 receptor, on the activity of the human breast cancer cell lines MCF-7, T47D, MDA-MB-231, and MDA-MB-453. To facilitate peptide delivery into breast cancer cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to these peptides. When breast cancer cells were treated with each of these synthetic Tat-conjugated peptides, the conjugated peptides penetrated into the cells and suppressed cell proliferation. An inhibitory effect of Tat-conjugated peptides against IGF-1-stimulated phosphorylation of IGF-1 receptors was observed. In addition, we found that combinations of these peptides suppressed phosphorylation of IGF-1 receptors to a greater extent than the peptides did individually. In conclusion, IGF-1 receptor autophosphorylation site-derived membrane-permeable peptides have the potential to suppress IGF-1 receptor function in breast cancer cells and to be developed into novel and useful agents for cancer therapy.
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http://dx.doi.org/10.1016/j.ejphar.2015.08.004DOI Listing
October 2015
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