Publications by authors named "Tomoka Takatani-Nakase"

33 Publications

Potentiating the Membrane Interaction of an Attenuated Cationic Amphiphilic Lytic Peptide for Intracellular Protein Delivery by Anchoring with Pyrene Moiety.

Bioconjug Chem 2021 05 16;32(5):950-957. Epub 2021 Apr 16.

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

We previously reported an approach for intracellular protein delivery by attenuating membrane-lytic activity of cationic amphiphilic peptides on cell surfaces. HAad is one such peptides that cytosolically delivers proteins of interest, including antibodies, by stimulating their endosomal escape. Additionally, HAad elicits ruffling of cell membrane, accompanied by transient membrane permeabilization, allowing for the efficient cytosolic translocation of proteins. In this study, we prepared a conjugate of HAad with pyrenebutyric acid as a membrane-anchoring unit (pBu-HAad). pBu-HAad demonstrated protein delivery into cells with only 1/20 concentration of HAad. However, the conjugates with cholesteryl hemisuccinate and aliphatic fatty acids (C = 3, 6, and 10) did not yield such marked effects. The results of time-course and inhibitor studies suggest that the membrane anchoring of HAad by a pyrene moiety leads to enhanced peptide-membrane interaction and to loosen lipid packing, thus facilitating cytosolic translocation through membranes.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00101DOI Listing
May 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

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

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

An influenza-derived membrane tension-modulating peptide regulates cell movement and morphology via actin remodeling.

Commun Biol 2019 26;2:243. Epub 2019 Jun 26.

1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 Japan.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.
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http://dx.doi.org/10.1038/s42003-019-0486-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594980PMC
May 2020

Application of Near-Infrared Spectrometry to Evaluate the Mechanism of Wet Granulation Using a High-Speed Mixer with Porous Calcium Silicate and Sugar Alcohols.

Chem Pharm Bull (Tokyo) 2018 ;66(11):1027-1034

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

The current study examined the mechanism of wet granulation with a high-speed mixer using porous calcium silicate (PCS) as the excipient and low-molecular-weight sugar alcohols (erythritol, mannitol, maltitol, or xylitol) as the binder. Granules did not form when erythritol or mannitol was used as the binder. No major changes in X-ray powder diffraction data and near-infrared (NIR) spectroscopy spectra were noted when erythritol was used in granulation. Meanwhile, granules formed when xylitol was used as the binder. The NIR spectra of the obtained granules had a widened band near 5100 cm due to hydroxyl groups. From the peak fitting near 4800 cm using the Gaussian-Lorentzian product function, the contribution of hydrogen bonds in water species increased during granulation. The NIR spectra of these potential binders revealed a band of hydroxyl groups for intramolecular hydrogen bonds near 6900 cm when maltitol or xylitol was used as the binder, whereas no band was observed for erythritol and mannitol. Changes in the NIR spectra assigned to water were useful in evaluating the progression of granulation as a process analytical technology. Moreover, X-ray powder diffraction illustrated that the peak due to xylitol crystals disappeared. Xylitol existed in an amorphous state in the granules, suggesting molecular interactions. Thus, hydroxyl groups in sugar alcohols facilitate hydrogen bonding between PCS (silanol groups) and molecules via water, and this is considered to be the mechanism by which granules are formed.
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http://dx.doi.org/10.1248/cpb.c18-00474DOI Listing
November 2018

Zinc Transporters and the Progression of Breast Cancers.

Biol Pharm Bull 2018 ;41(10):1517-1522

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

Zinc (Zn) is an essential heavy metal utilized in numerous biological processes in mammals, including its recently described role as a signaling mediator. The movement of Zn in and out of cells, across membranes, is regulated by two protein families: the zinc-regulated transporter (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) and the Zn transporter (ZnT) families. ZIPs and ZnTs maintain intracellular Zn homeostasis and control important cellular functions through Zn signaling. Recent studies have highlighted the role of Zn transporters and Zn in disease. ZIP6, 7, and 10 contribute to human breast cancer progression. ZIP6 is associated with breast tumor grade, size, and stage, suggesting that it is a potent driving force toward malignancy; ZIP7 plays an important role in tamoxifen-resistant breast cancer cells, and ZIP10 is involved in invasion and metastasis of breast cancer cells. These Zn transporters are key molecules in the malignant process; thus, understanding Zn transporters will lead to novel diagnostic and therapeutic strategies for breast cancer. This review discusses the emerging functional roles of Zn and Zn transporters in breast cancer.
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http://dx.doi.org/10.1248/bpb.b18-00086DOI Listing
January 2019

High-Glucose Conditions Promote Anchorage-Independent Colony Growth in Human Breast Cancer MCF-7 Cells.

Biol Pharm Bull 2018 ;41(9):1379-1383

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

Previous studies have shown that hyperglycemia is connected to the malignant progression of breast cancer; however, the effects of hyperglycemia on tumorigenic potential in breast cancer cells are largely unknown. Here, we demonstrated that the ability of the human breast cancer cell line MCF-7 to undertake anchorage-independent colony growth was significantly enhanced when cultured under high-glucose conditions compared with that under physiological glucose conditions. The high-glucose conditions also promoted phosphorylation of Akt, suggesting that MCF-7 cells cultured in these conditions acquired an increased ability to undergo anchorage-independent growth at least in part through Akt activation, which has been linked to the development of breast cancer. These results raise the possibility that regulation of Akt activity contributes to the tumorigenesis of breast cancer under high-glucose conditions, and we propose that additional analyses of high glucose-induced tumor formation would provide novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.
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http://dx.doi.org/10.1248/bpb.b18-00174DOI Listing
November 2018

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

Anti-inflammatory effects of water extract from bell pepper (. var. ) leaves .

Exp Ther Med 2017 Nov 5;14(5):4349-4355. Epub 2017 Sep 5.

Department of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan.

Fruits and vegetables have been recognized as natural sources of various bioactive compounds. Peppers, one such natural source, are consumed worldwide as spice crops. They additionally have an important role in traditional medicine, as a result of their antioxidant bioactivity via radical scavenging. However, there are no reports regarding the bioactivity of the bell pepper (. ), a commonly used edible vegetable. The present study aimed to evaluate the anti-inflammatory effect of water extract from bell pepper leaves on mouse spleen cells, and explore the potential mechanism underlying this effect. The extract was prepared through homogenization of bell pepper leaves in deionized water. The sterilized supernatant was added to a mouse spleen cell culture stimulated by concanavalin A. Following 72 h of culture, the levels of inflammatory cytokines in the culture supernatant were measured using a sandwich enzyme-linked immunosorbent assay system, and levels of inflammatory proteins were assessed using western blotting. The bell pepper leaf extract significantly inhibited inflammatory cytokine production, inhibited cell proliferation without producing cytotoxicity, and suppressed the expression of inflammatory proteins. These results suggest that components of the bell pepper leaf extract possess anti-inflammatory activity. The study of the anti-inflammatory mechanism of bell pepper leaf extract has provided useful information on its potential for therapeutic application.
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http://dx.doi.org/10.3892/etm.2017.5106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658767PMC
November 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

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells.

Angew Chem Int Ed Engl 2017 06;56(26):7644-7647

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

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.
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http://dx.doi.org/10.1002/anie.201703578DOI Listing
June 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

Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells.

BMC Neurosci 2017 01 17;18(1):14. Epub 2017 Jan 17.

Laboratory of Pathophysiology and Pharmacotherapeutics, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka, 584-8540, Japan.

Background: Poly(ADP-ribose) polymerase 1 (PARP-1), which catalyzes poly(ADP-ribosyl)ation of proteins by using NAD as a substrate, plays a key role in several nuclear events, including DNA repair, replication, and transcription. Recently, PARP-1 was reported to participate in the somatic cell reprogramming process. Previously, we revealed a role for PARP-1 in the induction of neural apoptosis in a cellular model of cerebral ischemia and suggested the possible use of PARP inhibitors as a new therapeutic intervention. In the present study, we examined the effects of PARP inhibitors on neural stem/progenitor cells (NSPCs) of the mouse brain.

Results: PARP-1 was more abundant and demonstrated higher activity in NSPCs than in mouse embryonic fibroblasts. Treatment with PARP inhibitors suppressed the formation of neurospheres by NSPCs through the suppression of cell cycle progression and the induction of apoptosis. In order to identify the genes responsible for these effects, we investigated gene expression profiles by microarray analyses and found that several genes in the p53 signaling pathway were upregulated, including Cdkn1a, which is critical for cell cycle control, and Fas, Pidd, Pmaip1, and Bbc3, which are principal factors in the apoptosis pathway. Inhibition of poly(ADP-ribosyl)ation increased the levels of p53 protein, but not p53 mRNA, and enhanced the phosphorylation of p53 at Ser18. Experiments with specific inhibitors and also shRNA demonstrated that PARP-1, but not PARP-2, has a role in the regulation of p53. The effects of PARP inhibitors on NSPCs were not observed in Trp53 NSPCs, suggesting a key role for p53 in these events.

Conclusions: On the basis of the finding that PARP inhibitors facilitated the p53 signaling pathway, we propose that poly(ADP-ribosyl)ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation.
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http://dx.doi.org/10.1186/s12868-016-0333-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240207PMC
January 2017

Photostable Solid Dispersion of Nifedipine by Porous Calcium Silicate.

Chem Pharm Bull (Tokyo) 2016 ;64(8):1218-21

Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University.

Nifedipine (NIF) is a typical light-sensitive drug requiring protection from light during manufacture, storage, and handling of its dosage forms. The purpose of this study was to evaluate the utility of porous calcium silicate (PCS) for maintaining the photostability of NIF in a solid dispersion formulation. Adsorption solid dispersion (ASD) prepared using NIF and PCS as an amorphous formulation was more stable to light irradiation than a physical mixture of NIF and microcrystalline cellulose (a control physical mixture) as a crystalline formulation. In addition, PCS in physical mixtures with NIF adequately protected NIF from photodegradation, suggesting that this protective effect could be because of some screening effect by the porous structure of PCS blocking the passage of light reaching NIF in pores of PCS. These findings suggest that PCS is useful for improving the solubility and photostability of NIF in solid dispersion formulation.
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http://dx.doi.org/10.1248/cpb.c15-00978DOI Listing
February 2017

Preparation and Evaluation of Solid Dispersion Tablets by a Simple and Manufacturable Wet Granulation Method Using Porous Calcium Silicate.

Chem Pharm Bull (Tokyo) 2016 ;64(4):311-8

Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University.

The aim of this study was to prepare and evaluate solid dispersion tablets containing a poorly water-soluble drug using porous calcium silicate (PCS) by a wet granulation method. Nifedipine (NIF) was used as the model poorly water-soluble drug. Solid dispersion tablets were prepared with the wet granulation method using ethanol and water by a high-speed mixer granulator. The binder and disintegrant were selected from 7 and 4 candidates, respectively. The dissolution test was conducted using the JP 16 paddle method. The oral absorption of NIF was studied in fasted rats. Xylitol and crospovidone were selected as the binder and disintegrant, respectively. The dissolution rates of NIF from solid dispersion formulations were markedly enhanced compared with NIF powder and physical mixtures. Powder X-ray diffraction (PXRD) confirmed the reduced crystallinity of NIF in the solid dispersion formulations. Fourier transform infrared (FT-IR) showed the physical interaction between NIF and PCS in the solid dispersion formulations. NIF is present in an amorphous state in granules prepared by the wet granulation method using water. The area under the plasma concentration-time curve (AUC) and peak concentration (C(max)) values of NIF after dosing rats with the solid dispersion granules were significantly greater than those after dosing with NIF powder. The solid dispersion formulations of NIF prepared with PCS using the wet granulation method exhibited accelerated dissolution rates and superior oral bioavailability. This method is very simple, and may be applicable to the development of other poorly water-soluble drugs.
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http://dx.doi.org/10.1248/cpb.c15-00838DOI Listing
December 2016

Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

Sci Rep 2015 Jun 3;5:10300. Epub 2015 Jun 3.

1] Keio Advanced Research Centers (KARC), Keio University, Tsukuba, Ibaraki 300-2611, Japan [2] Institute for Advanced Sciences, Toagosei Co., Ltd., Tsukuba, Ibaraki 300-2611, Japan.

Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.
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http://dx.doi.org/10.1038/srep10300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453128PMC
June 2015

Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA(2) activity.

Biochem Biophys Res Commun 2015 Jul 17-24;463(1-2):13-7. Epub 2015 May 13.

Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, 11-68, Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan. Electronic address:

Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA2, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death.
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http://dx.doi.org/10.1016/j.bbrc.2015.05.008DOI Listing
August 2015

High glucose level promotes migration behavior of breast cancer cells through zinc and its transporters.

PLoS One 2014 28;9(2):e90136. Epub 2014 Feb 28.

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

Background: The diabetes patients have been associated with an increased risk of mortality by breast cancer and there are difference between the breast cancer patients with diabetes, and their nondiabetic counterparts in the regimen choice and effects of breast cancer treatment. However, the pathophysiological relationships of diabetes and breast cancer have not yet been elucidated in detail. In this study, we investigate the breast cancer cell line, MCF-7 motility, which linked to invasion and metastasis, in high glucose level corresponding to hyperglycemia and the role of Zn and its transporter.

Methodology/principal Findings: We demonstrated the significant motility of MCF-7 cultured in hyperglycemic level (25 mM glucose) in comparison to normal physiological glucose level (5.5 mM glucose). The other hand, the osmotic control medium, 5.5 mM glucose with 19.5 mM mannitol or fructose had no effect on migratory, suggesting that high glucose level promotes the migration of MCF-7. Moreover, the activity of intracellular Zn(2+) uptake significantly increased in high glucose-treated cells in comparison to 5.5 mM glucose, and the mRNA expression of zinc transporters, ZIP6 and ZIP10, was upregulated in 25 mM glucose-treated cells. The deficiency of ZIP6 or ZIP10 and intracellular Zn(2+) significantly inhibited the high migration activity in 25 mM glucose medium, indicating that Zn(2+) transported via ZIP6 and ZIP10 play an essential role in the promotion of cell motility by high glucose stimulation.

Conclusion/significance: Zinc and its transporters, ZIP6 and ZIP10, are required for the motility stimulated with high glucose level. These findings provide the first evidence proposing the novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090136PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938647PMC
October 2014

[Artemisinin: a natural product for fighting against cancer].

Nihon Yakurigaku Zasshi 2014 Feb;143(2):61-4

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http://dx.doi.org/10.1254/fpj.143.61DOI Listing
February 2014

Migration behavior of breast cancer cells in the environment of high glucose level and the role of zinc and its transporter.

Yakugaku Zasshi 2013 ;133(11):1195-9

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

The diabetes patients have been associated with an increased risk of mortality by breast cancer, and there are differences in the regimen choice and effects of breast cancer treatment between the breast cancer patients with diabetes and their nondiabetic counterparts. However, the pathophysiological relationships of diabetes and breast cancer have not yet been elucidated in detail, therefore its evaluation is strongly required to achieve novel treatment strategies for breast cancer with hyperglycemia. Extracellular circumstances of cancer cells can influence the growth and behavior, resulting in invasion, metastasis and tumor development. We demonstrated that breast cancer cells, MCF-7, cultured in hyperglycemic level significantly promotes the motile activity in comparison to normal physiological glucose level. Moreover, Zn(2+) uptake activity into cellular cytosol and the mRNA expression of zinc transporters, ZIP6 and ZIP10, in the high glucose-exposed cells were shown to be especially higher than in the physiological glucose level. The depletion of intracellular Zn(2+) by zinc chelation and ZIP6 or ZIP10 knockdown blocked the high migration activity, indicating that Zn(2+) transported via ZIP6 and ZIP10 plays an essential role in the promotion of cell motility stimulated in high glucose level. These findings provide a proposing target of the novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.
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http://dx.doi.org/10.1248/yakushi.13-00197DOI Listing
November 2014

CXCR4 stimulates macropinocytosis: implications for cellular uptake of arginine-rich cell-penetrating peptides and HIV.

Chem Biol 2012 Nov;19(11):1437-46

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

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.
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http://dx.doi.org/10.1016/j.chembiol.2012.09.011DOI Listing
November 2012

Transcutaneous immunization system using a hydrotropic formulation induces a potent antigen-specific antibody response.

PLoS One 2012 24;7(10):e47980. Epub 2012 Oct 24.

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

Background: Transcutaneous immunization (TCI) is a novel vaccination strategy, which is expected to have therapeutic applications. However, to develop effective TCI systems, a simple, non-invasive and safe transdermal formulation is required. This study developed a novel TCI system utilizing the co-administration of a liposoluble absorption enhancer, propylene glycol monocaprylate (PGMC) and hydrosoluble protein antigen without pretreatment of any typical adjuvants and disruption of the skin. Novel transdermal formulations were also prepared with sodium salicylate (NaSal) as a hydrotropic agent to improve the solubility of poorly water-soluble substances.

Methodology/principal Findings: The TCI system, which used a transdermal formulation containing hen lysozyme (HEL) and PGMC, solubilized with NaSal, resulted in a substantial HEL-specific antibody response in an HEL dose-dependent manner even in the absence of potent adjuvants, such as cholera toxin (CT). We also investigated whether NaSal activates antigen-presenting cells in vitro to clarify the mechanisms of antibody production by the hydrotropic formulation. NaSal enhanced the expression of MHC class II molecules and increased the production of IL-12 and TNF-α in dendritic cells, which were stimulated by lipopolysaccharide in vitro, indicating that NaSal had an effective adjuvant-like property. Moreover, the use of NaSal in the TCI system did not induce an HEL-specific, IgE-dependent anaphylactic reaction.

Conclusion/significance: Our TCI system using a hydrotropic formulation effectively and safely induced the intended immune response, and this system thus represents a new advantageous method that will result in improved TCI strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047980PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480500PMC
April 2013

Curdione Plays an Important Role in the Inhibitory Effect of Curcuma aromatica on CYP3A4 in Caco-2 Cells.

Evid Based Complement Alternat Med 2011 13;2011:913898. Epub 2011 Mar 13.

Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, 9-11-68 koushien, Nishinomiya City, Hyogo, Japan.

Curcuma aromatica is a plant belonging to genus Curcuma of family Zingiberaceae and is widely used as supplements in Japan. Rhizomes of C. aromatica have curcumin as a major yellow pigment and curdione as a main ingredient of essential oils. In this study, we investigated the affect of C. aromatica on CYP3A4 using 1α,25-(OH)(2)-D(3)-treated Caco-2 clone cells. Caco-2 cells were treated with methanol extract (0.1 mg ml(-1)), its hexane soluble fraction (0.1 mg ml(-1)), curcumin (4 μM) and curdione (20 μM) for 72 hours. Nifedipine was used as a substrate of CYP3A4. Methanol extract, hexane fraction and curdione inhibited the formation of oxidized nifedipine by 50-70%, and curcumin showed no effect. The IC50s of methanol extract, hexane fraction and curdione to oxidized nifedipine formation were 21, 14 and 3.9 μg ml(-1) (16.9 μM), respectively. The content of curdione in methanol extract was 11.4%. Moreover, all of methanol extract, hexane fraction and curdione decreased CYP3A4 protein expression but had no affect on CYP3A4 mRNA expression. Our results showed that these drugs further decreased the CYP3A4 protein expression level after the protein synthesis was inhibited by cychroheximide. These findings suggest that curdione plays an important role in the CYP3A4 inhibitory activity of C. aromatica and curdione might inhibit the activity by accelerating the degradation of CYP3A4.
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http://dx.doi.org/10.1093/ecam/nep229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137788PMC
November 2011

Application of hydrotropy to transdermal formulations: hydrotropic solubilization of polyol fatty acid monoesters in water and enhancement effect on skin permeation of 5-FU.

J Pharm Pharmacol 2011 Aug 8;63(8):1008-14. Epub 2011 Jun 8.

Department of Pharmaceutics, School of Pharmaceutical Sciences, Mukogawa Women's University, Hyogo Department of Bioscience, Nagahama Institute of Bio-Science and Technology, Shiga, Japan.

Objectives: A hydrotropic formulation containing a percutaneous enhancer was developed for the transdermal formulation of a water-soluble drug and the solubilizing mechanisms of a percutaneous enhancer in water by a hydrotropic agent were investigated. The enhancement effect was also compared with the hydrotropic formulation and the other formulations using ethanol, propylene glycol or mixed micelles.

Methods: Sodium salicylate (SA) and sodium benzoate (BA) were selected as hydrotropic agents, and polyol fatty acid ester (POFE) and 5-fluorouracil (5-FU) were selected as a percutaneous enhancer and a water-soluble drug, respectively. Near-infrared (NIR) spectrophotometric and ¹H NMR spectroscopic studies were carried out to investigate the solubilizing mechanisms. The mean particle size in the hydrotropic formulation was measured. The in-vitro skin permeation of 5-FU and the accumulation in the skin of propylene glycol monocaprylate (PGMC), one of the monoesters of POFE, from the hydrotropic formulation or the other formulations were investigated by using Franz-type diffusion cell.

Key Findings: The presence of SA and BA had a visible effect on the O-H stretching band of water in the NIR region. The surface tension of SA and BA aqueous solutions was found to decrease with an increase in SA or BA concentration. Although SA interacted with PGMC in the presence of water, it did not interact with PGMC in the absence of water. Mean particle size in a solution consisting of 5% (v/v) PGMC and 30% SA aqueous solution was approximately 14 nm. ¹H NMR spectroscopic studies indicated that the hydrotropic salts formed aggregates with which PGMC interacted from the outside. The hydrotropic formulation prepared in this study enhanced skin permeation of 5-FU when compared with the other formulations.

Conclusions: SA and BA solubilized monoesters of POFE in water, and SA interacted with PGMC in water. The hydrotropic formulation prepared in this study significantly enhanced skin permeation of 5-FU compared with the other formulations. The results suggest that a hydrotropic formulation containing PGMC may be a useful transdermal formulation for water-soluble drugs.
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http://dx.doi.org/10.1111/j.2042-7158.2011.01308.xDOI Listing
August 2011
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