Elsevier Acta Medica Okayama 00223549 110 2021 Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells 1788 1798 EN Melissa Siaw Han Lim Yuki Nishiyama Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Graduate School of Natural Science and Technology, Okayama University Hirotsugu Kobuchi Kazuko Kobayashi Collaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eiji Matsuura The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2075-1729 11 2 2021 A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): “Lactosome” Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy 158 EN Melissa Siaw Han Lim Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Fumiaki Takenaka Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kazuko Kobayashi Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masaru Akehi Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hirotaka Uji Department of Material Chemistry, Graduate School of Engineering, Kyoto University Hirotsugu Kobuchi Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takanori Sasaki Collaborative Research Centre for OMIC, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Eiichi Ozeki Technology Research Laboratory, Shimadzu Corporation Eiji Matsuura Department of Cell Chemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University “Theranostics,” a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, “Lactosome” nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the “89Zr-labeled CPP and TPP-loaded Lactosome particles” and future directions based on important milestones and recent developments in this platform. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation e50082 EN Hirotsugu Kobuchi 1 Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koko Moriya Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University Tetsuya Ogino Department of Nursing Science, Faculty of Health and Welfare Science, Okayama Prefectural University Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Keiji Inoue Department of Urology, Kochi Medical School Taro Shuin Department of Urology, Kochi Medical School Tatsuji Yasuda 1 Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kozo Utsumi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science Toshihiko Utsumi Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation e50082 EN Hirotsugu Kobuchi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Koko Moriya Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Ogino Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keiji Inoue Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Taro Shuin Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tatsuji Yasuda Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kozo Utsumi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiko Utsumi Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0018-0661 150 1 2013 Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening 1 9 EN Hideki Hanada Hirotsugu Kobuchi Masanao Yamamoto Keiko Kashiwagi Kenjiro Katsu Toshihiko Utsumi Akihiko Kashiwagi Junzo Sasaki Masayasu Inoue Kozo Utsumi Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamor phosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for -oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3-induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3-treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 7 11 2012 Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation EN Hirotsugu Kobuchi Koko Moriya Tetsuya Ogino Hirofumi Fujita Keiji Inoue Taro Shuin Tatsuji Yasuda Kozo Utsumi Toshihiko Utsumi Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1995 Inhibition of Neutrophil Priming and Tyrosyl Phosphorylation by Cepharanthine, a Nonsteroidal Antiinflammatory Drug EN No potential conflict of interest relevant to this article was reported.