Publications by authors named "Mikihisa Takano"

97 Publications

Evaluation on epithelial-mesenchymal state and microRNAs focusing on isolated alveolar epithelial cells from bleomycin injured rat lung.

Toxicology 2021 Aug 20;461:152903. Epub 2021 Aug 20.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

Several studies using bleomycin (BLM)-induced lung injury rat model revealed that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. Conversely, microRNAs (miRNAs) are considered as useful markers of various diseases. In the present study, we aimed to characterize the EMT state through focusing on alveolar epithelial cells and identify the miRNAs that can be used as markers to predict pulmonary fibrosis using a BLM-induced lung injury rat model. Intratracheal administration of BLM increased hydroxyproline, a component of collagen, in lung tissues at day 14, but not at day 7. However, BLM induced EMT at day 7, which was accompanied with increased mRNA expression of α-smooth muscle actin, a representative EMT marker, in alveolar epithelium, thereby suggesting that EMT occurs prior to pulmonary fibrosis in alveolar epithelial cells. Using this rat model, the expression levels of several EMT-associated miRNAs were examined, and miR-222 was found to be upregulated in alveolar epithelial cells as well as bronchoalveolar lavage fluid from day 3. Our findings indicate that EMT in alveolar epithelial cells may occur before pulmonary fibrosis, and miR-222 may be used as a potential marker for early prediction of pulmonary fibrosis.
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http://dx.doi.org/10.1016/j.tox.2021.152903DOI Listing
August 2021

Differential mechanisms underlying methotrexate-induced cell death and epithelial-mesenchymal transition in A549 cells.

Toxicol Res 2021 Jul 27;37(3):293-300. Epub 2020 Oct 27.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553 Japan.

Epithelial-mesenchymal transition (EMT), a biological process through which epithelial cells transdifferentiate into mesenchymal cells, is involved in several pathological events, such as cancer progression and organ fibrosis. So far, we have found that methotrexate (MTX), an anticancer drug, induced EMT in the human A549 alveolar adenocarcinoma cell line. However, the relationship between EMT and the cytotoxicity induced by MTX remains unclear. In this study, we compared the processes of MTX-induced EMT and apoptosis in A549 cells. Q-VD-Oph, a caspase inhibitor, suppressed MTX-induced apoptosis, but not the increase in mRNA expression of α-smooth muscle actin (SMA), a representative EMT marker. In addition, SB431542, an EMT inhibitor, did not inhibit MTX-induced apoptosis. By using isolated clonal cells from wild-type A549 cells, the induction of EMT and apoptosis by MTX in each clone was analyzed, and no significant correlation was observed between the MTX-induced increase in α-SMA mRNA expression and the proportion of cells undergoing apoptosis. Furthermore, the increase in the mRNA expression of α-SMA was well correlated with cyclin-dependent kinase inhibitor 1A, a cell cycle arrest marker, but not with BCL-2 binding component 3 and Fas cell surface death receptor, which are both pro-apoptotic factors, indicating that the MTX-induced EMT may be related to cell cycle arrest, but not to apoptosis. These findings suggested that different mechanisms were involved in the MTX-induced EMT and apoptosis.
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http://dx.doi.org/10.1007/s43188-020-00067-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8249484PMC
July 2021

Direct N-Selective Alkylation of Hydantoins Using Potassium Bases.

Chem Pharm Bull (Tokyo) 2021 ;69(4):407-410

Department of Synthetic Organic Chemistry, Graduate School of Biomedical and Health Sciences, Hiroshima University.

Hydantoins, including the antiepileptic drug phenytoin, contain an amide nitrogen and an imide nitrogen, both of which can be alkylated. However, due to the higher acidity of its proton, N can be more easily alkylated than N under basic conditions. In this study, we explored methods for direct N-selective methylation of phenytoin and found that conditions using potassium bases [potassium tert-butoxide (BuOK) and potassium hexamethyldisilazide (KHMDS)] in tetrahydrofuran (THF) gave N-monomethylated phenytoin in good yield. The applicable scope of this reaction system was found to include various hydantoins and alkyl halides. To explore the function of methylated hydantoins, the effects of a series of methylated phenytoins on P-glycoprotein were examined, but none of methylated products showed inhibitory activity toward rhodamine 123 efflux by P-glycoprotein.
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http://dx.doi.org/10.1248/cpb.c20-00857DOI Listing
July 2021

miR-484: A Possible Indicator of Drug-Induced Pulmonary Fibrosis.

J Pharm Pharm Sci 2020 ;23:486-495

Graduate School of Biomedical and Health Sciences, Hiroshima University.

Background: Drug-induced lung injury leads to serious lung diseases, such as pulmonary fibrosis. We demonstrated in an alveolar epithelial cell line A549/ABCA3 that certain microRNAs were associated with bleomycin induced epithelial-mesenchymal transition (EMT) which is closely related to pulmonary fibrosis. In this study, we focused on the role of miR-484 in drug-induced EMT using A549/ABCA3 cells and a mouse lung injury model.

Methods: The expression of EMT-related genes and miR-484 was detected by real-time polymerase chain reaction. miR-484-targeted proteins were analyzed by Western blot. Pulmonary fibrosis mouse model was prepared by the intratracheal administration of BLM. As miR-484 is known to target SMAD2 and zinc finger E-box binding homeobox 1 (ZEB1), which are the well-known EMT-related transcription factors, we assessed the effects of a miR-484 inhibitor or mimic on the mRNA/protein expression of both the factors.

Results: We found that bleomycin significantly suppressed the intracellular expression and extracellular release of miR-484 in A549/ABCA3 cells. Moreover, the miR-484 mimic and inhibitor showed no drastic effects on the expression of the EMT-related transcription factors. In addition, the miR-484 mimic had no effect on the bleomycin-induced altered mRNA expression of the α-smooth muscle actin, a representative EMT marker. This suggested that miR-484 did not directly contribute to bleomycin-induced EMT in A549/ABCA3 cells. In contrast, the significant decrease in miR-484 expression in the lung tissue or plasma of bleomycin-administered mice suggested that miR-484 expression was closely correlated with bleomycin-induced lung injury.

Conclusions: These findings indicate that miR-484 could be a novel diagnostic indicator for drug-induced pulmonary fibrosis.
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http://dx.doi.org/10.18433/jpps31448DOI Listing
January 2020

Suppressive effect of quercetin against bleomycin-induced epithelial-mesenchymal transition in alveolar epithelial cells.

Drug Metab Pharmacokinet 2020 Dec 12;35(6):522-526. Epub 2020 Aug 12.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

Quercetin is a flavonol that is known to have numerous beneficial biological effects such as an anti-fibrotic effect. Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells is one of major causes of pulmonary fibrosis. However, the effect of quercetin on drug-induced EMT in alveolar type II cells is not known. In this study, we examined the effect of quercetin on bleomycin (BLM)-induced EMT using RLE/Abca3 cells having alveolar type II cell-like phenotype. BLM induced EMT-like morphological changes, downregulation of an epithelial marker E-cadherin, and upregulation of a mesenchymal marker α-smooth muscle actin in RLE/Abca3 cells. In addition, BLM increased the levels of phosphorylated Smad2 and Slug mRNA expression, and enhanced nuclear translocation of β-catenin, suggesting that BLM induced EMT in RLE/Abca3 cells via Smad and β-catenin signaling pathways. However, when the cells were co-treated with quercetin, quercetin suppressed all of these EMT-related changes induced by BLM. Furthermore, BLM increased the intracellular level of reactive oxygen species, which was also suppressed by quercetin. These results suggest that quercetin may be a possible candidate for preventing pulmonary fibrosis caused by drugs.
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http://dx.doi.org/10.1016/j.dmpk.2020.08.001DOI Listing
December 2020

Role of plasminogen activator inhibitor-1 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells.

Biochem Biophys Res Commun 2020 05 26;525(3):543-548. Epub 2020 Feb 26.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan. Electronic address:

There is increasing evidence that epithelial-mesenchymal transition (EMT) contributes to the development of organ fibrosis. We demonstrated that methotrexate (MTX) clearly induced EMT through the transforming growth factor (TGF)-β-related signaling pathway in human alveolar epithelial cell line, A549. However, critical factors associated with MTX-induced EMT have not yet been identified. In our study, we attempted to identify factors playing a crucial role in MTX-induced EMT in A549 cells. We focused on plasminogen activator inhibitor-1 (PAI-1) as the possible target for the prevention of MTX-induced EMT-related lung injury. Comprehensive gene expression analysis by microarray revealed that mRNA expression level of PAI-1 was clearly increased by MTX treatment. In addition, using several cloned A549 cells, we found a good correlation between MTX-induced increase in mRNA expression levels of α-smooth muscle actin (SMA), a representative EMT marker, and PAI-1. Furthermore, MTX upregulated mRNA and protein expression levels of PAI-1 in A549 cells; this upregulation was canceled by co-treatment with SB431542, a TGF-β-related signaling pathway inhibitor. Notably, tiplaxtinin, a PAI-1 inhibitor, and knockdown of urokinase-type plasminogen activator receptor (uPAR) prevented MTX-induced EMT in A549 cells. These findings indicate that MTX may induce EMT via upregulation of PAI-1 expression and interaction of PAI-1 with uPAR in A549 cells.
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http://dx.doi.org/10.1016/j.bbrc.2020.02.131DOI Listing
May 2020

Suppression of P-glycoprotein by cigarette smoke extract in human lung-derived A549/P-gp cells.

Drug Metab Pharmacokinet 2020 Apr 20;35(2):214-219. Epub 2019 Dec 20.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

Effect of long-term treatment with cigarette smoke extract (CSE) on the function and expression of P-glycoprotein (P-gp) in lung alveolar epithelial cells was examined using A549/P-gp cell line expressing P-gp. CSE treatment suppressed P-gp activity in a concentration- and treatment time-dependent manner. The suppression of P-gp activity by CSE was irreversible for at least 96 h after removal of CSE. In addition, CSE treatment suppressed the expression of P-gp mRNA and protein. In order to understand the mechanisms underlying P-gp suppression by CSE, the role of reactive oxygen species (ROS) was examined. CSE treatment increased intracellular ROS level, and suppressed catalase activity. α-Tocopherol suppressed ROS production by CSE, and ameliorated the suppression of P-gp activity by CSE, suggesting that ROS is involved in CSE-induced suppression of P-gp. The role of intracellular signaling pathways such as the nuclear factor κB and mitogen-activated protein kinase pathways was also examined. Among these pathways, the involvement of extracellular signal-regulated kinase (ERK) pathway was suggested. Taken together, long-term CSE treatment may suppress P-gp via modulation of ROS level and ERK pathway in alveolar epithelial cells.
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http://dx.doi.org/10.1016/j.dmpk.2019.12.001DOI Listing
April 2020

Effect of transforming growth factor-β1 on functional expression of monocarboxylate transporter 1 in alveolar epithelial A549 cells.

Naunyn Schmiedebergs Arch Pharmacol 2020 05 3;393(5):889-896. Epub 2020 Jan 3.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

Epithelial-mesenchymal transition (EMT) contributes to the development of severe lung diseases, such as pulmonary fibrosis. Recently, it has been reported that EMT involves complex metabolic reprogramming triggered by several factors including transforming growth factor (TGF-β1) and that monocarboxylate transporter (MCT1) plays an essential role in these metabolic changes. The aim of the present study was to clarify the functional expression of MCT1 during TGF-β1-induced EMT in alveolar epithelial A549 cells. The transport function of MCT1 in A549 cells was examined using [H]γ-hydroxybutyrate (GHB) and [H] lactic acid (LA) as substrates and α-cyano-4-hydroxycinnamate (CHC), lactic acid, phloretin, and AR-C155858 (AR) as inhibitors of MCT1. EMT was induced by treating the cells with TGF-β1. mRNA and protein expression levels were analyzed using real-time PCR and Western blotting, respectively. Time-, temperature-, and pH-dependent GHB and LA uptake were observed in A549 cells. CHC, lactic acid, phloretin, and AR significantly inhibited the uptake of GHB in a concentration-dependent manner, suggesting that MCT1 is primarily responsible for transport of monocarboxylates such as GHB and LA in A549 cells. TGF-β1 treatment significantly enhanced GHB and LA uptake as well as the mRNA and protein expression levels of MCT1 in A549 cells. These changes were neutralized by co-treatment with SB431542, an inhibitor for the TGF-β1 signaling pathway. CHC and AR had no effect on TGF-β1-induced EMT-related gene expression changes. Here, we have clearly characterized functional expression of MCT1 in A549 cells and have shown that MCT1 may be upregulated via the TGF-β1 signaling pathway.
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http://dx.doi.org/10.1007/s00210-019-01802-3DOI Listing
May 2020

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells.

J Pharm Pharm Sci 2019 ;22(1):516-524

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Purpose: Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model.

Methods: Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively.

Results: BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53.

Conclusions: These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells.
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http://dx.doi.org/10.18433/jpps30660DOI Listing
August 2020

Investigation on inhibitory effect of folic acid on methotrexate-induced epithelial-mesenchymal transition focusing on dihydrofolate reductase.

Drug Metab Pharmacokinet 2019 Dec 16;34(6):396-399. Epub 2019 Aug 16.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

Use of methotrexate (MTX) can induce serious adverse lung reactions, such as pulmonary fibrosis. Recently, we demonstrated that the epithelial-mesenchymal transition (EMT), which triggers pulmonary fibrosis, was induced by MTX, and folic acid (FA) suppressed MTX-induced EMT in A549 cells. In this study, the role of dihydrofolate reductase (DHFR), a target of MTX, in FA-mediated inhibition of MTX-induced EMT was evaluated. The inhibitory effects of FA and tetrahydrofolate (THF), a metabolite of FA produced by DHFR, on MTX-induced increases in mRNA expression of α-SMA, an EMT marker, were compared. The IC values of FA and THF for DHFR were 103.3 and 19.4 μM, respectively. In contrast, DHFR knockdown did not alter the mRNA expression of α-SMA. Notably, the inhibitory effect of FA, but not THF, on MTX-induced EMT was blunted in DHFR knockdown cells. These results suggested that DHFR may not directly contribute to MTX-induced EMT, but may contribute to suppression of MTX-induced EMT via production of THF in A549 cells.
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http://dx.doi.org/10.1016/j.dmpk.2019.08.003DOI Listing
December 2019

Association of cell cycle arrest with anticancer drug-induced epithelial-mesenchymal transition in alveolar epithelial cells.

Toxicology 2019 08 4;424:152231. Epub 2019 Jun 4.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

Many drugs exert serious cytotoxic effects on pulmonary tissues. Although several reports have shown an association of epithelial-mesenchymal transition (EMT) with anticancer drug-induced lung injury, mechanisms of these effects are poorly understood. In the present study, we evaluated mechanisms of anticancer drug-induced EMT, with a focus on involvement of cell cycle arrest. We found that methotrexate (MTX) altered mRNA expression levels of many genes as determined by microarray analysis. Gene set enrichment analysis revealed that cell cycle arrest pathways may be associated with MTX-induced EMT. In addition, thymidine (THY) and nocodazole (NOC), which induce cell cycle arrest at S-phase and G2/M-phase, increased mRNA expression levels of α-smooth muscle actin (SMA), an EMT marker. Furthermore, α-SMA protein expression in cells arrested at S- and G2/M-phases by MTX and paclitaxel (PTX) was significantly higher than that in cells at G1. Notably, co-treatment of cells with THY or NOC and EMT-inducing anticancer drugs did not result in additional upregulation of α-SMA mRNA expression. These findings suggested that cell cycle arrest may be closely associated with anticancer drug-induced EMT in alveolar epithelial cells.
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http://dx.doi.org/10.1016/j.tox.2019.06.002DOI Listing
August 2019

Role of peptide transporter 2 and MAPK signaling pathways in the innate immune response induced by bacterial peptides in alveolar epithelial cells.

Life Sci 2019 Jul 16;229:173-179. Epub 2019 May 16.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Aims: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549.

Main Methods: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium.

Key Findings: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response.

Significance: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.
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http://dx.doi.org/10.1016/j.lfs.2019.05.042DOI Listing
July 2019

Reduced folate carrier-mediated methotrexate transport in human distal lung epithelial NCl-H441 cells.

J Pharm Pharmacol 2019 Feb 15;71(2):167-175. Epub 2018 Oct 15.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

Objectives: We had previously found that reduced folate carrier (RFC; SLC19A1) is mainly involved in an influx of transport of methotrexate (MTX), a folate analogue, using alveolar epithelial A549 cells. Therefore, we examined MTX uptake in NCl-H441 (H441) cells, another in vitro alveolar epithelial model, focusing on the localization of RFC in the present study.

Methods: Transport function of RFC in H441 cells was studied using [ H]MTX.

Key Findings: The uptake of MTX was increased remarkably after pretreatment of the cell monolayer with ethylenediaminetetraacetic acid (EDTA) in H441 cells but not in A549 cells, indicating the contribution of the basolaterally located transporter. In addition, folic acid and thiamine monophosphate, RFC inhibitors, inhibited the uptake of MTX from the basolateral side of the H441 cells. In order to compare the function of RFC on the apical and basolateral sides of the cells, the uptake of MTX from each side was examined using a Transwell chamber. Intracellular MTX amounts from the basolateral side were found to be significantly higher than those from the apical side.

Conclusions: These findings suggest that the distribution of MTX in the lung alveolar epithelial cells may be mediated by basolaterally located RFC in alveolar epithelial cells.
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http://dx.doi.org/10.1111/jphp.13022DOI Listing
February 2019

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell.

J Pharm Pharm Sci 2018 ;21(1):247-255

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

Purpose: Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549.

Methods: FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively.

Results: FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation.

Conclusion: AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
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http://dx.doi.org/10.18433/jpps29905DOI Listing
September 2019

Folic acid prevents methotrexate-induced epithelial-mesenchymal transition via suppression of secreted factors from the human alveolar epithelial cell line A549.

Biochem Biophys Res Commun 2018 02 13;497(1):457-463. Epub 2018 Feb 13.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan. Electronic address:

Methotrexate (MTX) often induces serious lung diseases such as pulmonary fibrosis. Although MTX is known to be a folic acid (FA) antagonist, the effect of FA on MTX-induced lung injury remains unclear. Recent studies indicate that epithelial-mesenchymal transition (EMT) is involved in pulmonary fibrosis. Here, we aimed to clarify the effect of FA on MTX-induced EMT in human alveolar epithelial cell line A549 using conditioned medium (CM). CM was prepared from the supernatants of A549 cells treated with MTX in the absence (CMM) or presence (CMMF) of FA. FA suppressed EMT-like morphological changes and elevated mRNA/protein expression levels of α-smooth muscle actin induced by MTX in A549 cells. In addition, CMM induced EMT-like phenotypical changes, whereas CMMF had no effect on the phenotype of A549 cells, indicating that FA may suppress MTX-induced EMT via inhibiting the secretion of certain factors into the supernatant of the cells. Furthermore, FA also prevented CMM-induced EMT-like phenotypical changes in A549 cells. These findings indicate that FA may be a useful pharmaceutical for MTX-induced lung injury.
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http://dx.doi.org/10.1016/j.bbrc.2018.02.111DOI Listing
February 2018

Nicotine transport in lung and non-lung epithelial cells.

Life Sci 2017 Nov 31;188:76-82. Epub 2017 Aug 31.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Aims: Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7.

Materials And Methods: Characteristics of [H]nicotine uptake was studied using these cell lines.

Key Findings: Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations.

Significance: The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed.
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http://dx.doi.org/10.1016/j.lfs.2017.08.030DOI Listing
November 2017

Effect of COA-Cl on transforming growth factor-β1-induced epithelial-mesenchymal transition in RLE/Abca3 cells.

Drug Metab Pharmacokinet 2017 Aug 17;32(4):224-227. Epub 2017 May 17.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

Transforming growth factor (TGF)-β1 has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) in pathological conditions such as cancer and organ fibrosis. In this study, we examined the effect of a novel nucleic acid analog, COA-Cl, on TGF-β1-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Changes in the cell morphology consistent with EMT were induced by TGF-β1, whereas, this response was suppressed by co-treatment of the cells with COA-Cl. In addition, co-treatment with COA-Cl abolished TGF-β1-induced downregulation of cytokeratin 19 and upregulation of α-smooth muscle actin transcripts. In order to delineate the mechanism underlying the inhibitory effect of COA-Cl on TGF-β1-induced EMT in RLE/Abca3 cells, we examined the role of zinc finger E-box binding homeobox (ZEB) family transcription factors in this phenomenon. Our results demonstrated that the treatment of cells with COA-Cl suppressed the TGF-β1 mediated increase in the mRNA levels of ZEB2. Overall, it was concluded that COA-Cl may have an inhibitory effect on TGF-β1-induced EMT-like phenotypical changes in RLE/Abca3 cells via suppression of ZEB2 mRNA expression.
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http://dx.doi.org/10.1016/j.dmpk.2017.05.001DOI Listing
August 2017

Current Progress Toward a Better Understanding of Drug Disposition Within the Lungs: Summary Proceedings of the First Workshop on Drug Transporters in the Lungs.

J Pharm Sci 2017 09 15;106(9):2234-2244. Epub 2017 Apr 15.

Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

The School of Pharmacy and Pharmaceutical Sciences at Trinity College Dublin hosted the "1 Workshop on Drug Transporters in the Lungs" in September 2016 to discuss the impact of transporters on pulmonary drug disposition and their roles as drug targets in lung disease. The workshop brought together about 30 scientists from academia and pharmaceutical industry from Europe and Japan and addressed the primary questions: What do we know today, and what do we need to know tomorrow about transporters in the lung? The 3 themes of the workshop were: (1) techniques to study drug transporter expression and actions in the lungs; (2) drug transporter effects on pulmonary pharmacokinetics-case studies; and (3) transporters as drug targets in lung disease. Some of the conclusions of the workshop were: suitable experimental models that allow studies of transporter effects are available; data from these models convincingly show a contribution of both uptake and efflux transporters on pulmonary drug disposition; the effects of transporters on drug lung PK is now better conceptualized; some transporters are associated with lung diseases. However, more work is needed to establish which of the available models best translate to the clinical situation.
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http://dx.doi.org/10.1016/j.xphs.2017.04.011DOI Listing
September 2017

Role of miR-34a in TGF-β1- and Drug-Induced Epithelial-Mesenchymal Transition in Alveolar Type II Epithelial Cells.

J Pharm Sci 2017 09 10;106(9):2868-2872. Epub 2017 Apr 10.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Epithelial-mesenchymal transition (EMT) of alveolar type II epithelial cells may play an important role in the pulmonary fibrosis induced by drugs such as bleomycin (BLM) and methotrexate (MTX). In this study, we examined the role of microRNAs (miRNAs) in drug-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Based on the screening using miRNA microarray analysis, it was found that the expression of some miRNAs, such as miR-34a, was increased by transforming growth factor (TGF)-β1 and BLM. An increase in miR-34a expression due to TGF-β1, BLM, and MTX was also observed in real-time PCR analysis. Therefore, miR-34a was focused upon in further studies. The expression of nectin-1 mRNA and protein, a possible target of miR-34a, was decreased by the treatment with TGF-β1, BLM, and MTX. In addition, when RLE/Abca3 cells were transfected with miR-34a mimic, the expression of nectin-1 mRNA and Abca3 mRNA, another target of miR34a, decreased significantly. Furthermore, the mRNA expression of cytokeratin 19, an epithelial marker, decreased, whereas that of α-smooth muscle actin, a mesenchymal marker, increased in the cells transfected with miR-34a mimic. These results suggest that miR-34a is involved in drug-induced EMT in alveolar epithelial cells, and possibly in lung fibrosis.
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http://dx.doi.org/10.1016/j.xphs.2017.04.002DOI Listing
September 2017

Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

Biol Pharm Bull 2017 ;40(1):82-87

Laboratory of Pharmaceutics, Osaka University of Pharmaceutical Sciences.

The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.
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http://dx.doi.org/10.1248/bpb.b16-00684DOI Listing
February 2017

Effect of cigarette smoke extract on P-glycoprotein function in primary cultured and newly developed alveolar epithelial cells.

Drug Metab Pharmacokinet 2016 Dec 31;31(6):417-424. Epub 2016 Aug 31.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

The effect of cigarette smoke extract (CSE) on P-glycoprotein (P-gp) function in the distal lung is unclear. In this study, we first examined the expression and function of P-gp and the effect of CSE in rat primary cultured alveolar epithelial cells. The expression of P-gp protein was observed in type I-like cells, but not in type II cells. In type I-like cells, rhodamine 123 (Rho123) accumulation was enhanced by various P-gp inhibitors such as verapamil and cyclosporine A. In addition, the expression of P-gp mRNAs, mdr1a and mdr1b, as well as P-gp activity increased along with the transdifferentiation. When type I-like cells were co-incubated with CSE, P-gp activity was suppressed. Next, we attempted to clarify the effect of CSE on P-gp function in human-derived cultured alveolar epithelial cells. For this purpose, we isolated an A549 clone (A549/P-gp) expressing P-gp, because P-gp expression in native A549 cells was negligible. In A549/P-gp cells, P-gp was functionally expressed, and the inhibitory effect of CSE on P-gp was observed. These results suggested that smoking would directly suppress P-gp activity, and that A549/P-gp cell line should be a useful model to further study the effect of xenobiotics on P-gp function in the alveolar epithelial cells.
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http://dx.doi.org/10.1016/j.dmpk.2016.08.006DOI Listing
December 2016

Methotrexate-Induced Epithelial-Mesenchymal Transition in the Alveolar Epithelial Cell Line A549.

Lung 2016 12 7;194(6):923-930. Epub 2016 Sep 7.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734-8553, Japan.

Purpose: Methotrexate (MTX) therapy of certain cancers and rheumatoid arthritis often induces serious interstitial lung complications including pulmonary fibrosis. In this study, we investigated the epithelial-mesenchymal transition (EMT) induced by MTX and by transforming growth factor (TGF)-β1 in the human alveolar epithelial cell line A549 in order to develop new strategies for the prevention of EMT.

Methods: First, we examined the effect of TGF-β1 and MTX on cell morphology and the expression of EMT-related mRNAs in A549 cells. Then, the effects of SB431542 (SB), a potent inhibitor of TGF-β receptor kinase, and a neutralizing antibody for TGF-β1 on the phenotypic changes of A549 cells induced by TGF-β1 and MTX were examined.

Results: After incubation with TGF-β1 and MTX, the mRNA expression of epithelial markers such as cytokeratin 19 was reduced, while that of mesenchymal markers such as α-smooth muscle actin was increased. SB suppressed the development of morphological changes and partially rescued alterations in mRNA expression of EMT markers induced by MTX. In addition, the enhancement of SMAD2 phosphorylation by MTX was also prevented by SB. On the other hand, EMT-related changes induced by MTX were not affected by a neutralizing antibody for TGF-β1.

Conclusion: We have demonstrated that phenotypic changes of A549 cells induced by MTX are partly mediated by a TGF-β1-related intracellular signaling pathway, although TGF-β1 itself is not directly involved in this process.
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http://dx.doi.org/10.1007/s00408-016-9935-7DOI Listing
December 2016

Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

J Pharm Sci 2016 Feb 12;105(2):982-988. Epub 2016 Jan 12.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan.

Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.
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http://dx.doi.org/10.1002/jps.24627DOI Listing
February 2016

Phase I dose-finding and pharmacokinetic study of docetaxel and gefitinib in patients with advanced or metastatic non-small-cell lung cancer: evaluation of drug-drug interaction.

Cancer Chemother Pharmacol 2015 Oct 2;76(4):713-21. Epub 2015 Aug 2.

Department of Pharmacy, National Cancer Center Hospital, Tokyo, Japan.

Purpose: Docetaxel and gefitinib play key roles in the treatment of non-small-cell lung cancer (NSCLC), and their combination could be of interest. Both drugs are mainly metabolized by CYP3A4, and drug-drug interactions are a major concern. This phase I dose-finding study was designed to assess the tolerability and drug-drug interactions in this combination using full pharmacokinetic (PK) samplings.

Methods: Docetaxel was intravenously administered on days 1 and 22 at a dose of 45 or 60 mg/m(2). Gefitinib (250 mg/day) was orally administrated starting on day 2. Ten PK samplings of docetaxel were performed on days 1 and 22. Seven PK samplings of gefitinib were performed on day 18 ± 3 and on day 22.

Results: Twelve patients with advanced or metastatic NSCLC were enrolled without considering EGFR mutation status. The major toxicity was neutropenia. Two patients withdrew from this study due to dose-limiting toxicities; however, the toxicity profiles in this combination were generally acceptable. The docetaxel AUC0-24 and C max did not differ whether administered alone or with gefitinib, and the geometric mean ratios (GMRs) of AUC0-24 and C max (co-administrated/administrated alone) were 0.95 (90 % CI 0.85-1.06) and 0.95 (90 % CI 0.85-1.05), respectively. Furthermore, the GMRs of the steady state gefitinib AUC0-24 and C max were 0.93 (90 % CI 0.84-1.03) and 0.98 (90 % CI 0.88-1.09), respectively.

Conclusion: The tolerability of 60 mg/m(2) docetaxel with 250 mg/day gefitinib was confirmed, and we observed no drug-drug interaction in this combination.
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http://dx.doi.org/10.1007/s00280-015-2837-1DOI Listing
October 2015

Methotrexate influx via folate transporters into alveolar epithelial cell line A549.

Drug Metab Pharmacokinet 2015 Aug 16;30(4):276-81. Epub 2015 May 16.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address:

Methotrexate (MTX), a drug used for the treatment of certain cancers as well as rheumatoid arthritis, sometimes induces serious interstitial lung injury. Although lung toxicity of MTX is related to its accumulation, the information concerning MTX transport in the lungs is lacking. In this study, we investigated the mechanisms underlying MTX influx into human alveolar epithelial cell line A549. MTX influx into A549 cells was time-, pH-, and temperature-dependent and showed saturation kinetics. The influx was inhibited by folic acid with IC50 values of 256.1 μM at pH 7.4 and 1.6 μM at pH 5.5, indicating that the mechanisms underlying MTX influx would be different at these pHs. We then examined the role of two folate transporters in MTX influx, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). The expression of RFC and PCFT mRNAs in A549 cells was confirmed by reverse transcription polymerase chain reaction. In addition, MTX influx was inhibited by thiamine monophosphate, an RFC inhibitor, at pH 7.4, and by sulfasalazine, a PCFT inhibitor, at pH 5.5. These results indicated that RFC and PCFT are predominantly involved in MTX influx into A549 cells at pH 7.4 and pH 5.5, respectively.
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http://dx.doi.org/10.1016/j.dmpk.2015.04.005DOI Listing
August 2015

Functional Expression of PEPT2 in the Human Distal Lung Epithelial Cell Line NCl-H441.

Pharm Res 2015 Dec 14;32(12):3916-26. Epub 2015 Jul 14.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

Purpose: The peptide transporter PEPT2 is expressed in alveolar type II epithelial cells. So far, however, no appropriate alveolar epithelial cell line for studying PEPT2 function has been known. In this study, we examined the functional expression of PEPT2 in the human distal lung epithelial cell line NCl-H441 (H441).

Methods: Expression of PEPT2 mRNA and protein was examined in H441 cells. Transport function of PEPT2 was studied using glycylsarcosine (Gly-Sar) as a substrate.

Results: Lamellar bodies were well developed in H441 cells and mRNA expression of type II cell markers and PEPT2 increased during time in culture. PEPT2 protein expression was confirmed in H441 cells, but not in A549 cells, by immunostaining and Western blotting. The uptake of Gly-Sar in H441 cells was inhibited by cefadroxil, and the cefadroxil-sensitive uptake was pH-dependent and peaked at pH 6.5. Gly-Sar uptake in H441 cells showed saturation kinetics with a Km value of 112.5 μM. In addition, apical-to-basal, but not basal-to-apical, transport of cephalexin across H441 cell monolayers was sensitive to cefadroxil.

Conclusions: PEPT2 is functionally expressed in H441 cells, making the cell line a good in vitro model to study PEPT2 function and its regulation in human distal lung.
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http://dx.doi.org/10.1007/s11095-015-1751-xDOI Listing
December 2015

Analysis of TGF-β1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3.

Drug Metab Pharmacokinet 2015 Feb 4;30(1):111-8. Epub 2014 Nov 4.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

In this study, we examined the induction of epithelial-mesenchymal transition (EMT) by transforming growth factor (TGF)-β1 and drugs in genetically engineered type II alveolar epithelial cell line RLE/Abca3. Treatment of RLE/Abca3 cells with TGF-β1 induced marked changes in cell morphology from epithelial-like to elongated fibroblast-like morphology. With these morphological changes, mRNA expression of epithelial markers such as cytokeratin 19 (CK19) decreased, while that of mesenchymal markers such as α-smooth muscle actin (α-SMA) increased. TGF-β1 treatment also decreased the mRNA expression of Abca3, a type II cell marker, and formation of lamellar body structures. Interestingly, the effect of TGF-β1 on Abca3 mRNA expression was observed in RLE/Abca3 cells, but not in wild-type RLE-6TN, A549, and H441 cells. Treatment of RLE/Abca3 cells with bleomycin (BLM) and methotrexate (MTX) induced similar morphological and mRNA expression changes. In addition, the increase in α-SMA and the decrease in Abca3 mRNA expression by these drugs were observed only in RLE/Abca3 cells. These findings suggest that, like TGF-β1, BLM and MTX induce EMT in RLE/Abca3 cells, and RLE/Abca3 cells would be a good model to study drug-induced EMT. The effect of pirfenidone, an antifibrotic and anti-inflammatory drug, on EMT induced by TGF-β1 was also discussed.
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http://dx.doi.org/10.1016/j.dmpk.2014.10.007DOI Listing
February 2015

Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins.

Life Sci 2015 Mar 24;124:31-40. Epub 2015 Jan 24.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

Aims: Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use.

Main Methods: Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells.

Key Findings: A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression.

Significance: The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin.
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http://dx.doi.org/10.1016/j.lfs.2015.01.011DOI Listing
March 2015

Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar epithelial cells.

Expert Opin Drug Deliv 2015 May 12;12(5):813-25. Epub 2014 Dec 12.

Hiroshima University, Graduate School of Biomedical and Health Sciences, Department of Pharmaceutics and Therapeutics , 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan +81 82 257 5315 ; +81 82 257 5319 ;

Introduction: Pulmonary delivery is an attractive administration route for therapeutic proteins and peptides. In this context, endocytosis/transcytosis at the distal lung epithelial barrier is an important process in the pulmonary absorption of therapeutic macromolecules. The alveolar epithelium is comprised of type I and type II cells. Understanding the transport mechanisms in these cells is essential for the development of efficient pulmonary delivery systems of therapeutic macromolecules.

Areas Covered: Endocytic pathways for albumin and insulin in alveolar epithelial cells and possible receptors for the endocytosis are discussed. Strategies to enhance the endocytosis and pulmonary absorption of macromolecules are also discussed, by focusing on the effects of cationic poly(amino acid)s.

Expert Opinion: Although the surface area occupied by type II cells in alveoli is much smaller than that covered by type I cells, type II cells may significantly contribute to the endocytosis/transcytosis of macromolecules such as albumin. Identification of the receptors involved in the cellular uptake of each macromolecule is prerequisite for the understanding and regulation of its transport into and across alveolar epithelial cells. Establishment of novel in-vitro culture cell models of type I and type II cells would be a great help for the future advance of this research field.
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http://dx.doi.org/10.1517/17425247.2015.992778DOI Listing
May 2015

Fatty acid-bearing albumin but not fatty acid-depleted albumin induces HIF-1 activation in human renal proximal tubular epithelial cell line HK-2.

Biochem Biophys Res Commun 2014 Jul 9;450(1):476-81. Epub 2014 Jun 9.

Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

Recently, we found that albumin overload induces expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) protein and several HIF-1 target genes in human renal proximal tubular epithelial cell line HK-2. In this study, the role of albumin-bound fatty acids in the albumin-induced HIF-1 activation was studied. The enhancing effect of fatty acid-bearing human serum albumin [FA(+)HSA] treatment on HIF-1α protein expression was much greater than that of fatty acid-depleted human serum albumin [FA(-)HSA] treatment. The FA(+)HSA treatment induced HIF-1 target gene mRNAs such as those of glucose transporter 1 (GLUT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and breast cancer resistance protein (BCRP) in concentration-dependent manners, while FA(-)HSA caused no significant increases in these mRNAs. Consistent with increased GLUT1 mRNA, GLUT1 protein expression and GLUT inhibitor cytochalasin B-sensitive d-[(3)H]glucose uptake activity were significantly enhanced by treatment with FA(+)HSA, but not with FA(-)HSA. These findings indicate that fatty acids bound to albumin play a crucial role in albumin-induced HIF-1 activation followed by changes in HIF-1 target gene expression and protein product activity.
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http://dx.doi.org/10.1016/j.bbrc.2014.05.146DOI Listing
July 2014
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