MDPI AG Acta Medica Okayama 1422-0067 27 5 2026 Fgf10 Gene Dosage from a Single Allele Is Insufficient for Forming Multilayered Epithelial Cells in the Murine Lacrimal Gland 2113 EN Shiori Ikeda Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yuki Tajika Department of Radiological Technology, Gumma Prefectural College of Health Sciences Hirofumi Fujita Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Tetsuya Bando Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Tsutomu Nohno Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Satoru Miyaishi Department of Legal Medicine, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Mutations in the fibroblast growth factor 10 (FGF10) gene in humans cause aplasia of the lacrimal and salivary glands (ALSG). In patients with ALSG, heterozygous loss-of-function mutations are found, and FGF10 haploinsufficiency results in the absence of these secretory organs. Lacrimal glands (LGs) are formed through epithelial thickening, budding, and branching morphogenesis. To compare the variable phenotypes of the Fgf10+/�| Harderian glands (HGs) previously reported, we examined the development of LGs in wild-type (WT), Fgf10+/�|, and Fgf10-null mice. Pax6 immunostaining was performed to visualize the LG primordia from embryonic day 15.5 (E15.5) onwards. In situ hybridization of the genes encoding the epithelial receptor of FGF10, FGFR2b, and its other ligands was performed to determine their potential involvement in LG development. LG primordia were not observed in Fgf10+/�| mice bilaterally at E16.5 or later stages. At E15.5, budding from the developing conjunctival epithelium (CE) was observed in a small fraction of the Fgf10+/�| LG primordia. In contrast, the Fgf10-null CE failed to promote budding. Among Fgf1, Fgf3, Fgf7, Fgf10, and Fgf22, Fgf10 was expressed in the mesenchyme surrounding developing LG epithelial cells, whereas Fgf1 was expressed in the LG epithelium of WT mice. Fgf7 was initially expressed in the mesenchyme surrounding the nascent LG epithelium, but its expression subsequently became diffused. Thus, we conclude that among the FGFR2b ligands, initial LG formation is dependent on the mesenchymal factors FGF10 and FGF7, and FGF1 is likely to function as an epithelial factor in the LG primordia. A single allele of Fgf10 was found to be insufficient to support the budding process during LG morphogenesis. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2221-3759 12 2 2024 Harderian Gland Development and Degeneration in the Fgf10-Deficient Heterozygous Mouse 16 EN Shiori Ikeda Department of Cytology and Histology, Medical School, Okayama University Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirofumi Fujita Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hitomi Ono-Minagi Department of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Satoru Miyaishi Department of Legal Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tsutomu Nohno Department of Cytology and Histology, Medical School, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10(+/-) heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10(+/-) mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10(+/-) mice. We found that more than half of the Fgf10(+/-) mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10(+/-) embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 17 11 2022 Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption e0277307 EN Hirofumi Fujita Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Aoi Ando Faculty of Medicine, Okayama University Medical School Yohei Mizusawa Faculty of Medicine, Okayama University Medical School Mitsuaki Ono Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Takako Hattori Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Munenori Habuta Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshitaka Oohashi Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1(Delta 105)). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D-4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D-4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D-4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4425 13 2 2022 Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice 285 EN Ruizhi Xue Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Wenfeng Lin Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jingkai Sun Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Ochiai Laboratory of Veterinary Hygiene, Nippon Veterinary and Life Science University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Zhengyan Tang Department of Urology, Xiangya Hospital, Central South University Peng Huang Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasutomo Nasu Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 +/- 8.12% vs. 23.26 +/- 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 +/- 0.69 vs. 8.27 +/- 0.87, x10(6)), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 +/- 1.55% vs. 72.50 +/- 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 15 10 2020 Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung e0240333 EN Munenori Habuta Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Yasue Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Ken-Ichi T. Suzuki Department of Mathematical and Life Sciences, Hiroshima University Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitomi Kono Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ayuko Takayama Center for the Development of New Model Organisms, National Institute for Basic Biology Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Miyaishi Department of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University Eiji Tanaka Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known thatFibroblast growth factor 10(Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in theFgf10-CRISPR F0 mice. However, how the lung phenotype in theFgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in theFgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functionalFgf10genotypes. Firstly, according to a previous report,Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of theFgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 +/- 6.2% in type I, 25.3 +/- 2.7% in type II, and 54.3 +/- 9.5% in type III (mean +/- standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in theFgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functionalFgf10genotypes. These data suggest theFgf10gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, theFgf10-CRISPR F0 mouse would present an ideal experimental system to explore it. No potential conflict of interest relevant to this article was reported.

Association for Research in Vision and Ophthalmology Acta Medica Okayama 0146-0404 61 5 2020 KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells 38 EN Yuki Kanzaki Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hirofumi Fujita Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Mio Hosokawa Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroshi Matsumae Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Fumio Shiraga Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuki Morizane Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hideyo Ohuchi Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. </br> Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. </br> Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. </br> Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 3 2020 Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland 199 208 EN Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute for Genome Research, University of Tokushima Kazuhiko Ochiai Department of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/59950 Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland. 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.

Okayama University Medical School Acta Medica Okayama 0386-300X 67 3 2013 Improvement of the Efficacy of 5-aminolevulinic Acid-mediated Photodynamic Treatment in Human Oral Squamous Cell Carcinoma HSC-4 153 164 EN Masanao Yamamoto Hirofumi Fujita Naoki Katase Keiji Inoue Hitoshi Nagatsuka Kozo Utsumi Junzo Sasaki Hideyo Ohuchi Original Article 10.18926/AMO/50408 Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1206 2008 alpha-lipoic acid suppresses 6-hydroxydoparnine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1 1 12 EN Hirofumi Fujita Masahiko Shiosaka Tetsuya Ogino Yuya Okimura Toshihiko Utsumi Eisuke F. Sato Reiko Akagi Masayasu Inoue Kozo Utsumi Junzo Sasaki <p>We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. in addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.</p> No potential conflict of interest relevant to this article was reported.