Springer Science and Business Media LLC Acta Medica Okayama 2051-5960 13 1 2025 Rotenone targets midbrain astrocytes to produce glial dysfunction-mediated dopaminergic neurodegeneration 234 EN Ikuko Miyazaki Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nami Isooka Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ryo Kikuoka Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fuminori Imafuku Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaori Masai Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kana Tomimoto Department of Medical Neurobiology, 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 Chiharu Sogawa Department of Food and Health Sciences, Faculty of Environmental Studies, Hiroshima Institute of Technology Norio Sogawa Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshihisa Kitamura Department of Pharmacotherapy, School of Pharmacy, Shujitsu University Masato Asanuma Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Exposure to pesticides, such as rotenone or paraquat, is an environmental factor that plays an important role in the pathogenesis of Parkinson's disease (PD). Rotenone induces PD-like pathology and is therefore used to develop parkinsonian animal models. Dopaminergic neurotoxicity caused by rotenone has been attributed to the inhibition of mitochondrial complex I, oxidative stress and neuroinflammation; however, the mechanisms underlying selective dopaminergic neurodegeneration by rotenone remain unclear. To resolve this, we focused on glial diversity and examined whether the brain region-specific glial response to rotenone could determine the vulnerability of dopaminergic neurons using primary cultured neurons, astrocytes and microglia from the midbrain and striatum of rat embryos and rotenone-injected PD model mice. Direct neuronal treatment with low-dose rotenone failed to damage dopaminergic neurons. Conversely, rotenone exposure in the presence of midbrain astrocyte and microglia or conditioned media from rotenone-treated midbrain glial cultures containing astrocytes and microglia produced dopaminergic neurotoxicity, but striatal glia did not. Surprisingly, conditioned media from rotenone-treated midbrain astrocytes or microglia monocultures did not affect neuronal survival. We also demonstrated that rotenone targeted midbrain astrocytes prior to microglia to induce dopaminergic neurotoxicity. Rotenone-treated astrocytes produced secreted protein acidic and rich in cysteine (SPARC) extracellularly, which induced microglial proliferation, increase in IL-1β and TNF-α, and NF-κB (p65) nuclear translocation in microglia, resulting in dopaminergic neurodegeneration. In addition, rotenone exposure caused the secretion of NFAT-related inflammatory cytokines and a reduction in the level of an antioxidant metallothionein (MT)-1 from midbrain glia. Furthermore, we observed microglial proliferation and a decrease in the number of MT-positive astrocytes in the substantia nigra, but not the striatum, of low-dose rotenone-injected PD model mice. Our data highlight that rotenone targets midbrain astrocytes, leading to SPARC secretion, which promotes the neurotoxic conversion of microglia and leads to glial dysfunction-mediated dopaminergic neurodegeneration. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 26 20 2025 Neurofibromin Encoded by the Neurofibromatosis Type 1 (NF1) Gene Promotes the Membrane Translocation of SPRED2, Thereby Inhibiting the ERK Pathway in Breast Cancer Cells 10072 EN Nang Thee Su Pwint Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Neurofibromin (NF) inhibits the RAS/RAF/ERK pathway through its interaction with SPRED1 (Sprouty-related EVH1 domain-containing protein 1). Here, we investigated the functional relationship between NF and SPRED2 in breast cancer (BC). Human BC cell lines were transfected to downregulate or overexpress NF and SPRED2 and subsequently subjected to functional assays. Protein and mRNA levels were analyzed by Western blotting and RT-qPCR, respectively. Protein–protein interactions were examined by immunoprecipitation. Database analyses and immunohistochemistry (IHC) of BC tissues were performed to validate the in vitro findings. Downregulating NF or SPRED2 expression in BC cells enhanced cell proliferation, migration and invasion accompanied by RAF/ERK activation, whereas overexpression produced opposite effects. NF formed a protein complex with SPRED2 and facilitated its translocation to the plasma membrane. By IHC, SPRED2 membrane localization was absent in NF-negative luminal A and triple-negative BC (TNBC) but present in a subset of luminal A BC. By database analyses, both NF1 and SPRED2 mRNA levels were reduced in BC tissues, and luminal A BC patients with high expression of both NF1 and SPRED2 mRNA exhibited improved relapse-free survival. These results suggest a critical role for the NF–SPRED2 axis in BC progression and highlight it as a potential therapeutic target. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 2025 S100A8/A9-MCAM signaling promotes gastric cancer cell progression via ERK-c-Jun activation EN Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Xu Yang Department of Pathology, The First Affiliated Hospital, Zhejiang University School of Medicine Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Bo Pan The First Affiliated Hospital, Zhejiang University School of Medicine Fangping Wu School of Pharmaceutical Sciences, Zhejiang Chinese Medical University Xu Zhang Department of Urology, The First Affiliated Hospital, Zhejiang University School of Medicine Kazumi Sagayama Faculties of Educational and Research Management Field, Okayama University Bei Sun Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences S100 protein family members S100A8 and S100A9 function primarily as a heterodimer complex (S100A8/A9) in vivo. This complex has been implicated in various cancers, including gastric cancer (GC). Recent studies suggest that these proteins play significant roles in tumor progression, inflammation, and metastasis. However, the exact mechanisms by which S100A8/A9 contributes to GC pathogenesis remain unclear. This study investigates the role of S100A8/A9 and its receptor in GC. Immunohistochemical analysis was performed on GC tissue samples to assess the expression of the S100A8/A9 receptor melanoma cell adhesion molecule (MCAM). In vitro transwell migration and invasion assays were used to evaluate the motility and invasiveness of GC cells. Cell proliferation was assessed using a growth assay, and Western blotting (WB) was employed to examine downstream signaling pathways, including ERK and the transcription factor c-Jun, in response to S100A8/A9–MCAM interaction. S100A8/A9 stimulation enhanced both proliferation and migration through MCAM binding in GC cell lines. These cellular events were accompanied by ERK activation and c-Jun induction. Downregulation of MCAM suppressed both ERK phosphorylation and c-Jun expression, highlighting the importance of the S100A8/A9‒MCAM‒ERK‒c-Jun axis in promoting GC progression. These findings indicate that S100A8/A9 contributes to GC progression via MCAM, which activates the ERK‒c-Jun pathway. The S100A8/A9‒signaling axis may represent a novel therapeutic target in GC. No potential conflict of interest relevant to this article was reported.

Universitas Airlangga Acta Medica Okayama 2716-0920 34 2 2024 Depletion of Lysyl Oxidase-Like 4 (LOXL4) Attenuates Colony Formation in vitro and Collagen Deposition in vivo Breast Cancer Model 67 73 EN Ni Luh Gede Yoni Komalasari Department of Cell Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama University I Gde Haryo Ganesha Department of Histology, Faculty of Medicine, Udayana University I Gusti Nyoman Sri Wiryawan Department of Histology, Faculty of Medicine, Udayana University Nahoko Tomonobu Department of Cell Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama University Background: Lysyl oxidase (LOX) family proteins have recently become a topic in cancer progression. Our recent study found a high expression of LOX-like 4 (LOXL4) in MDA-MB-231 cells. Objective: To reveal the impact of depleted LOXL4 in both in vitro and in vivo breast cancer models from a histological perspective. Material and Method: Endogenous LOXL4 was depleted using the CRISPR/Cas9 on MDA-MB-231 parental cells. Based on the LOXL4 protein expression, the clone was determined for the next experiment, thus generating MDA-MB-231 LOXL4 KO. Cell assay was conducted using colony formation assay (n=3) followed by crystal violet staining. The indicated cells were inoculated orthotopically to female BALB/c nude mice (n=5). At the end of the experiment, tumors were isolated, fixed, and prepared for Masson Trichrome staining. Result: CRISPR/Cas9 completely depleted LOXL4 expression on clone number #2-22. Depletion of LOXL4 reduced the colony size formed by MDA-MB-231 cells. MDA-MB-231 LOXL4 KO #2-22 derived tumors showed depressed tumor volume compared to the parental group. Reduced collagen was also observed from the Masson Trichrome staining (p<0.001). Conclusion: Depletion of LOXL4 downregulates the growth of MDA-MB-231 cells in vitro and collagen deposition in vivo. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 NCF-1 plays a pivotal role in the survival of adenocarcinoma cells of pancreatic and gastric origins 1151 1159 EN Chiemi Furuya-Ikude Division of Tumor Pathology, NIR-PIT Research Institute, Kansai Medical University Akane Kitta Division of Tumor Pathology, NIR-PIT Research Institute, Kansai Medical University Naoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yoshihiro Kawasaki Division of Tumor Pathology, NIR-PIT Research Institute, Kansai Medical University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Eisaku Kondo Division of Tumor Pathology, NIR-PIT Research Institute, Kansai Medical University Reactive oxygen species (ROS) play a pivotal biological role in cells, with ROS function differing depending on cellular conditions and the extracellular environment. Notably, ROS act as cytotoxic factors to eliminate infectious pathogens or promote cell death under cellular stress, while also facilitating cell growth (via ROS-sensing pathways) by modifying gene expression. Among ROS-related genes, neutrophil cytosolic factor-1 (NCF-1; p47phox) was identified as a ROS generator in neutrophils. This product is a subunit of a cytosolic NADPH oxidase complex activated in response to pathogens such as bacteria and viruses. NCF-1 has been examined primarily in terms of ROS-production pathways in macrophages and neutrophils; however, the expression of this protein and its biological role in cancer cells remain unclear. Here, we report expression of NCF-1 in pancreatic and gastric cancers, and demonstrate its biological significance in these tumor cells. Abundant expression of NCF-1 was observed in pancreatic adenocarcinoma (PDAC) lines and in patient tissues, as well as in gastric adenocarcinomas. Accumulation of the protein was also detected in the invasive/metastatic foci of these tumors. Unexpectedly, BxPC-3 underwent apoptotic cell death when transfected with a small interfering RNA (siRNA) specific to NCF-1, whereas the cells treated with a control siRNA proliferated in a time-dependent manner. A similar phenomenon was observed in HSC-58, a poorly differentiated gastric adenocarcinoma line. Consequently, the tumor cells highly expressing NCF-1 obtained coincident accumulation of ROS and reduced glutathione (GSH) with expression of glutathione peroxidase 4 (GPX4), a quencher involved in ferroptosis. Unlike the conventional role of ROS as a representative cytotoxic factor, these findings suggest that NCF-1-mediated ROS generation may be required for expansive growth of PDAC and gastric cancers. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells 1215 1227 EN Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Tetta Takahashi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Toshiki Ochi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Kazumi Sagayama Organization for Research and Innovation Strategy, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector’s shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2076-3921 14 7 2025 Carnosol, a Rosemary Ingredient Discovered in a Screen for Inhibitors of SARM1-NAD+ Cleavage Activity, Ameliorates Symptoms of Peripheral Neuropathy 808 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Ogawa Tama Biochemical Co., Ltd. Yu Yasui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, 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 Yoji Wada Tama Biochemical Co., Ltd. Hiromichi Nakamura Tama Biochemical Co., Ltd. Masahiro Nishibori Department of Translational Research and Drug Development, 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 Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+) hydrolase involved in axonal degeneration and neuronal cell death. SARM1 plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents the degeneration; as a result, SARM1 has been attracting attention as a potent therapeutic target. In recent years, the development of several SARM1 inhibitors derived from synthetic chemical compounds has been reported; however, no dietary ingredients with SARM1 inhibitory activity have been identified. Therefore, we here focused on dietary ingredients and found that carnosol, an antioxidant contained in rosemary, inhibits the NAD+-cleavage activity of SARM1. Purified carnosol inhibited the enzymatic activity of SARM1 and suppressed neurite degeneration and cell death induced by the anti-cancer medicine vincristine (VCR). Carnosol also inhibited VCR-induced hyperalgesia symptoms, suppressed the loss of intra-epidermal nerve fibers in vivo, and reduced the blood fluid level of phosphorylated neurofilament-H caused by an axonal degeneration event. These results indicate that carnosol has a neuroprotective effect via SARM1 inhibition in addition to its previously known antioxidant effect via NF-E2-related factor 2 and thus suppresses neurotoxin-induced peripheral neuropathy. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 15 1 2025 Plasma S100A8/A9 level predicts response to immune checkpoint inhibitors in patients with advanced non-small cell lung cancer 2577 EN Tadahiro Kuribayashi Department of Hematology, Oncology and Respiratory Medicine, 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 Kiichiro Ninomiya Department of Allergy and Respiratory Medicine, Okayama University Hospital Go Makimoto Department of Allergy and Respiratory Medicine, Okayama University Hospital Toshio Kubo Department of Allergy and Respiratory Medicine, Okayama University Hospital Kammei Rai Department of Allergy and Respiratory Medicine, Okayama University Hospital Eiki Ichihara Center for Clinical Oncology, Okayama University Hospital Katsuyuki Hotta Department of Allergy and Respiratory Medicine, Okayama University Hospital Masahiro Tabata Department of Allergy and Respiratory Medicine, Okayama University Hospital Yoshinobu Maeda Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Katsuyuki Kiura Department of Allergy and Respiratory Medicine, Okayama University Hospital Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, 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 Kadoaki Ohashi Department of Allergy and Respiratory Medicine, Okayama University Hospital Blood-based predictive markers for the efficacy of immune checkpoint inhibitors (ICIs) have not yet been established. We investigated the association of the plasma level of S100A8/A9 with the efficacy of immunotherapy. We evaluated patients with unresectable stage III/IV or recurrent non-small cell lung cancer (NSCLC) who were treated with ICIs at Okayama University Hospital. The pre-treatment plasma levels of S100A8/A9 were analyzed. Eighty-one eligible patients were included (median age, 69 years). Sixty-two patients were men, 54 had adenocarcinoma, 74 had performance status (PS) 0–1, and 47 received ICIs as first-line treatment. The median time to treatment failure (TTF) for ICIs was 5.7 months, and the median overall survival (OS) was 19.6 months. The TTF and OS were worse in patients with high plasma S100A8/A9 levels (≥ 2.475 µg/mL) (median TTF: 4.3 vs. 8.5 months, p = 0.009; median OS: 15.4 vs. 38.0 months, p = 0.001). Multivariate analysis revealed that PS ≥ 2, liver metastasis, and high plasma S100A8/A9 levels were significantly associated with short TTF and OS. In conclusion, plasma S100A8/A9 level may have a limited effect on ICI therapy for NSCLC. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway 1138 1149 EN Jin Zhou Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naoko Mizuta Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuko Yamakawa Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, 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 Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11’s nature in colorectal cancers and others. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis 1371307 EN Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Nahoko Tomonobu Department of Cell Biology, 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 Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Kazumi Ninomiya Faculty of Science and Technology, Division of Molecular Science, Gunma University Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, 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 Background Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-beta 1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-beta 1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-kappa B (NF-kappa B). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-beta activated kinase 1 (TAK1) were required for the activation of NF-kappa B through I kappa beta kinase kinase (IKK alpha/beta) phosphorylation. Conclusion Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-beta 1-TRAF4-TAK1-IKK alpha/beta-I kappa beta alpha-NF-kappa B-MMP9, is crucial for TNBC cell invasiveness. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 25 11 2024 SPRED2 Is a Novel Regulator of Autophagy in Hepatocellular Carcinoma Cells and Normal Hepatocytes 6269 EN Tianyi Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0946-2716 101 4 2023 Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells 431 447 EN Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Léna Audebert Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Youyi Chen Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Chikako Yoshizawa Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masami Watanabe Department of Urology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3―namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0021-924X 174 6 2023 Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration 533 548 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Tha Zin Phoo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, 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 Ikuko Miyazaki Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Translational Research and Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masato Asanuma Department of Medical Neurobiology, 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 Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson’s disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface 1371342 EN Tetta Takahashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, 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 Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshito Zamami Department of Pharmacy, Okayama University Hospital Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein–protein interactions by an immunoprecipitation–Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0007-0920 130 9 2024 PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours 1493 1504 EN Shota Takihira Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Daisuke Yamada Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tatsunori Osone Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomoka Takao Department of Regenerative Science, 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 Michiyuki Hakozaki Department of Orthopedic Surgery, Fukushima Medical University School of Medicine Takuto Itano Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eiji Nakata Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomohiro Fujiwara Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyuki Kunisada Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshifumi Ozaki Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takeshi Takarada Department of Regenerative Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. Methods: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. Results: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1–TOP2A interaction. Conclusion: Targeting the PRRX1–TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 135 3 2023 日本組織培養学会第95回大会報告 181 182 EN Masakiyo Sakaguchi Department of Cell Biology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 13 2023 Lysyl oxidase-like 4 exerts an atypical role in breast cancer progression that is dependent on the enzymatic activity that targets the cell-surface annexin A2 1142907 EN Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, 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 Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, TheFourth Affiliated Hospital of Harbin Medical University Yoshihiko Sakaguchi Department of Microbiology, Kitasato University School of Medicine Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ich Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of Dalian University of Technology Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, 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 Background: LOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer. Methods: We established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells’ activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach. Results: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin β-1, resulting in the locking of integrin β-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth. Conclusions: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin β-1 on the cell surface. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 13 2023 LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells 1142886 EN Daisuke Hirabayashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Maruyama Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, 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 Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (Delta Zn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (Delta Zn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. Conclusions: These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 24 5 2023 SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells 4996 EN Tong Gao Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Xu Yang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tianyi Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nahoko Tomonobu Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44(+)CD90(+) and CD44(-)CD90(-) populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44(+)CD90(+) cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2306-5354 9 11 2022 Functional Blockage of S100A8/A9 Ameliorates Ischemia-Reperfusion Injury in the Lung 673 EN Kentaro Nakata Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikio Okazaki Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomohisa Sakaue Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuhei Komoda Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Dai Shimizu Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruchika Yamamoto Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaroh Miyoshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, 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 Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury. Mice were assigned to sham and IR groups with or without the injection of anti-S100A8/A9 neutralizing monoclonal antibody (mAb). (3) Results: Anti-S100A8/A9 mAb treatment significantly attenuated plasma S100A8/A9 levels compared with control IgG. As evaluated by oxygenation capacity and neutrophil infiltration, the antibody treatment dramatically ameliorated the IR injury. The gene expression levels of cytokines and chemokines induced by IR injury were significantly reduced by the neutralizing antibody. Furthermore, the antibody treatment significantly reduced TUNEL-positive cells, indicating the presence of apoptotic cells. (4) Conclusions: We identified S100A8/A9 as a novel therapeutic target against lung IR injury. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 18 2022 Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells 10300 EN Nahoko Tomonobu Department of Cell Biology, 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 Hidenori Wake Department of Pharmacology, Kindai University Faculty of Medicine Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University I. Made Winarsa Ruma Faculty of Medicine, Udayana University Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Faculty of Medicine, Udayana University Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Cell Biology, 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 The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1465-5411 24 1 2022 Exosomal Wnt7a from a low metastatic subclone promotes lung metastasis of a highly metastatic subclone in the murine 4t1 breast cancer 60 EN Chunning Li Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teizo Yoshimura Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miao Tian Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuze Wang Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takamasa Kondo Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-Ichi Yamamoto Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayoshi Fujisawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akihiro Matsukawa Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Background Patients with triple-negative breast cancer (TNBC) often have poorer prognosis than those with other subtypes because of its aggressive behaviors. Cancer cells are heterogeneous, and only a few highly metastatic subclones metastasize. Although the majority of subclones may not metastasize, they could contribute by releasing factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Here, we analyzed the interclonal communication in TNBC which leads to efficient cancer progression, particularly lung metastasis, using the polyclonal murine 4T1 BC model. Methods We isolated two 4T1 subclones, LM.4T1 and HM.4T1 cells with a low and a high metastatic potential, respectively, and examined the effects of LM.4T1 cells on the behaviors of HM.4T1 cells using the cell scratch assay, sphere-forming assay, sphere invasion assay, RT-qPCR, and western blotting in vitro. We also examined the contribution of LM.4T1 cells to the lung metastasis of HM.4T1 cells and TME in vivo. To identify a critical factor which may be responsible for the effects by LM.4T1 cells, we analyzed the data obtained from the GEO database. Results Co-injection of LM.4T1 cells significantly augmented lung metastases by HM.4T1 cells. LM.4T1-derived exosomes promoted the migration and invasion of HM.4T1 cells in vitro, and blocking the secretion of exosome abrogated their effects on HM.4T1 cells. Analyses of data obtained from the GEO database suggested that Wnt7a might be a critical factor responsible for the enhancing effects. In fact, a higher level of Wnt7a was detected in LM.4T1 cells, especially in exosomes, than in HM.4T1 cells, and deletion of Wnt7a in LM.4T1 cells significantly decreased the lung metastasis of HM.4T1 cells. Further, treatment with Wnt7a increased the spheroid formation by HM.4T1 cells via activation of the PI3K/Akt/mTOR signaling pathway. Finally, infiltration of alpha SMA-positive fibroblasts and angiogenesis was more prominent in tumors of LM.4T1 cells and deletion of Wnt7a in LM.4T1 cells markedly reduced angiogenesis. Conclusions We demonstrated, for the first time, that a low metastatic subclone can enhance lung metastasis of highly metastatic subclone via exosomal Wnt7a and propose Wnt7a as a molecular target to treat TNBC patients. 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.

MDPI Acta Medica Okayama 1422-0067 22 23 2021 Multifaceted Analysis of IL-23A-and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes 12659 EN Kota Tachibana Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Nina Tang Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Hitoshi Urakami Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Ai Kajita Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Mina Kobashi Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Hayato Nomura Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Minori Sasakura Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Satoru Sugihara Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Mamoru Ouchida Department of Molecular Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Shin Morizane Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-alpha, IL-17A, and interferon (IFN)-gamma, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA. Acta Medica Okayama 2234-943X 11 2021 RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion (vol 11, 665273, 2021) 729192 EN Fang He Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshinori Matsumoto Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yosuke Asano Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuriko Yamamura Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takayuki Katsuyama Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jose La Rose Princess Margaret Cancer Center, University Health Network, University of Toronto Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, 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 Robert Rottapel Princess Margaret Cancer Center, University Health Network, University of Toronto Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A. Acta Medica Okayama 2234-943X 11 2021 RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion 665273 EN Fang He Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshinori Matsumoto Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yosuke Asano Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuriko Yamamura Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takayuki Katsuyama Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jose La Rose Princess Margaret Cancer Center, University Health Network, University of Toronto Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, 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 Robert Rottapel Princess Margaret Cancer Center, University Health Network, University of Toronto Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Activity of transcription factors is normally regulated through interaction with other transcription factors, chromatin remodeling proteins and transcriptional co-activators. In distinction to these well-established transcriptional controls of gene expression, we have uncovered a unique activation model of transcription factors between tyrosine kinase ABL and RUNX2, an osteoblastic master transcription factor, for cancer invasion. We show that ABL directly binds to, phosphorylates, and activates RUNX2 through its SH2 domain in a kinase activity-dependent manner and that the complex formation of these proteins is required for expression of its target gene MMP13. Additionally, we show that the RUNX2 transcriptional activity is dependent on the number of its tyrosine residues that are phosphorylated by ABL. In addition to regulation of RUNX2 activity, we show that ABL transcriptionally enhances RUNX2 expression through activation of the bone morphogenetic protein (BMP)-SMAD pathway. Lastly, we show that ABL expression in highly metastatic breast cancer MDA-MB231 cells is associated with their invasive capacity and that ABL-mediated invasion is abolished by depletion of endogenous RUNX2 or MMP13. Our genetic and biochemical evidence obtained in this study contributes to a mechanistic insight linking ABL-mediated phosphorylation and activation of RUNX2 to induction of MMP13, which underlies a fundamental invasive capacity in cancer and is different from the previously described model of transcriptional activation. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 09145087 78 1 2020 Inhibitory effects of RAGE-aptamer on development of monocrotaline-induced pulmonary arterial hypertension in rats 12 16 EN Kazufumi Nakamura Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Akagi Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaro Ejiri Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masashi Yoshida Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toru Miyoshi Department of Cardiovascular Medicine, 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 Naofumi Amioka Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Luh Oliva Saraswati Suastika Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Megumi Kondo Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Nakayama Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoichi Takaya Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuichiro Higashimoto Department of Chemistry, Kurume University School of Medicine Kei Fukami Division of Nephrology, Department of Medicine, Kurume University School of Medicine Hiromi Matsubara Department of Cardiology, National Hospital Organization Okayama Medical Center Hiroshi Ito Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: The receptor for advanced glycation end products (RAGE), a transmembrane receptor belonging to the immunoglobulin superfamily, is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with pulmonary arterial hypertension (PAH) and is implicated in the etiology of PAH. Recently, we reported that RAGE-aptamer, a short and single-stranded DNA directed against RAGE, inhibited an inappropriate increase in cultured PASMCs in PAH. The aim of this study was to determine the efficacy of RAGEaptamer in monocrotaline-induced PAH in rats. Methods and Results: Rats were assigned to either an untreated control group, a group that received continuous subcutaneous administration of RAGE-aptamer immediately after monocrotaline injection, or a group that received control-aptamer immediately after monocrotaline injection. All rats survived 21 days after injection of monocrotaline and control-aptamer or RAGE-aptamer. Injection of monocrotaline with continuous subcutaneous delivery of control-aptamer resulted in higher right ventricular systolic pressure compared with controls. This increase was attenuated by continuous subcutaneous delivery of RAGE-aptamer. The proportion of small pulmonary arteries with full muscularization was greater in the monocrotaline and control-aptamer group than in the control group. Continuous subcutaneous delivery of RAGE-aptamer significantly reduced the percentage of small pulmonary arteries with full muscularization Conclusions: Continuous subcutaneous delivery of RAGE-aptamer suppresses development of monocrotaline-induced PAH in rats. Inhibition of RAGE ameliorates muscularization of 3 small pulmonary arteries. Treatment with RAGE-aptamer might be a new therapeutic option for PAH. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 22 2020 Neuroplastinβ-mediated upregulation of solute carrier family 22 member 18 antisense (SLC22A18AS) plays a crucial role in the epithelial-mesenchymal transition, leading to lung cancer cells' enhanced motility 100768 EN Karolina Bajkowska Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, 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 Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School I. Made Winarsa Ruma University of Surrey Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Our recent study revealed an important role of the neuroplastin (NPTN)β downstream signal in lung cancer dissemination in the lung. The molecular mechanism of the signal pathway downstream of NPTNβ is a serial activation of the key molecules we identified: tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) adaptor, nuclear factor (NF)IA/NFIB heterodimer transcription factor, and SAM pointed-domain containing ETS transcription factor (SPDEF). The question of how dissemination is controlled by SPDEF under the activated NPTNβ has not been answered. Here, we show that the NPTNβ-SPDEF-mediated induction of solute carrier family 22 member 18 antisense (SLC22A18AS) is definitely required for the epithelial-mesenchymal transition (EMT) through the NPTNβ pathway in lung cancer cells. In vitro, the induced EMT is linked to the acquisition of active cellular motility but not growth, and this is correlated with highly disseminative tumor progression in vivo. The publicly available data also show the poor survival of SLC22A18AS-overexpressing lung cancer patients. Taken together, these data highlight a crucial role of SLC22A18AS in lung cancer dissemination, which provides novel input of this molecule to the signal cascade of NPTNβ. Our findings contribute to a better understanding of NPTNβ-mediated lung cancer metastasis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 4 2020 Cytotoxic Effects of Alcohol Extracts from a Plastic Wrap (Polyvinylidene Chloride) on Human Cultured Liver Cells and Mouse Primary Cultured Liver Cells 327 334 EN Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroko Kagawa Okayama University Hospital Sakae Arimoto Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Xian Wen Tan Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kento Yasui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiyuki Oshiki Department of Applied Chemistry, Graduate School of Natural Science and Technology, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/60371 An increasing accumulation of microplastics and further degraded nanoplastics in our environment is suspected to have harmful effects on humans and animals. To clarify this problem, we tested the cytotoxicity of two types of plastic wrap on human cultured liver cells and mouse primary cultured liver cells. Alcohol extracts from plastic wrap, i.e., polyvinylidene chloride (PVDC), showed cytotoxic effects on the cells. Alcohol extracts of polyethylene (PE) wrap were not toxic. The commercially available PVDC wrap consists of vinylidene chloride, epoxidized soybean oil, epoxidized linseed oil as a stiffener and stabilizer; we sought to identify which component(s) are toxic. The epoxidized soybean oil and epoxidized linseed oil exerted strong cytotoxicity, but the plastic raw material itself, vinylidene chloride, did not. Our findings indicate that plastic wraps should be used with caution in order to prevent health risks. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 23 6 2020 Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A 101180 EN Shangze Gao Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidenori Wake Department of Pharmacology, 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 Dengli Wang Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youhei Takahashi Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kiyoshi Teshigawara Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hui Zhong Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuji Mori Department of Pharmacology, School of Pharmacy, Shujitsu University Keyue Liu Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideo Takahashi Department of Pharmacology, Faculty of Medicine, Kindai University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences High-mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG), a 75 kDa plasma protein, was demonstrated to improve the survival rate of septic mice through the regulation of neutrophils and endothelium barrier function. As the relalionship of HRG and HMGB1 remains poorly understood, we investigated the effects of HRG on HMGB1-mediated pathway in endothelial cells, focusing on the involvement of specific receptors for HRG. HRC potently inhibited the HMGB1 mobilization and effectively suppressed rHMGB1-induced inflammatory responses and expression of all three HMGB1 receptors in endothelial cells. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Thus, current study elueiates protective effects of HRG on vascular endothelial cells through inhintion of HMGB1-mediated pathways may contribute to the therapeutic effects of HRG on severe sepsis. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 19365233 13 4 2020 S100 Soil Sensor Receptors and Molecular Targeting Therapy Against Them in Cancer Metastasis 100753 EN Nahoko Tomonobu Department of Cell Biology, 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 Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The molecular mechanisms underlying the 'seed and soil' theory are unknown. S100A8/A9 (a heterodimer complex of S100A8 and S100A9 proteins that exhibits a 'soil signal') is a ligand for Toll-like receptor 4, causing distant melanoma cells to approach the lung as a 'seeding' site. Unknown soil sensors for S100A8/A9 may exist, e.g., extracellular matrix metalloproteinase inducer, neuroplastin, activated leukocyte cell adhesion molecule, and melanoma cell adhesion molecule. We call these receptor proteins 'novel S100 soil sensor receptors (novel SSSRs).' Here we review and summarize a crucial role of the S100A8/A9-novel SSSRs' axis in cancer metastasis. The binding of S100A8/A9 to individual SSSRs is important in cancer metastasis via upregulations of the epithelial-mesenchymal transition, cellular motility, and cancer cell invasiveness, plus the formation of an inflammatory immune suppressive environment in metastatic organ(s). These metastatic cellular events are caused by the SSSR-featured signal transductions we identified that provide cancer cells a driving force for metastasis. To deprive cancer cells of these metastatic forces, we developed novel biologics that prevent the interaction of S100A8/A9 with SSSRs, followed by the efficient suppression of S100A8/A9-mediated lung-tropic metastasis in vivo. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0009-2797 324 2020 Xylitol acts as an anticancer monosaccharide to induce selective cancer death via regulation of the glutathione level 109085 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, 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 Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Herbal medicines and their bioactive compounds are increasingly being recognized as useful drugs for cancer treatments. The parasitic fungus Cordyceps militaris is an attractive anticancer herbal since it shows very powerful anticancer activity due to its phytocompound cordycepin. We previously discovered and reported that a high amount of xylitol is present in Cordyceps militaris extract, and that xylitol unexpectedly showed anticancer activity in a cancer-selective manner. We thus hypothesized that xylitol could become a useful supplement to help prevent various cancers, if we can clarify the specific machinery by which xylitol induces cancer cell death. It is also unclear whether xylitol acts on cancer suppression in vivo as well as in vitro. Here we show for the first time that induction of the glutathione-degrading enzyme CHAC1 is the main cause of xylitol-induced apoptotic cell death in cancer cells. The induction of CHAC1 is required for the endoplasmic reticulum (ER) stress that is triggered by xylitol in cancer cells, and is linked to a second induction of oxidative stress in the treated cells, and eventually leads to apoptotic cell death. Our in vivo approach also demonstrated that an intravenous injection of xylitol had a tumor-suppressing effect in mice, to which the xylitol-triggered ER stress also greatly contributed. We also observed that xylitol efficiently sensitized cancer cells to chemotherapeutic drugs. Based on our findings, a chemotherapeutic strategy combined with xylitol might improve the outcomes of patients facing cancer. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 25890042 23 2 2020 PLOD2 is essential to functional activation of integrin β1 for invasion/metastasis in head and neck squamous cell carcinomas. 100850 EN Yushi Ueki Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Izumi Sakamoto ACROSCALE Inc. Yasuhiro Kanda Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Arata Horii Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 14765586 21 12 2019 PODXL1 promotes metastasis of the pancreatic ductal adenocarcinoma by activating the C5aR/C5a axis from the tumor microenvironment 1121 1132 EN Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Satoshi Maruyama Oral Pathology Section, Department of Surgical Pathology, Niigata University Hospital I. Wayan Sumardika Department of Cell Biology, 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 Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 18 2019 Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue 100619 EN Nahoko Tomonobu Department of Cell Biology, 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 I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, 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 Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1522-8002 21 7 2019 Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness 627 640 EN Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, 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 Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Yosuke Mitsui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences I Made Winarsa Ruma Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroki Sato Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuta Tomida Department of Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichi Soh Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miyoko Kubo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endy Widya Putranto Department of Pediatrics, Dr. Sardjito Hospital/Faculty of Medicine, Universitas Gadjah Mada Takashi Murakami Department of Microbiology, Faculty of Medicine, Saitama Medical University Ming Liu Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Toshihiko Hibino Department of Dermatology, Tokyo Medical University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Departments of Thoracic, Breast and Endocrinological Surgery, 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 Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Ltd. Acta Medica Okayama 1107-3756 32 4 2013 Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 938 944 EN Endy Widya Putranto Hitoshi Murata Ken-Ichi Yamamoto Ken Kataoka Hidenori Yamada Jun-Ichiro Futami Masakiyo Sakaguchi Nam-Ho Huh The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 68 1 2014 Preclinical Evaluation of MicroRNA-34b/c Delivery for Malignant Pleural Mesothelioma 23 26 EN Tsuyoshi Ueno Shinichi Toyooka Takuya Fukazawa Takafumi Kubo Junichi Soh Hiroaki Asano Takayuki Muraoka Norimitsu Tanaka Yuho Maki Kazuhiko Shien Masashi Furukawa Masakiyo Sakaguchi Hiromasa Yamamoto Kazunori Tsukuda Shinichiro Miyoshi Original Article 10.18926/AMO/52140 The microRNA-34s (miR-34s) have p53 response elements in their 5ʼ-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0020-7136 126 7 2010 Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3 1562 1569 EN Ryuta Tanimoto Masakiyo Sakaguchi Fernando Abarzua Ken Kataoka Kaoru Kurose Hitoshi Murata Yasutomo Nasu Hiromi Kumon Nam-Ho Huh We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC. No potential conflict of interest relevant to this article was reported.

The American Society for Biochemistry and Molecular Biology, Inc. Acta Medica Okayama 0021-9258 284 21 2009 Overexpression of REIC/Dkk-3 in Normal Fibroblasts Suppresses Tumor Growth via Induction of Interleukin-7 14236 14244 EN Masakiyo Sakaguchi Ken Kataoka Fernando Abarzua Ryuta Tanimoto Masami Watanabe Hitoshi Murata Swe Swe Than Kaoru Kurose Yuji Kashiwakura Kazuhiko Ochiai Yasutomo Nasu Hiromi Kumon Nam-ho Huh We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1 alpha, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 66 1 2012 Preclinical Safety and Efficacy of in Situ REIC/Dkk-3 Gene Therapy for Prostate Cancer 7 16 EN Keiichiro Kawauchi Masami Watanabe Haruki Kaku Peng Huang Kasumi Sasaki Masakiyo Sakaguchi Kazuhiko Ochiai Nam-ho Huh Yasutomo Nasu Hiromi Kumon Original Article 10.18926/AMO/48076 The preclinical safety and therapeutic efficacy of adenoviral vectors that express the REIC/Dkk-3 tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer effects in vitro and in vivo, and we herein report the results of two in vivo studies. First, intra-tumor Ad-hREIC administration was examined for toxicity and therapeutic effects in a subcutaneous tumor model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration was tested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed. Both experiments indicated that there was no significant difference in the examined parameters between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In the in vitro analysis using PC3 cells, a significant apoptotic effect was observed after Ad-hREIC treatment. Confirming this observation, the robust anti-tumor efficacy of Ad-hREIC was demonstrated in the in vivo subcutaneous prostate cancer model. Based on the results of these preclinical experiments, we consider the adenovirus-mediated REIC/Dkk-3 in situ gene therapy to be safe and useful for the clinical treatment of prostate cancer. No potential conflict of interest relevant to this article was reported.

Academic Press Inc Elsevier Science Acta Medica Okayama 0006-291X 412 2 2011 Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1 391 395 EN Kazuhiko Ochiai Masami Watanabe Hideo Ueki Peng Huang Yasuyuki Fujii Yasutomo Nasu Hirofumi Noguchi Takeshi Hirata Masakiyo Sakaguchi Nam-ho Huh Yuji Kashiwakura Haruki Kaku Hiromi Kumon Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than ORB. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the ORB and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 56 1 2002 Localization of S100C immunoreactivity in various human tissues. 31 34 EN Asami Kondo Masakiyo Sakaguchi Eiichi Makino Masayoshi Namba Shigeru Okada Nam-ho Huh Article 10.18926/AMO/31719 Using 2-dimensional gel electrophoresis, we previously demonstrated that the S100C protein remarkably decreased after immortalization of normal human fibroblasts, and that this protein caused growth inhibition of human tumor cells when forcibly expressed in these cells, suggesting that S100C plays a significant role in tumor suppression. The present study was carried out to determine what type of human tissues express S100C protein, and, subsequently, whether the S100C content in these tissues changes after normal cells have been transformed into cancer cells. We found that ductal cells in various tissues were positively stained with the S100C protein. In comparison, epithelial cells in digestive organs such as the stomach, small intestine, and colon were not stained as strongly. When 14 pairs of human normal and cancerous tissues were stained with the antibody, decreases in the staining levels of S100C were observed in 6 kinds of cancerous tissues--from the bronchus, mammary duct, renal tubule, prostate, uterus, and testis--in comparison with staining in their normal counterparts. These results suggest that S100C is a new tumor marker protein, the expression of which significantly decreases after malignant transformation of human tissues. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 62 6 2008 Mechanistic Analysis of Resistance to REIC/Dkk-3-induced Apoptosis in Human Bladder Cancer Cells 393 401 EN Tomoko Kobayashi Masakiyo Sakaguchi Ryuta Tanimoto Fernando Abarzua Mikiro Takaishi Haruki Kaku Ken Kataoka Takashi Saika Yasutomo Nasu Masahiro Miyazaki Hiromi Kumon Nam-ho Huh Original Article 10.18926/AMO/30945 We have recently shown that a new therapeutic modality using the REIC/Dkk-3 gene (Ad-REIC) is effective against various human cancers, including those of prostate, testis and breast origins. The aim of the present study was to examine the sensitivity of bladder cancers to Ad-REIC and to clarify the molecular mechanisms that determine sensitivity/resistance. We found that 2 human bladder cancer cell lines, T24 and J82, are resistant to Ad-REIC. In T24 and J82 cells, the ER stress response and activation of JNK were observed in a manner similar to that in the sensitive PC3 cells. Translocation of Bax to mitochondria occurred in PC3 cells but not in T24 and J82 cells. Bcl-2 was remarkably overexpressed in T24 and J82 compared with the expression levels in sensitive cell lines. Treatment of T24 and J82 cells with a Bcl-2 inhibitor sensitized the cells to Ad-REIC-induced apoptosis. The results indicate that some human bladder cancers are resistant to apoptosis induced by overexpression of REIC/Dkk-3, which is at least in part due to up-regulation of Bcl-2. These results provide a basis for possible use of Bcl-2 as a marker of sensitive cancers and to try to sensitize resistant cancers to Ad-REIC by down-regulation of Bcl-2. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2001 Relationship Between Contact Inhibition and Intranuclear S100C of Normal Human Fibroblasts EN No potential conflict of interest relevant to this article was reported.