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Tatsuya Yoshida,1 Takuya Miura,1 Tomoh Matsumiya,2 Hidemi Yoshida,2 Hajime Morohashi,1 Yoshiyuki Sakamoto,1 Akira Kurose,3 Tadaatsu Imaizumi,2 Kenichi Hakamada1 1Department of Gastroenterological Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562, Japan; 2Department of Vascular Biology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562, Japan; 3Department of Anatomic Pathology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562, JapanCorrespondence: Kenichi HakamadaDepartment of Gastroenterological Surgery, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, JapanTel +81 172 39 5079Fax +81 172 39 5080Email hakamada@hirosaki-u.ac.jpPurpose: Colorectal cancer (CRC) often recurs after curative resection. Identification of major risk factors for CRC recurrence is important for effective prevention and treatment. In this study, we examined the potential relationship between CRC and TLR3 as this remains unclear.Patients and Methods: Correlations between TLR3 immunostaining and clinicopathological factors and prognosis were examined in 50 samples that were randomly extracted from 264 patients with CRC from January 2010 to December 2011. Chemokines induced by TLR3 agonist stimulation were also examined using TLR3-positive human CRC cell lines. Furthermore, the association between TLR3 and chemokine expression was assessed by analyzing the immunohistochemistry of surgical specimens.Results: Of the 50 patients, 14 (28%) were TLR3-negative. In the comparison of clinicopathological factors between the TLR3-negative and -positive groups, there were more lymph node metastasis-positive cases in the TLR3-negative group, and this difference was significant. Furthermore, there was no difference in overall survival rates between the two groups, but the 5-year recurrence-free survival (RFS) was significantly lower in the TLR3-negative group (46.2%) than in the TLR3-positive group (78.1%). Analysis of 5-year RFS using factors thought to be related to recurrence identified a high tumor budding and a TLR3-negative status as independent risk factors for recurrence. TLR3 activation of CRC cell lines induced expression of C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), and interleukin-8. The expressions of CCL2, CCL5, and IL-8 were observed in the TLR3-positive tumor cells of surgical specimens.Conclusion: Non-expression of TLR3 in CRC cells was associated with lymph node metastasis and was an independent risk factor for recurrence. These results suggest that TLR3 may not only be used as a prognostic factor and a risk factor for recurrence, but further studies on the involvement of TLR3 with tumor growth may provide new therapeutic strategies.Keywords: colorectal cancer, toll-like receptor 3, recurrence, CCL2, CCL5, IL-8

Toxic amyloid-beta （Aβ） is known to generate symptoms of Alzheimer’s disease （AD）; however, less is known regarding the neurotoxicity of Aβ at lower concentrations. Moreover, the neuroprotective potential of combination treatment with plant biophenols and existing drugs is not well understood. In this study, we estimated the no-observed adverse effect level （NOAEL） of Aβ 1–42 （Aβ42） against cultured human neuroblastoma SHSY5Y cells, and examined the neuroprotective effect of combination pretreatment with 10 μM carnosic acid, 30 nM rebamipide, 10 μM edaravone, and 10 μM of resveratrol （the “CRER” blend） on weak but toxic Aβ42-treated SH-SY5Y cells. We evaluated the NOAEL of Aβ42 at 500 nM in these cells. Aβ42 at 1–8 μM reduced cell viability; however, the “CRER” blend ameliorated this Aβ42-induced decrease in viability. The “CRER” blend induced the expression of Aβ-degrading enzymes including matrix metalloproteinase-14 （MMP-14） and neprilysin, while also enhancing the expression of the inducible α-secretase TACE （tumor necrosis factor-α-converting enzyme）, sirtuin 1 （SIRT1）, and brain-derived neurotrophic factor（ BDNF）. Collectively, our results indicate that the “CRER” may aid in the prevention of Aβ toxicity by enhancing MMP-14, neprilysin, TACE, SIRT1, and BDNF. Thus, the “CRER” blend may prove to be a promising strategy for the prevention of Aβ-mediated disorders, particularly AD.

Purpose: Retinoic acid-inducible gene-I （RIG-I） is a member of cytoplasmic viral sensors which plays an important role in inflammation of biliary epithelial cells（ BECs）. The aim of this study is to examine if RIG-I and C-X-C motif chemokine 10（ CXCL10） are involved in the etiology of human biliary atresia（ BA）. Methods: Immunohistochemical study was performed on surgically resected tissues obtained（ June 1994 to March 2016）from 30 infants with BA and non-inflamed hepatic tissues from 7 infants with hepatoblastoma. A semiquantitative scoring system was designed to evaluate the staining with an antibodies to the RIG-I and CXCL10. The expression of RIG-I and CXCL10 in HuCCT1 cholangiocarcinoma cell line were studied by western blotting, ELISA and RT-PCR analyses. Results: Intense immunoreactivity for RIG-I and CXCL10 was detected in BECs in tissues resected from BA patients.The expression of RIG-I and CXCL10 in the hilar tissue was significantly stronger than in the hepatic tissue.Transfection of HuCCT1 cells with poly（I:C）, a synthetic analog of viral dsRNA, induced the expression of RIG-I, and knockdown of RIG-I inhibited the induction of CXCL10 in HuCCT1 cells transfected with poly（I:C）. Conclusion: These results suggest that RIG-I-CXCL10 cascade may be involved in the etiology of human BA.

Dysregulation of iron homeostasis in brain causes various neurodegenerative disorders. In fact, high concentration of iron is present in brains of patients with Alzheimer’s disease. It was previously reported that CXCL8 protects human neurons from amyloid-β-induced neurotoxicity and that astrocytes have the potential to play important roles in Alzheimer’s disease. In the present study, we examined the effect of desferrioxamine, an iron chelator, on the expression of CXCL8 in U373MG human astrocytoma cells used as a model of astrocytes. Treatment of the cells with desferrioxamine induced the expression of CXCL8. Pretreatment of the cells with FeSO4 counteracted the positive effect of desferrioxamine on CXCL8 production, suggesting that the effect of desferrioxamine was due to iron chelation. RNA interference experiments showed that HIF-1α was not involved in desferrioxamine-induced CXCL8 expression. We conclude that desferrioxamine induces CXCL8 in astrocytes and the chelation of iron may be a new therapeutic strategy for Alzheimer’s disease., 弘前医学. 66, 2016, p.127-134

The CXC chemokine growth-related oncogene protein-α （GRO-α） has a wide variety of biological activities including as neutrophil trafficking or migration of vascular endothelial cells. In addition, studies have shown a crosstalk between tumor cells and vascular endothelial cells; GRO-α released by endothelial cells induces invasion of tumor cells toward endothelial cells, indicating an importance of GRO-α in a tumor environment. Oral squamous cells are reported to produce GRO-α in response to cytokines such as tumor necrosis factor-α （TNF-α）. However, little is known about how GRO-α is involved in oral cancer. Here, we investigated the biological role of GRO-α for both tumor growth and angiogenesis in oral squamous cell carcinoma cells. We first evaluated the effect of TNF-α on GRO-α expression in three oral cancer cells from different origins. Among the cell lines we used, KOSC-2 cells expressed the highest amount of GRO-α mRNA in response to TNF-α. TNF-α-treated condition medium from KOSC-2 cells enhanced endothelial cell chemotaxis and the chemotactic activity was partially inhibited by the addition of neutralizing anti-GRO-α antibody. In addition, GRO-α exerted tumor cell migration of KOSC-2. From these results, we conclude that GRO-α may contribute to both angiogenesis and proliferation in oral cancer., 弘前医学. 65(2-4), 2014, p.147-155

Viral infection is important in renal pathology both as a trigger of chronic inflammatory diseases and as a complication associated with organ transplantation. Glomerular mesangial cells produce a variety of functional molecules potentially involved in immune reactions, and we investigated anti-viral responses in normal human mesangial cells. Human mesangial cells were treated with polyinosinic-polycytidylic acid （poly IC）, an authentic double-stranded RNA that mimics viral RNA. Treatment of cells with poly IC induced interferon-β （IFN-β）, retinoic acid-inducible gene-I（ RIG-I）, CC chemokine ligand 5（ CCL5）, differentiated embryo-chondrocyte 2（ DEC2） and IFN-stimulated gene 20 （ISG20）. Knockdown of toll-like receptor 3 （TLR3）, by RNA interference （RNAi）, abolished the poly IC-induced expression of these molecules. RNAi against IFN-β inhibited the induction of RIG-I, CCL5 and ISG20, but not of DEC2. Knockdown of RIG-I resulted in the reduced expression of CCL5. RNAi against DEC2 enhanced the poly IC-induced expression of IFN-β, RIG-I and CCL5. Transfection of cells with a poly IC/cationic lipid complex induced IFN-β, RIG-I and ISG20. Knockdown of RIG-I decreased the expression of IFN-β and ISG20 induced by transfection with poly IC/cationic lipid. TLR3 and RIG-I may function as recognition receptors against double-stranded RNA, which induce IFN-β and its downstream IFN-inducible genes. In the signaling elicited by poly IC, the IFN-inducible genes include RIG-I and effector molecules as CCL5 with leukocyte chemotactic activity and ISG20 with exonuclease activity on single-stranded RNA. The poly IC-induced expression of DEC2 is independent on IFN-β and it may control the signaling elicited by double-stranded RNA. The poly IC-inducible molecules may mediate anti-viral innate responses in renal mesangial cells., 弘前医学. 64(Suppl.), 2013, p.S70-73

The circadian rhythms in mammals are regulated by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. Five clock-gene families, i.e. Clock, Bmal, Per, Cry and Dec, have been found to be involved in a transcription-translation feedback loop that generates the circadian rhythm at the intracellular level. In this study, we examined functional analysis of the Dec gene. DEC1 and DEC2 are basic-helix-loop-helix （bHLH） transcription factors, involved in cellular diff erentiation, responses to hypoxia, and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was upregulated by hypoxia, however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor （VEGF） is upregulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1 α （HIF-1α） and arylhydrocarbon receptor nuclear translocator 1 （ARNT1）. In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1α but not with ARNT1. The binding of HIF-1α to the hypoxia response element（ HRE） in the VEGF promoter was decreased by DEC2 overexpression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved., 弘前医学. 61(Suppl.), 2010, p.S43-S52

Optimization of neuronal function and survival is an important goal in the treatment of cerebrovascular diseases in order to avoid or improve devastating long-term sequelae. Nerve growth factor （NGF） is essential for neuronal growth and survival in the central nervous system （CNS）. Vascular endothelial growth factor （VEGF） is a potent mitogen specifi c for endothelial cells and a stimulator of neovascularization. VEGF also enhances vascular permeability, which may promote the development of brain edema during cerebral ischemia. These molecules aff ect the outcome of ischemia/reperfusion injury in the CNS. Edaravone, a brain-penetrant, free radical scavenger, is known to ameliorate postischemic neuronal dysfunction. Transcription factor Nrf2（ nuclear factor-erythroid 2-related factor 2）, a master regulator of antioxidant responses, plays an important role in the coordinated expressions of stress-inducible genes. Astrocytes express various genes involved in the regulation of neuronal functions, and the regulation of astrocyte gene expressions may be a potential therapeutic target in brain injury. This review aims to appraise the eff ects of radical scavenger edaravone and a natural Nrf2-inducer as neuroprotective agents in human astrocytes, particularly under an experimental model for hypoxia/reoxygenation., 弘前医学. 61(Suppl.), 2010, p.S147-S156

TDP-43 proteinopathy （amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions） is a newly categorized group of neurodegenerative disorders characterized by abnormal accumulation and mislocalization of nuclear TDP-43 protein in the neuronal cytoplasm. 15-deoxy-Δ12,14-prostaglandin J2 （15d-PGJ2） is non-enzymatically produced from PGD2, and plays roles in infl ammation and oxidative stress responses. Indeed, 15d-PGJ2 is up-regulated in the spinal motor neurons in ALS. In this study, biochemical and fluorescent staining analyses showed that 15d-PGJ2 modifi es expression, solubility, and subcellular localization of TDP-43. This alteration was at least partly related to a cyclopentenone ring structure containing an electrophilic carbon of 15d-PGJ2, because 15d-PGJ2 analogue, which lacks an cyclopentenone ring structure, had almost no eff ect on TDP-43 protein. Finally in vitro binding experiment indicated that 15d-PGJ2 is covalently bound to TDP-43 protein. These fi ndings suggest that a sustained high level of 15d-PGJ2 is involved in the pathogenesis of neurodegenerative disorders related to abnormal TDP-43 protein., 弘前医学. 61(Suppl.), 2010, p.S204-S210