酸化型HMGB-1は間葉系幹細胞/間葉系細胞を介して大腸癌の転移性を促進する

High mobility group box-1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post-translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three-fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM-MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM-MSCs pretreated with oxidized HMGB1 were co-cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co-culture with reduced HMGB1-pretreated BM-MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1–MSC axis may be an important target for cancer therapy.博士（医学）・甲第874号・令和5年3月15

Role of CD10 in the Metastasis of Colorectal Cancer to the Liver.

CD10 is a widely expressed endopeptidase that is present in human colorectal cancer (CRC), which shows a high frequency of liver metastasis. CD10 expression in CRC cells is associated with liver metastasis in rodent models, and CD10 expression enhances the phosphorylation of epidermal growth factor (EGF) receptor (EGFR) and extracellular signalregulated kinase (ERK) l/2. Met-enkephalin (MENK), a CD10 substrate, activates its specific receptor δ-opioid receptor (DOR), which is expressed in CRCs. DOR is a partial agonist of ERK1/2, which suppresses EGF-induced phosphorylation of EGFR and ERK1/2. CD10 retains EGF-induced EGFR activation by degrading MENK. Paradoxically, CRCs express MENK at a high frequency. Since MENK suppresses T lymphocytes, CD10-expressing CRCs can escape from T-cell immunity without exhibiting auto-inhibition. CD10 is strongly associated with the metastasis of CRCs to the liver via an immunosuppressive mechanism. Additionally, CD10 may be an excellent serum marker for liver metastasis in patients with CRC and could represent a potential molecular target for antimetastatic treatment in patients with CRC

胃癌におけるクローディン4標的化によるシスプラチン化学療法感受性の向上

Claudins are major tight-junction proteins that mediate cellular polarity and differentiation. The present study investigated whether the 4D3 antibody to the human CLDN4 extracellular domain (that we previously established) is capable of modulating chemotherapeutic sensitivity in gastric cancer (GC). The results of the present study showed that CLDN4 was overexpressed in 137 of the 192 analyzed GC cases, and that CLDN4 expression was retained in tumors of a lower histological grade (more differentiated), and/or those that were caudal-type homeobox protein 2 (CDX2)-positive, but was reduced in more highly undifferentiated, and CDX2-negative GC cases. The study also compared the synergic effects of combining 4D3 with CDDP treatment and knocking down CLDN4 expression in MKN74 and TMK-1 human GC cells. Co-treatment with 4D3 increased anti-tumor effects of CDDP, whereas CLDN4 knockdown did not. In the TMK-1 cells, non-tight junction CLDN4 associated with integrin β1, increasing stem cell-associated proteins via FAK-c-SRC signals. The anti-tumoral effect of CDDP and 4D3 was examined in a nude mouse subcutaneous tumor model. In the two GC cell lines, concurrent treatment with 4D3 and CDDP synergistically inhibited cell proliferation and increased tumor necrosis and apoptosis to a greater degree than CDDP treatment alone. These findings suggest that 4D3 might increase chemotherapeutic sensitivity by evoking structural disintegration of tight-junction CLDN4 expressed in gastric cancer.博士（医学）・甲第713号・令和元年6月26日Copyright: Nishiguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0 https://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

中鎖脂肪酸と糖質の併用摂取は癌関連骨格筋萎縮から保護する

Skeletal muscle volume is associated with prognosis of cancer patients. Maintenance of skeletal muscle is an essential concern in cancer treatment. In nutritional intervention, it is important to focus on differences in metabolism between tumor and skeletal muscle. We examined the influence of oral intake of glucose (0%, 10%, 50%) and 2% medium-chain fatty acid (lauric acid, LAA, C12:0) on tumor growth and skeletal muscle atrophy in mouse peritoneal metastasis models using CT26 mouse colon cancer cells and HT29 human colon cancer cells. After 2 weeks of experimental breeding, skeletal muscle and tumor were removed and analyzed. Glucose intake contributed to prevention of skeletal muscle atrophy in a sugar concentration-dependent way and also promoted tumor growth. LAA ingestion elevated the level of skeletal muscle protein and suppressed tumor growth by inducing tumor-selective oxidative stress production. When a combination of glucose and LAA was ingested, skeletal muscle mass increased and tumor growth was suppressed. Our results confirmed that although glucose is an important nutrient for the prevention of skeletal muscle atrophy, it may also foster tumor growth. However, the ingestion of LAA inhibited tumor growth, and its combination with glucose promoted skeletal muscle integrity and function, without stimulating tumor growth. These findings suggest novel strategies for the prevention of skeletal muscle atrophy.博士（医学）・甲第733号・令和2年3月16日© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License(https://creativecommons.org/licenses/by-nc/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes

Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein

Effects of a high-sodium diet on renal tubule Ca2+ transporter and claudin expression in Wistar-Kyoto rats

Background: Urinary Ca^{2+} excretion increases with dietary NaCl. NaCl-induced calciuria may be associated with hypertension, urinary stone formation and osteoporosis, but its mechanism and long-term effects are not fully understood. This study examined alterations in the expressions of renal Ca^{2+} transporters, channels and claudins upon salt loading to better understand the mechanism of salt-induced urinary Ca^{2+} loss. Methods: Eight-week old Wistar-Kyoto rats were fed either 0.3% or 8% NaCl diet for 8 weeks. Renal cortical expressions of Na+/Ca2+ exchanger 1 (NCX1), Ca^{2+} pump (PCMA1b), Ca^{2+} channel (TRPV5), calbindin-D28k, and claudins (CLDN-2, -7, -8, -16 and −19) were analyzed by quantitative PCR, western blot and/or immunohistochemistry. Results: Fractional excretion of Ca^{2+} increased 6.0 fold with high-salt diet. Renal cortical claudin-2 protein decreased by approximately 20% with decreased immunological staining on tissue sections. Claudin-16 and −19 expressions were not altered. Renal cortical TRPV5, calbindin-D28k and NCX1 expressions increased 1.6, 1.5 and 1.2 fold, respectively. Conclusions: Chronic high-salt diet decreased claudin-2 protein and increased renal TRPV5, calbindin-D28k, and NCX1. Salt loading is known to reduce the proximal tubular reabsorption of both Na+ and Ca^{2+}. The reduction in claudin-2 protein expression may be partly responsible for the reduced Ca^{2+} reabsorption in this segment. The concerted upregulation of more distal Ca^{2+}-transporting molecules may be a physiological response to curtail the loss of Ca^{2+}, although the magnitude of compensation does not seem adequate to bring the urinary Ca^{2+} excretion down to that of the normal-diet group

The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression

Attitudes toward and current status of disclosure of secondary findings from next-generation sequencing: a nation-wide survey of clinical genetics professionals in Japan

The management of secondary findings (SFs), which are beyond the intended purpose of the analysis, from clinical comprehensive genomic analysis using next generation sequencing (NGS) presents challenges. Policy statements regarding their clinical management have been announced in Japan and other countries. In Japan, however, the current status of and attitudes of clinical genetics professionals toward reporting them are unclear. We conducted a questionnaire survey of clinical genetics professionals at two time points (2013 and 2019) to determine the enforcement of the SF management policy in cases of comprehensive genetic analysis of intractable diseases and clinical cancer genome profiling testing. According to the survey findings, 40% and 70% of the respondents stated in the 2013 and 2019 surveys, respectively, that they had an SF policy in the field of intractable diseases, indicating that SF policy awareness in Japan has changed significantly in recent years. Furthermore, a total of 80% of respondents stated that their facility had established a policy for clinical cancer genome profiling testing in the 2019 survey. In both surveys, the policies included the selection criteria for genes to be disclosed and the procedure to return SFs, followed by recommendations and proposals regarding SFs in Japan and other countries. To create a better list of the genes to be disclosed, further examination is needed considering the characteristics of each analysis

膀胱癌における抗クローディン4細胞外ドメイン中和抗体の化学療法増感効果

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.博士（医学）・甲第649号・平成28年3月15日Copyright © 2015 Elsevier Ireland Ltd. All rights reserved