尿中Nerve Growth Factorは、過活動膀胱症状を呈する患児の治療効果の予測因子になる

Objective: To assess urinary nerve growth factor (NGF) in children with overactive bladder (OAB) and to investigate the relationship between urinary NGF/creatinine (Cr) levels and OAB. Patients and methods: Thirty-five children (27 boys and 8 girls) with OAB and 11 children (6 boys and 5 girls) without OAB or any other urinary symptoms, who served as controls, were included in this study. Urinary NGF levels were measured using enzyme-linked immunosorbent assay. The total urinary NGF levels were adjusted with the concentration of urinary creatinine (NGF/Cr level). Refractory OAB was defined as little improvement in OAB symptoms despite at least 3 months of urotherapy and anticholinergic agent treatment. Urinary NGF/Cr was compared between the children with OAB and the controls. The relationship between urinary NGF/Cr and treatment outcomes was also evaluated. Results: Urinary NGF/Cr was significantly higher in the children with OAB when compared with those in the control group (0.65 ± 0.82 vs 0.11 ± 0.09, P = .0007). Improvement of OAB symptoms was observed in 26 out of 35 children (74%). The remaining 9 children showed refractory OAB symptoms (the refractory group). Urinary NGF/Cr was significantly higher in the refractory group than in the improved group (1.28 ± 1.34 vs 0.44 ± 0.39, P = .027). Conclusion: Urinary NGF/Cr was significantly higher in the children with OAB than in the controls, and was significantly higher in the refractory group than in the improved group. Urinary NGF/Cr could not only be a potential biomarker for children with OAB, but also a predictor of therapeutic efficacy in children with OAB.博士（医学）・乙第1454号・令和2年3月16日Copyright © 2017 Elsevier Inc. All rights reserved

前立腺癌マウスモデルにおけるジクロフェナク局所投与によるCOX-2発現の阻害はTRAILの増幅を介して放射線感受性を増強させる

BACKGROUND: COX-2 inhibitors have an antitumor potential and have been verified by many researchers. Treatment of cancer cells with external stressors such as irradiation can stimulate the over-expression of COX-2 and possibly confer radiation resistance. In this study, we tested if topical diclofenac, which inhibits both COX-1 and COX-2, administration rendered prostate tumor cells sensitize to the effects of radiation. METHODS: LNCaP-COX-2 and LNCaP-Neo cells were treated with 0 to 1000 μM diclofenac. Next, a clonogenic assay was performed in which cells were subjected to irradiation (0 to 4 Gy) with or without diclofenac. COX-2 expression and other relevant molecules were measured by real-time PCR and immunohistochemistry after irradiation and diclofenac treatment. In addition, we assessed the tumor volumes of xenograft LNCaP-COX-2 cells treated with topical diclofenac with or without radiation therapy (RT). RESULTS: LNCaP-COX-2 and LNCaP-Neo cell lines experienced cytotoxic effects of diclofenac in a dose related manner. Clonogenic assays demonstrated that LNCaP-COX-2 cells were significantly more resistant to RT than LNCaP-Neo cells. Furthermore, the addition of diclofenac sensitized LNCaP-COX-2 not but LNCaP-Neo cells to the cytocidal effects of radiation. In LNCaP-COX-2 cells, diclofenac enhanced radiation-induced apoptosis compared with RT alone. This phenomenon might be attributed to enhancement of RT-induced TRAIL expression as demonstrated by real-time PCR analysis. Lastly, tumor volumes of LNCaP-COX-2 cells xenograft treated with diclofenac or RT alone was >4-fold higher than in mice treated with combined diclofenac and radiation (p<0.05). CONCLUSIONS: These in vitro and in vivo findings suggest that conventional COX inhibitor, diclofenac enhances the effect of RT on prostate cancer cells that express COX-2. Thus, diclofenac may have potential as radiosensitizer for treatment of prostate cancer.博士（医学）・甲第606号・平成25年11月27日© 2013 Inoue et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

N-ブチル-N-（4-ヒドロキシブチル）ニトロソアミン誘発膀胱癌マウスモデルを用いた膀胱内化学療法による局所及び全身の免疫応答

Intravesical bacillus Calmette-Guerin (BCG) treatment is the most common therapy to prevent progression and recurrence of non-muscle invasive bladder cancer (NMIBC). Although the immunoreaction elicited by BCG treatment is well documented, those induced by intravesical treatment with chemotherapeutic agents are much less known. We investigated the immunological profiles caused by mitomycin C, gemcitabine, adriamycin and docetaxel in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced orthotopic bladder cancer mouse model. Ninety mice bearing orthotopic bladder cancer induced by BBN were randomly divided into six groups and treated with chemotherapeutic agents once a week for four weeks. After last treatment, bladder and serum samples were analyzed for cell surface and immunological markers (CD4, CD8, CD56, CD204, Foxp3, and PD-L1) using immunohistochemistry staining. Serum and urine cytokine levels were evaluated by ELISA. All chemotherapeutic agents presented anti-tumor properties similar to those of BCG. These included changes in immune cells that resulted in fewer M2 macrophages and regulatory T cells around tumors. This result was compatible with those in human samples. Intravesical chemotherapy also induced systemic changes in cytokines, especially urinary interleukin (IL)-17A and granulocyte colony stimulating factor (G-CSF), as well as in the distribution of blood neutrophils, lymphocytes, and monocytes. Our findings suggest that intravesical treatment with mitomycin C and adriamycin suppresses protumoral immunity while enhancing anti-tumor immunity, possibly through the action of specific cytokines. A better understanding of the immunoreaction induced by chemotherapeutic agents can lead to improved outcomes and fewer side effects in intravesical chemotherapy against NMIBC.博士（医学）・甲第695号・平成31年3月15日Copyright: © 2017 Hori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

ラット膀胱での水吸収におけるアクアポリン-2の役割

AIM: We investigated the role of the bladder wall in permeating water, focusing on aquaporins. METHODS: Female Sprague-Dawley rats weighing 300 g were used to investigate the role of the bladder wall in saline permeation. Changes in intravesical fluid volume and sodium concentration were measured in the desmopressin acetate hydrate-loaded and control groups 3 h after administration. Bladders were resected to measure aquaporin-1, 2, and 3 gene expression using qRT-PCR. Additionally, the change of aquaporin-2 expression was measured using Western blotting and immunohistochemistry in intravesical aquaporin-2 siRNA-treated and control groups. RESULTS: Although the intravesical fluid volume and sodium concentration significantly decreased from 0 to 3 h (1.00 ± 0.00 vs 0.83 ± 0.08 mL, 157.80 ± 1.30 vs 146.8 ± 1.92 mEq/mL, P < 0.01, respectively in the control group), administration of desmopressin did not affect the extent of volume change. Aquaporin-2 expression was significantly higher in the 3-h distended bladders than in the empty bladder. Aquaporin-2 siRNA treatment suppressed aquaporin-2 expression and the change of intravesical fluid volume from 0 to 3 h (1.00 ± 0.00 and 0.99 ± 0.02 mL), which was related to the suppression of sodium concentration change in comparison with control siRNA treatment (149.6 ± 2.4 vs 143.6 ± 3.67 mEq/mL, P < 0.05). CONCLUSIONS: The rat urinary bladder absorbs water and salts under the full-filled condition. Aquaporin-2 plays an important role in the transport of water, accompanied by sodium concentration change. We demonstrated a part of the bladder absorption mechanism, which may lead to development of a new method for regulating bladder storage function.博士（医学）・甲第697号・平成31年3月15日© 2018 Wiley Periodicals, Inc.This is the pre-peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1002/nau.23715], which has been published in final form at [http://dx.doi.org/10.1002/nau.23715]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

Life sciences

Aims: To examine the circadian expression changes in bladder clock genes in Dahl salt-sensitive rats following high salt intake. Main methods: Eighteen rats were divided into three groups: the high-salt diet group (HS group), the normal-salt diet group (NS group), and the salt-load interruption group (from a 4 % salt diet to a normal diet; salt-load interruption group [SI group]). Each rat was placed in an individual metabolic cage for 24 h twice weekly. Water intake, urine production, voiding frequency, and voided volume per micturition were recorded. Furthermore, 108 control rats were prepared. Bladders were harvested every 4 h at six time points. Furthermore, the mRNA expression of clock genes and mechanosensors was analyzed. Key findings: In the HS group, the bladder clock genes showed lower mRNA levels than in the NS group. The amplitude of circadian expression changes in bladder clock genes in the HS group was lower than that in the NS group. However, after changing from a 4 % salt diet to a normal diet, the waveforms of the clock gene expression in the SI group were closer to those of the NS group. The 24-h water intake and urinary volume of the SI group decreased to levels comparable to those of the NS group. Significance: Reduced salt intake partially restored the circadian rhythms of bladder clock genes.博士（医学）・甲第857号・令和4年12月22日Copyright © 2022 Elsevier Inc. All rights reserved

Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer

Background To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. Methods SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. Results Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. Conclusion Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression

去勢抵抗性前立腺癌におけるKlothoγのドセタキセル抵抗性との関連と新規治療としての可能性

The Klotho (KL) gene was first identified as a potent aging suppressor. The KL family currently comprises of three proteins: α-Klotho (KLA), β-Klotho (KLB), and γ-Klotho (KLG). Many studies have shown that KLA and KLB participate in tumor progression or suppression, depending on the type of cancer; however, the relationship between KLG and prostate cancer has not yet been studied. Some studies have claimed that KL is correlated to sensitivity to chemotherapy. Here, we investigated the oncogenic potential of KLG in castration-resistant prostate cancer (CRPC). Immunohistochemical analysis using prostate biopsy specimens revealed that patients with high KLG expression in primary prostate cancer tissue had a significantly poor prognosis for overall survival. In addition, the prostate-specific antigen response rate after docetaxel (DTX) therapy in patients with high KLG expression was lower than that in patients with low KLG expression. To evaluate the potential of KLG as a therapeutic target in human prostate cancer, we generated a xenograft model of human CRPC cell line (PC-3) in male athymic mice. The animals were randomly divided into four groups as follows: i) control group (vehicle only); ii) DTX group (intraperitoneal administration); iii) small interfering RNA targeting KLG (KLG siRNA) group (intratumoral administration); and iv) a combination group (DTX plus KLG siRNA). After 3 weeks of treatment, the tumor weight and tumor Ki-67 labeling index were significantly lower in the KLG siRNA group and the combination group than in the control group. Sensitivity to DTX was increased upon treatment with KLG siRNA. These findings suggest that KLG expression in primary prostate cancer lesions is associated with resistance to DTX in CRPC and has potential as a diagnostic and therapeutic target for patients with CRPC.博士（医学）・甲第740号・令和2年3月16日Copyright: © Onishiet al. This is an open access article distributed under theterms of CreativeCommons Attribution License(https://creativecommons.org/licenses/by-nc-nd/4.0/)

尿中剥離前立腺癌細胞における5-ALA を用いた光力学的診断の有用性

BACKGROUND:Past attempts at detecting prostate cancer (PCa) cells in voided urine by traditional cytology have been impeded by undesirably low sensitivities but high specificities. To improve the sensitivities, we evaluate the feasibility and clinical utility of photodynamic diagnosis (PDD) of prostate cancer by using 5-aminolevulinic acid (5-ALA) to examine shed prostate cancer cells in voided urine samples. METHODS:One hundred thirty-eight patients with an abnormal digital rectal exam (DRE) and/or abnormal prostate-specific antigen (PSA) levels were recruited between April 2009 and December 2010. Voided urine specimens were collected before prostate biopsy. Urine specimens were treated with 5-ALA and imaged by fluorescence microscopy and reported as protoporphyrin IX (PPIX) positive (presence of cells demonstrating simultaneous PPIX fluorescence) or PPIX negative (lack of cells demonstrating fluorescence). RESULTS:Of the 138 patients, PCa was detected on needle biopsy in 81 patients (58.7%); of these 81 patients with PCa, 60 were PPIX-positive (sensitivity: 74.1%). Although 57 patients did not harbor PCa by conventional diagnostic procedures, 17 of these at-risk patients were found to be PPIX-positive (specificity: 70.2%). PPIX-PDD was more sensitive compared with DRE and transrectal ultrasound and more specific compared with PSA and PSA density. The incidence of PPIX-PDD positivity did not increase with increasing total PSA levels, tumor stage or Gleason score.CONCLUSIONS:To our knowledge, this is the first successful demonstration of PPIX in urine sediments treated with 5-ALA used to detect PCa in a noninvasive yet highly sensitive manner. However, further studies are warranted to determine the role of PPIX-PPD for PCa detection.博士（医学）・甲第633号・平成27年3月16日© 2014 Nakai et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

膀胱癌細胞株において、ヘパラナーゼを阻害することにより、細胞浸潤、遊走、接着能を抑制する

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.博士（医学）・乙第1506号・令和3年3月15日© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)