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

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/)

PDE5阻害薬であるタダラフィルは糖尿病ラットの膀胱血流を改善し、初期段階に低下する下部尿路機能を回復する

AIMS: To investigate the effect of tadalafil on bladder blood flow and lower urinary tract function in a rat model of diabetes. MATERIALS AND METHODS: We studied female Sprague-Dawley rats and induced diabetes in some using a single intraperitoneal injection of streptozotocin. We divided the rats into nondiabetes (ND), diabetes (D), and diabetes with tadalafil (DT) groups. The rats were raised for an additional 7 weeks after diabetes induction. The DT group received oral tadalafil (2 mg/kg/day) for 7 days before the experiments. At 7 weeks after diabetes induction, we performed cystometry, resected the bladders for immunohistochemistry (hypoxia-inducible factor-1α [HIF-1α] and 8-oxo-2'-deoxyguanosine [8-OHdG] staining), and measured bladder blood supply using a laser blood flow meter. RESULTS: The opening pressure, when the urethra opens and urine flow starts, was significantly lower in the DT group than in the D group (24.9 ± 5.9 vs 43.6 ± 12.3 cmH2 O). The inter-contraction interval was significantly longer in the D group than in the ND and DT groups (1566.2 ± 168.7 vs 702.9 ± 165.2 and 787.4 ± 148.8 s). Immunohistochemistry showed positive staining of the urothelial layer for both HIF-1α and 8-OHdG in the D group, but not in the ND or DT groups. Bladder blood flow was significantly lower in the D group than in the ND or DT groups. CONCLUSIONS: Tadalafil improves bladder blood supply and lower urinary tract function in diabetic rats. Tadalafil may be a promising drug that restores lower urinary tract dysfunction in the early phase of diabetes.博士（医学）・甲第677号・平成30年3月15日© 2017 Wiley Periodicals, Inc.This is the peer reviewed version of the following article: "http://dx.doi.org/10.1002/nau.23372", which has been published in final form at [Link to final article using the DOI]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

Pretreatment Platelet-to-Lymphocyte Ratio as Biomarker for Neoadjuvant Chemotherapy Prior to Radical Cystectomy in Muscle-Invasive Bladder Cancer.

Objectives : To evaluate the clinical benefit of neoadjuvant chemotherapy with gemcitabine and cisplatin (GC) in patients with muscle-invasive bladder cancer treated with radical cystectomy and to identify patients who may benefit from neoadjuvant chemotherapy and predictors of therapeutic response to it. Methods : In this prospective study, we enrolled 37 patients with muscle-invasive bladder cancer (cT2-4aNanyM0). The primary endpoint was the pathological response rate at cystectomy after receiving neoadjuvant GC chemotherapy. Univariable and multivariable analyses were used to determine predictive factors of pT0N0 and ≦pT1N0. The secondary endpoints were adverse events during chemotherapy, surgical complications, as well as overall, disease-specific, and recurrence-free survival. Results : A mean of 2.7 cycles of neoadjuvant GC was administered. Pathological complete response (pT0N0), partial response (pTisN0/pT1N0), and pathological response (≦pT1N0) rates were 24.3%, 27.0%, and 5l.3%, respectively. Grade 3 or 4 non-hematologic adverse events were rare. Three-year overall, disease-specific, and recurrence-free survival rates were 70.7%, 8l.3%, and 63.9%, respectively. Patients with pathological response (≦pT1N0) demonstrated a significantly improved 3-year overall survival rate (94.7% vs. 42.8%), disease-specific survival rate (94.7% vs.62.9%), and recurrence-free survival rate (80.6% vs.45.5%), compared with pathological non-responders (≦pT2Nany). Clinical stage cT2 and low pre-chemotherapy platelet-to-lymphocyte ratios were significant indicators of favorable pathological response to neoadjuvant Gc. Conclusions : Neoadjuvant chemotherapy using GC is safe and effective in patients with muscle-invasive bladder cancer, Pretreatment clinical T2 stage and low platelet-to-lymphocyte ratios were predictive markers for successful neoadjuvant treatment of muscle-invasive bladder cancer with GC