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Expression of Stathmin in Urothelial carcinoma
No full text研究背景：
Stathmin是一種存在於細胞質內的ubiquitous磷酸化蛋白，stathmin對細胞週期的調控是藉由對微小管（microtubule）聚合（polymerization）的調控，已知stathmin在許多惡性腫瘤會高度表現且與其預後有關， stathmin的表現也與taxanes類化學治療的反應有關。目前對於侵犯性泌尿上皮癌的化學治療中，paclitaxel為一重要藥物且stathmin在泌尿上皮癌的表現在過去並無相關研究，故設計此實驗。
研究目的：
探討stathmin在不同抗藥性的泌尿上皮癌細胞株的表現情形。另外，stathmin在泌尿上皮癌腫瘤組織上的表現情形。其表現是否與病人之臨床、病理參數及預後有關？是否與化學治療的反應有關？s-16 stathmin (Ser16 phosphorylated stathmin) 的表現是否與total stathmin不同？
研究方法：
利用不同抗藥性泌尿上皮癌的細胞株 (NTU-B1, NTU-B1/P, NTU-B1/T)，利用兩株stathmin的抗體（s-16 stathmin及total stathmin）進行免疫螢光染色及西方點墨法偵測其表現。為確定其細胞內染色的位置 (intracellular localization)及在不同細胞週期中的表現，吾人利用細胞核質分離及nocodazole synchronized cell cycle來研究。並利用臨床上收集之泌尿上皮癌之組織，以此兩種抗體進行免疫組織化學染色，與病人之年齡，性別，臨床資料（如是否合併慢性腎衰竭及末期腎病變、是否抽煙、是否居住於烏腳病盛行區、腫瘤原發部位、病理分級，臨床病理分期、預後及化學治療的反應，以chi-square test及Fischer’s exact test作雙尾檢定，p<0.05為具有統計上之顯著意義。
結果：
在泌尿上皮癌細胞株的total stathmin及s-16 stathmin表現，可見total stathmin及s-16 stathmin在三株不同的細胞株中均有高度表現。特別的是，S-16 stathmin除了在細胞質內，在細胞核內也有高度表現，且在各個不同的細胞週期內均有表現，並不侷限於G2-M期。而total stathmin的表現則主要細胞質中，在細胞核中也有表現，也可見表現於不同的細胞週期。
Stathmin在泌尿上皮癌組織中之表現及其與臨床病理參數的關係顯示：
1.在85位泌尿上皮癌病人中：
Total stathmin在所有泌尿上皮癌組織中均有表現（100％），其中弱陽性為10.6％，中度陽性為16.5％，高度陽性為72.9％，total stathmin的表現與腫瘤之病理期別 (pT staging) 有統計上的相關（p=0.002）。在S-16 stathmin方面，在泌尿上皮癌組織中均有表現（100％），其中弱陽性為21.4％，中度陽性為34.6％，高度陽性為44.0％，其表現強度除了與腫瘤之病理期別 (staging)有關，也與腫瘤細胞惡性度分級(grading)有關。
2.在44位侵犯性泌尿上皮癌病人中：
僅有s-16 stathmin表現與腫瘤病理期別有關（p=0.065）也與化學治療的反應有關（p=0.03），而total stathmin的表現與化學治療的反應無關（p=0.26）。
3.在41位表淺性泌尿上皮癌腫瘤中：
僅有total stathmin的表現與表淺性的腫瘤進行（progression）成侵犯性的病變有關（p=0.014）。
結論：
S-16 stathmin的表現在泌尿上皮癌與腫瘤侵犯病理期別及病理組織分級有關。侵犯性泌尿上皮癌的比表淺性泌尿上皮癌表現要強，高惡性度（high grade）比低惡性度（low grade）的表現更強。而在侵犯性泌尿上皮癌的化學治療（以TP-HDFL處方治療）上，表現較強者，似乎其化學治療的反應較差，與過去在乳癌的研究相符。Total stathmin僅與腫瘤侵犯病理期別與表淺性的腫瘤進行（progression）成侵入性的病變有關。而在泌尿上皮癌細胞株的實驗上，是否s-16 stathmin在細胞週期的不同階段，有除了調控紡錘絲的形成及分解之外的不同的功能，而能表現在細胞核內，仍須進一步研究。Purpose:
Stathmin is a ubiquitous cytosolic phosphoprotein that regulates dynamics of microtubule polymerization. The activity of stathmin is regulated via phosphorylation and dephosphrylation by various protein kinase. Stathmin has been proved to play a role in human cancers. including lung, breast, ovary, leukemia, lymphoma, neuroblastoma etc. However, no relevant data in urothelial carcinoma was reported. In this way, we hope to analyze the expression of stathmin in human urothelial carcinoma (UC) cell lines and tissue samples. We also hope to study the potential of stathmin as predictor in tumor behavior and response to chemotherapy. The differential expression of Ser-16 phosphorylated stathmin (s-16 stathmin) and total stathmin was also studied.
Materials and methods:
We collected 3 urothelial cell lines NTUB1 (high grade urothelial carcinoma), NTUB1/P (cisplatin resistant), NTUB1/T (taxol resistant) and checked the total stathmin and s-16 stathmin expression by immunofluorescence staining and western blot analysis.
Besides, we collected 85 urothelial carcinoma surgical specimens and analyzed the stathmin expression by immunohistochemical staining in paraffin-embedded cancer tissue samples. Correlation between stathmin expression and clinicopathologic features, such as age, sex, primary tumor site, black foot disease endemic area residency, chronic renal insufficiency, tumor grading and pathological staging, overall survival, disease free survival and response to TP-HDFL regimen (paclitaxel, cisplatin, high dose fluorouracil, and leucovorin) were examined.
Results:
Total stathmin and s-16 stathmin were both highly expressed in 3 cell lines without significant quantitative diffenence. The expression present in both cytoplasm and nucleus was proved by nuclear cytoplasmic fractionation and western blot analysis. Stathmin was also expressed in all stages of cell cycle.
In urothelial carcinoma tissue, s-16 stathmin expression was significantly associated with tumor grading( p=0.022) and pathological staging (p=0.006). The response to TP-HDFL in metastatic invasive urothelial carcinoma was significantly correlated with s-16 stathmin expression. There was no difference in overall and progression free survival in reference to the expression of stathmin. Total stathmin expression was significantly associated with pathological staging. Total stathmin expressin is significantly related to the risk of superficial urothelial carcinoma progression to invasive disease.
Conclusions:
In our preliminary data, we observed high expression rate of stathmin in urothelial carcinoma cell lines and tumor tissue. Stathmin expression is related to disease status and tumor behavior of urothelial carcinoma. Further study will be necessary to determine the role of stathmin in urothelial carcinoma and the potential application for prediction of chemotherapy response.一、中文摘要--------------------------------------------------------------------------III
二、英文摘要-------------------------------------------------------------------------- V
三、緒論-------------------------------------------------------------------------------- 7
四、研究方法與材料----------------------------------------------------------------- 13
五、結果------------------------------------------------------------------------------- 17
六、討論-------------------------------------------------------------------------------- 20
七、參考文獻-------------------------------------------------------------------------- 22
八、圖表-------------------------------------------------------------------------------- 27
九、附錄-------------------------------------------------------------------------------- 3
The Pathophysiological Role of Endoplasmic Reticulum Stress in Urothelial Carcinoma and Testicular Torsion
No full text此篇論文包括了泌尿科兩個重要的疾病，尿路上皮癌 (Urothelial Carcinoma; UC) 及睪丸扭轉 (Testicular torsion)，這兩個疾病在臨床治療上，都面臨了一些亟待解決的問題。論文的第一及第二部份，乃針對尿路上皮癌的研究。台灣地區的流行病學研究發現，尿路上皮癌發生率逐年上升，其中上泌尿道 (upper urinary tract) 的尿路上皮癌發生率特別高，且常合併慢性腎臟疾病或末期腎病變。此外，轉移性的尿路上皮癌，病患需接受化學治療，化學治療的反應率為40-70%。但在臨床上，尿路上皮癌常見於中老年人，國內的尿路上皮癌患者，常合併腎功能障礙，加以早期上泌尿道尿路上皮癌的病患，常需手術切除患側的腎臟及輸尿管，更進一步造成腎功能的下降。當腫瘤復發或轉移時需接受化學治療，這些腎功能不好的病患，對化學治療常有較大的副作用及較差的療效。更重要的是，這些病人最後會因化學抗藥性 (chemoresistance)而終至病患死亡，如何降低藥物治療的副作用，並提高其療效及克服化學抗藥性也成為臨床治療的重要課題。
Celecoxib是一種環氧化酵素-2抑制劑 (cyclooxygenase-2 inhibitor)，也是是臨床上常用的消炎止痛用藥。Celecoxib已被報告對不同的癌症有治療效果，但其抑癌機轉仍未確認。此論文的第一及第二部份，即是在探討celecoxib對尿路上皮癌的治療效果及機轉，我們主要著眼在內質網壓力 (endoplasmic reticulum stress; ER stress)及細胞自噬作用（autophagy），並研究如何透過這些機轉，調控celecoxib的細胞毒性，希望能發展新的治療模式。
本論文的第三部份則是研究內質網壓力在睪丸扭轉扮演的角色。臨床上，當懷疑睪丸扭轉時需儘早進行手術，最好在4-6小時內手術，回復血流及施行睪丸固定，期能保留睪丸的功能。但在臨床上，即使手術保留睪丸，仍常見睪丸功能的傷害，嚴重者甚至造成睪丸萎縮，影響生殖能力。睪丸扭轉造成睪丸傷害的主因是缺血-再灌流(ischemia-reperfusion)，其機轉主要是氧化壓力(oxidative stress)。內質網壓力與睪丸扭轉造成睪丸傷害之間的關係，迄今未有相關的研究。目前臨床上仍無藥物，用以治療睪丸扭轉的併發症，包括睪丸功能傷害及日後的不孕。我們希望釐清內質網壓力在睪丸扭轉中扮演的角色。另外，我們也將利用一木蘭科植物厚朴 (Magnolia officinalis)之成分─異厚朴酚 (honokiol)，其已被報告能減輕腦部缺血-再灌流的傷害，研究其是否能降低睪丸扭轉後的睪丸傷害。
第一部份
負調控(down-regulation) 葡萄糖調控蛋白(glucose-regulated protein; GRP78) 能增強celecoxib對尿路上皮癌細胞的毒殺作用
在此部份的研究中我們主要研究內質網壓力在celecoxib引發的細胞毒性中扮演的角色。我們發現celecoxib確實會抑制尿路上皮癌細胞的增生，造成細胞凋亡 (apoptosis)，引發細胞週期G1的停滯，並同時引起內質網壓力。藉由調降伴隨蛋白 (chaperone protein) GRP78的表現或以綠茶的兒茶素(−)-epigallocatechin gallate (EGCG) 抑制GRP78的作用，均會增強celecoxib對尿路上皮癌細胞的毒殺作用。此外蛋白酶體抑制劑（proteasome inhibitor）MG132 也會增強celecoxib對尿路上皮癌細胞的細胞毒殺作用。這些發現將對發展出克服化學抗藥性的方法，有相當大的助益。
第二部分
抑制細胞自噬作用(Autophagy)能增加celecoxib在尿路上皮癌細胞的細胞凋亡
我們發現celecoxib引起細胞毒殺作用的同時，也會引起細胞自噬作用，引發LC3及autophagolysosome的表現。3-methyladenine (3-MA)是一細胞自噬作用的抑制劑，合併使用會提高celecoxib引起的細胞毒殺作用，而m-TOR抑制劑rapamycin則會增加自噬作用，而減輕celecoxib引起的細胞毒殺作用。利用LC3-GFP-transfection 的LC3的過度表現(overexpression)，則會減少celecoxib引起的細胞毒殺作用。吾人能藉調控自噬作用，來增加celecoxib對尿路上皮癌細胞的細胞毒殺作用，據此發展出新的治療模式。
第三部份
異厚朴酚(honokiol)減輕睪丸扭轉/回復之缺氧及再灌流傷害，與內質網壓力引發的細胞凋亡有關
睪丸扭轉是泌尿科急症，若無及時處理常會導致睪丸萎縮及不孕，我們建立睪丸扭轉的動物模式，並研究其機轉及異厚朴酚 (honokiol) 能否減輕睪丸扭轉/回復之缺氧及再灌流傷害。我們發現在扭轉2小時後恢復灌流，在扭轉後第6小時即可見急性組織學傷害，在第24小時更為明顯。而扭轉後第24小時，細胞凋亡的分子指標如PARP、caspases便會表現，同時合併表現內質網壓力的分子如 (phospho-eIF2α and CHOP)。在恢復灌流前給予honokiol (10mg/kg)能減輕這些急性傷害及內質網壓力。尤其重要的是，honokiol能改善扭轉後第三個月睪丸的造精功能 (spermatogenesis)，這有助於減少因睪丸扭轉造成的不孕。This dissertation combines results from studies of two urological diseases that present special challenges in clinical treatment, and in which the role of endoplasmic reticulum stress (ER stress) is not well understood, urothelial carcinoma (UC) and testicular torsion. The UC study explores some novel strategies for treatment of UC with the cyclooxygenase-2 (COX-2) inhibitor celecoxib, via the modulation of ER stress and autophagy.
Epidemiological studies show that the incidence of upper urinary tract (UUT) UC in Taiwan is much higher than in the western world. This may be a consequence of special carcinogens exposure, such as arsenic-contaminated drinking water or aristolochic acid. Moreover, UC patients in Taiwan have a higher risk of concurrent chronic kidney disease and advanced renal disease. Patients with metastatic UCs need chemotherapy, but due to chemoresistance the achieved response rate is limited, ranging from 40-70%, and the chemoresistance reaction is normally fatal to affected patients. In addition, there are substantial toxicities in current chemotherapeutic regimens such as myelosuppression and nephrotoxicity. Higher rates of chemotherapy-related toxicities and lower rates of treatment efficacy have been noted in patients with concurrent metastatic UCs and late-stage chronic kidney disease. These facts make it imperative to explore novel chemotherapeutic strategies for lowering toxicity, improving efficacy, and controlling chemoresistance.
Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) and is widely used for anti-inflammation or pain control. Considerable preclinical evidence supports the potential of celecoxib against several types of malignancies; however, the utility of celecoxib by itself or in combination with other therapies for treating UC has not been fully explored. We seek clarification of the roles of ER stress and autophagy in celecoxib-induced cytotoxicity, and we search for novel strategies to potentiate the cytotoxic effects of celecoxib by the modulation of ER stress and autophagy.
The testicular torsion study concerns a condition of medical emergency that can cause impairment of semen quality, permanent testicular atrophy, or outright loss of the affected testicle. We explore the role of ER stress in the testicular injury caused by ischemia-reperfusion, and investigate the protective effect of honokiol, a phytochemical used in traditional medical treatment of testicular torsion.
Part 1
Down-regulation of glucose-regulated protein (GRP)78 Potentiates Cytotoxic Effect of Celecoxib in Human Urothelial Carcinoma Cells
Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role of endoplasmic reticulum (ER) stress play in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and endoplasmic reticulum (ER) stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP)78 knockdown (siRNA), (−)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate endoplasmic reticulum (ER) stress-related chaperons (IRE-1α and GRP78), caspase-4 and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA , co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces G1 arrest, ER stress and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.
Part 2
Autophagy Inhibition Enhances Celecoxib-induced Apoptosis in Human Urothelial Carcinoma Cells
In this study, we attempted to clarify the role of autophagy in celecoxib-induced cytotoxicity in human urothelial carcinoma (UC) cells. Cell viability and apoptosis of UC cells were determined by MTT assay and flow cytometry; and the formation of autophagy was examined by immunofluorescence staining, lysotracker, and immunoblotting analysis for LC3, an autophagosome marker. The present results showed that celecoxib induced cell death and apoptosis in human UC cells. Besides, autophagy was detected concomitantly in celecoxib-induced apoptosis as revealed by membrane-bound LC3-II in cells with fragmented Hoechst staining, as well as the formation of lipidized LC3 in immunoblotting analysis. Co-treatment with 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the apoptotic effect of celecoxib in human UC cells by suppression of autophagy. Rapamycin, an inhibitor of mTOR, could alleviate celecoxib-induced apoptosis via the enhancement of autophagy. Consistently, upregulation of autophagy by LC3-GFP-transfection decreased cytotoxicity of celecoxib in human UC cells. In summary, our data indicates that inhibition of autophagy enhances celecoxib induced-apoptosis, suggesting a novel therapeutic strategy against UC.
Part 3
Honokiol Attenuates Torsion/Detorsion-Induced Testicular Injury in Rat Testis via Suppressing Endoplasmic Reticulum Stress-Related Apoptosis
Testicular torsion is a medical emergency that can cause impairment of semen quality, permeant testicular atrophy or loss. In this study, we investigated the protective effect of honokiol, a phytochemical used in traditional medicine, on testicular injury after torsion/detorsion (T/D) in a rat model. Male Wistar rats were randomized to each time point of each group (n=6/time point/group). After 2 h of torsion, testes were counter-rotated to the natural position. Rats in each group underwent a sham operation, T/D, T/D with honokiol treatment (5 mg/kg and 10mg/kg, i.p., immediately before detorsion). Bilateral orchiectomy was performed at 6 h, 24 h, and 3 months after detorsion. Testes were examined histologically. The apoptosis and endoplasmic reticulum (ER) stress was detected by western blot. Histological examination revealed that testicular T/D induced acute injury after 6 and 24 h, and spermatogenesis was decreased by 3 month. 24 h after T/D, there were increases in the activations of apoptosis-related molecules (PARP, and caspases 3 and 7), as well as in the expression levels of ER stress-associated molecules (phospho-eIF2α and CHOP). These increases were significantly reversed by honokiol treatment. Furthermore, honokiol effectively reversed the inhibition of spermatogenesis in testes treated with T/D for 3 months. The study proves the ER stress-related apoptotic pathway is involved in testicular injury following testicular torsion/detorsion. It remains to be determined if alterations in this pathway would have a protective affect against reperfusion damage
Characteristics of Female Non-Muscle Invasive Bladder Cancer in Taiwan: an Association with Upper Tract Urothelial Carcinoma and End-Stage Renal Disease
No full textComparison of 6- and 12-Core Prostate Biopsy in Taiwanese Men: Impact of Total Prostate-Specific Antigen, Prostate-Specific Antigen Density and Prostate Volume on Prostate Cancer Detection
No full textThe Role of Lymphovascular Invasion in Predictiong the Prognosis of clinically Localized Upper Tract Urothelial (pT1-3cN0M0)
No full textScrotal Mass with Bladder Outlet Obstruction
No full textA 71-year-old man presented with a 6-cm scrotal mass and urinary retention. He had had progressive voiding difficulty after an untreated urethral injury in a traffic accident 15 years before presentation. Retrograde urethrography showed an anterior urethral diverticulum with stone formation. He underwent open diverticulectomy and recovered well. Pathologic examination revealed the diverticulum was lined by granulation tissue. Male urethral diverticula are rare and can be congenital or acquired. An acquired urethral diverticulum, as shown in this case, often results from infection, stricture, or trauma. The diagnosis requires a detailed history, physical examination, imaging studies, and pathologic examination
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