內質網壓力在尿路上皮癌及睪丸扭轉之病理生理角色探討

The Pathophysiological Role of Endoplasmic Reticulum Stress in Urothelial Carcinoma and Testicular Torsion

黃國皓(Kuo-How Huang)

指導教授 ： 劉興華

國立臺灣大學/醫學院/毒理學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.03154

摘要

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。

關鍵字

內質網壓力 ； 細胞自噬 ； 尿路上皮癌 ； 睪丸扭轉 ； 環氧化酵素-2抑制劑 ； celecoxib ； 細胞凋亡 ； 葡萄糖調控蛋白GRP78 ； 內質網蛋白不良摺疊效應 ； 異厚朴酚 ； 不孕

並列摘要

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.

並列關鍵字

Endoplasmic reticulum stress ； Autophage ； Urothelial carcinoma ； Testicular torsion ； Cyclooxygenase-2 inhibitor ； Celecoxib ； Apoptosis ； Glucose-regulated protein78 ； Unfolded protein response ； Honochiol ； Infertility

參考文獻

Adams, J., Kauffman, M., 2004. Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest 22, 304-311.

Adhim, Z., Matsuoka, T., Bito, T., Shigemura, K., Lee, K.M., Kawabata, M., Fujisawa, M., Nibu, K., Shirakawa, T., 2011. In vitro and in vivo inhibitory effect of three Cox-2 inhibitors and epithelial-to-mesenchymal transition in human bladder cancer cell lines. Br J Cancer 105, 393-402.

Amaravadi, R.K., Thompson, C.B., 2007. The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin.Cancer Res. 13, 7271-7279.

Amaravadi, R.K., Yu, D., Lum, J.J., Bui, T., Christophorou, M.A., Evan, G.I., Thomas-Tikhonenko, A., Thompson, C.B., 2007. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117, 326-336.

Arap, M.A., Lahdenranta, J., Mintz, P.J., Hajitou, A., Sarkis, A.S., Arap, W., Pasqualini, R., 2004. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell 6, 275-284.