探討SPZ1引起人類腎細胞癌之integrin相關轉移和標靶藥物sorafenib抗藥性的分子角色

Molecular role of SPZ1 mediated integrin-related metastasis in human renal cell carcinoma and sorafenib resistance

林玉涵(Yu-Han Lin)

指導教授 ： 侯自銓

高雄醫學大學/醫學院/醫學系生物化學科碩士班/碩士(2015年)

摘要

腎細胞癌 (renal cell carcinoma, RCC) 為腎臟最常見的惡性腫瘤。是由近端腎小管細胞 (proximal tubular cell) 發展而成，早期的RCC主要以外科手術治療為主，而在接受化學治療的RCC則具有高度抗藥性，近年來標靶藥物的發展已做為晚期轉移型的RCC的治療方式，然而RCC患者最終產生對其藥物的抗藥性。因此，研究高度抗藥性的惡性RCC的治療方法是一重要課題。 Sorafenib為用來治療晚期腎細胞癌的標靶藥物，透過抑制細胞生長與血管新生，達到抑制腫瘤生長，但在治療後其病患逐漸發展出對其的抗藥性。SPZ1 (Spermatogenic leucine zipper 1) 屬於鹼性螺旋-圈環-螺旋的轉錄因子 (Basic helix-loop-helix transcription factor , bHLH)，文獻研究指出SPZ1扮演原致癌基因 (proto-oncogene)的角色，並參與細胞的發育、生長、分化等重要控。Integrin可通過傳遞特定的信號或誘導基因表達來調控腫瘤細胞的分化、生長、侵襲和轉移以及凋亡。本研究中，建立ACHN 之Sorafenib抗藥株，利用MTT assay檢測原非性抗藥株與抗藥株之IC50，確認抗藥株IC50比原非性抗藥株高約3倍。以Western blot比較其原非性抗藥株與抗藥株之SPZ1與其它分子蛋白表現量，發現在抗藥株中SPZ1、 integrin β5、磷酸化EGFR、磷酸化ERK及磷酸化AKT的蛋白表現皆比原非性抗藥株要來的高， 而將抗藥株之integrin β5基因靜默其抗藥株磷酸化ERK及磷酸化AKT蛋白表現隨之減少，且抗藥株會恢復對sorafenib敏感性，細胞存活、增生及轉移能力也隨之下降，而將SPZ1基因靜默抗藥株也會恢復對藥物之敏感性，可以得知SPZ1與integrin β5在人類腎細胞癌標靶藥物之抗性中扮演一重要角色。

關鍵字

腎臟癌 ； 抗藥性

並列摘要

Renal cell carcinoma (RCC), which arises from proximal tubular cells, is one of the most common malignant tumors of the kidney. Surgical operation is presently the main treatment method for primary RCC since in clinical practice. RCC is highly resistant to chemotherapy. But recently, target drugs have been explored as treatments for malignant or advanced RCC and the drug-resistant mechanism is an important research topic for renal cell carcinoma. Sorafenib is a target drug for the treatment of advanced renal cell carcinoma since it suppresses tumor growth and angiogenesis, although patients gradually develop drug-resistance after treatment. SPZ1 is a member of the bacis helix-loop helix transcription factor (bHLH) family. Recent study has demonstrated that SPZ1 is a proto oncogene and has an important role in the regulation of cell growth, proliferation and differentiation. Integrins are crucial for cell invasion and migration, not only for physically tethering cells to the matrix, but also for sending and receiving molecular signals that regulate these processes. In this study, having established the sorafenib-resistant cells from ACHN cell lines, referred to as ACHN-SR 4.0, we compare the parental cell and resistant cell survival rate by MTT assay, finding that ACHN-SR 4.0 cells also manifested higher resistance to sorafenib. Western blot results showed that the expression of SPZ1, integrin β5, phosphorylated-EGFR, p-ERK and p-AKT were moderately higher in ACHN-SR 4.0 cells. Integrin β5 knocked down by siRNA in ACHN-SR 4.0 cells, down-regulated the p-ERK and p-AKT protein and also reduced cell migration. Furthermore, ACHN-SR 4.0 cells were restored to sorafenib sensitiv. SPZ1 knocked down by SPZ1i plasmid showed that the resistant cells were also restored to sorafenib sensitiv. Overall, SPZ1 and integrin β5 have an important role in the resistance to sorafenib in renal cell carcinoma.

並列關鍵字

SPZ1 ； Integrin

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