鑑定胰臟癌誘發新生糖尿病之致糖尿病因子

Abstract

胰臟癌是世界上致死率最高的癌症之一，其五年內存活率不到百分之五，目前大部份胰臟癌治療方式為手術切除，然而約85%的病人就診時已是胰臟癌晚期，無法適用手術切除腫瘤，因此目前急需要有效的早期診斷方法如生物標記，來幫助早期胰臟癌之診斷。據統計約有40%的胰臟癌病人，在胰臟癌前兩年伴隨著新生糖尿病的現象，新生糖尿病發生的時間為我們提供一個絕佳早期診斷胰臟癌的機會，新生糖尿病是由於胰臟癌細胞分泌某些致糖尿病因子 (diabetogenic factors)所造成。目前形成的機制仍未知，但如果我們可以找到這些致糖尿病因子，除了可以幫助我們釐清其機轉，作為生物標記也可為我們區分新生糖尿病和第二型糖尿病患者，達到早期診斷胰臟癌的目的。 我們收集了兩種胰臟癌細胞MIAPaCa-2和PANC-1的分泌性蛋白，經濃縮去鹽後以LC-MS/MS鑑定，將結果以Ingenuity Pathway Analysis (IPA)將蛋白依相關疾病、生理現象和功能分類後，與胰臟癌病人表現量較高之基因互相交集比較，以IPA分析有潛力成為致糖尿病因子，並觀察到候選蛋白在高糖環境下MIAPaCa-2的分泌量上升，即選定galectin-1和galectin-3為目標蛋白。接著以分泌性蛋白、galectin-1、galectin-3分別和老鼠肌肉細胞進行培養，發現皆會影響其葡萄糖攝取能力，干擾肌肉細胞的胰島素敏感程度。而在病人檢體中，可以看到galectin-1含量在新生糖尿病、糖尿病、胰臟癌病人、健康人檢體中分佈平均，無法將病人做區隔，因此不能作為有效的生物。反觀galectin-3在病人檢體中含量有顯著差別，可以將病人區隔成新生糖尿病、糖尿病與胰臟癌、健康人兩組。而為了知道這些目標蛋白如何影響胰島素之傳遞，我們觀察在有目標蛋白存在時，胰島素訊息傳遞路徑是否有受到抑制，發現IRS-1和Akt的磷酸化皆明顯減弱。倘若繼續找到其他糖尿病或新生糖尿病生物標記，則可以將四群病人完全分開，以複合型生物標記形式可有效達到胰臟癌的早期診斷，並供後續研究致糖尿病因子的發展機轉。

Pancreatic Cancer (PC) is one of the leading cause of cancer death in the world with a 5-year survival less than 5%. Surgical resection of tumor is the only potential therapy, but the PC-specific symptoms occur late and 85% of the tumors are diagnosed when a surgical cure is impossible. Thus, the discovery of biomarkers for early detection is urgent necessary. There are approximately 40% of PC patients followed with PC-associated new-onset diabetes (PCDM), occurring 2 years before the tumor is diagnosed, which provides a potential opportunity to detect PC in early stages. PCDM is mediated by some unknown tumor-secreted diabetogenic factors. Identifying the diabetogenic factors may provide novel insights into its pathogenesis and discover a novel biomarker to distinguish PCDM from type 2 diabetes (T2DM). To discover the diabetogenic factors and potential serum PC biomarkers, we collected the condition medium (CM) from two PC cell lines (MIAPaCa-2 and PANC-1). Potential factors in CM further identified by gel-based LC-MS/MS and analyzed by Ingenuity Pathway Analysis (IPA). We also found some candidate proteins, which were up-regulated in PC patients from microarray analysis. Comparison of data in proteomics and microarray, we found the secretion proteins of target (galectin-1 and galectin-3) from MIPaCa-2 under high glucose condition were increased. The glucose uptake was reduced in C2C12 mouse skeletal muscle cells under the incubation with CM, galectin-1 or galectin-3. For clinical validation of the potential biomarkers, we confirmed the levels of galectin-1 and galectin-3 among patients of PC, diabetes, and healthy controls. The data revealed only galectin-3 can distinguish patients of PCDM and T2DM from PC and healthy controls. To verify how the factors affect glucose uptake by regulating insulin signaling, we observed the phosphorylation levels of insulin receptor substrate-1 (p-IRS-1) and Akt by Western blotting after treated C2C12 with CM, galectin-1 or galectin-3. Our results shown both p-IRS-1 and p-Akt were attenuated. Other target proteins in our finding are also potential candidates for diabetogenic factors. We will construct these potential biomarkers into a multiple biomarker panel, and look forward to using as early diagnosis tool for PC. Identification of diabetogenic factors is necessary not only for early detection of PC, but also for clarifying the mechanism of PC associated new-onset diabetes.