1.探討EP4調控Ras訊息表現在人類結腸組織癌化之作用 2.荷葉水萃物改善動物高脂飲食所引發的肥胖與肝脂肪堆積之作用

1.EP4 Upregulation of Ras Signaling and Feedback Regulation of Ras in Human Colon Tissue and Cancer cell 2.Improvement in High-fat Diet-induced Obesity and Liver Fat Accumulation by a Nelumbo nucifera Leaf Flavonoid-rich Extract in Mice

吳政訓(Cheng-Hsun Wu)

指導教授 ： 王朝鐘

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2010年)

https://doi.org/10.6834/CSMU.2010.00130

摘要

1.
腫瘤的發展過程中，原型致癌基因的活化與抑癌基因的不活化是必需的。K-ras的突變在結腸直腸癌中佔有相當高的比率。COX-2在癌化過程中扮演了一個非常重要的角色。先前的研究指出結腸上皮細胞的癌化是透過前列腺素E2 (prostaglandin E2；PGE2) 接受器EP4及活化其下游的訊息傳遞路徑。但目前為止，並沒有研究確切指出K-ras的突變與PGE2 /EP4 訊息傳遞路徑之間是否具有相關性及其分子機制為何。我們在這篇論文中主要的目的是找出K-ras與PGE2 接受器之間的關聯性。我們將pcDNA 3.1載體及pcDNA-K-ras轉殖到HT29大腸癌細胞中，以MTT及西方墨點法進行分析。根據我們的研究結果推測K-ras會活化PI3K/Akt路徑，進而促使GSK3磷酸化與COX-2的產生，故我們推測磷酸化的GSK3在不具活性下，無法降解β-catenin，而β-catenin會進入細胞核內與TCF4轉錄因子結合，誘導COX-2的產生，進而分泌PGE2，而PGE2活化接受器EP4，EP4又開始活化PI3K/Akt訊息傳遞路徑；我們也以COX-2抑制劑(NS398)及EP4抑制劑(AH23848)來驗證這個路徑是否調控著腸癌細胞的增殖；而以流式細胞儀分析，發現K-ras會使S期延長與G2/M增加。同時我們也收集了過度表現Ras 蛋白的結腸直腸癌檢體，其COX-2、pAkt 及EP4蛋白與正常組織比較之下也有過度表現的情形。我們的發現提供了在治療由K-ras突變所引起的結腸直腸癌一個新思考點；透過EP4抑制劑的使用對於將來結腸直腸癌的預防也是一個可以考量的選擇。
2.
根據文獻及統計數據顯示，肥胖經常伴隨肝臟堆積大量的脂肪，正常人類肝臟組織含有少量脂肪如三酸甘油脂、磷脂、醣脂及膽固醇，其重量約佔肝總重量的4~5 %，肝臟內脂肪含量如超過肝總重量5 %以上，或以肝組織切片診斷，顯微視野下肝組織切片含有脂肪空泡堆積的範圍若超過10 %以上，即所謂脂肪肝。雖然脂肪肝沒有太明顯的病狀，若進一步成脂肪性肝炎則會導致一連串病變的發生，使肝臟纖維化與肝硬化，進而導致肝癌的形成。本研究以動物試驗探討荷葉水萃物 (Nelumbo nucifera leaf flavonoid-rich extract)的抗肥胖功效。實驗動物餵食高脂肪食物誘導其肥胖，結果顯示荷葉水萃物降低了體重、體脂肪的堆積，並抑制FAS (fatty acid synthase)、GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase)的活性。荷葉水萃物也抑制FAS、acetyl-CoA carboxylase 和HMG-CoA還原酶(HMGCoA reductase)的表現，同時提昇肝臟中AMPK (AMP-activated protein kinase)的磷酸化。總而言之，荷葉水萃物可調控脂肪合成酶，並能有效抑制體脂肪的堆積，進而預防肥胖。

關鍵字

結腸直腸癌 ； K-ras ； 前列腺素接受器EP4 ； 肝醣合成激酶3β

並列摘要

1.
Recent advances in molecular genetics have revealed that multiple genetic alterations including activation of oncogenes and inactivation of tumor suppressor genes are required for tumor development and progression. K-ras is frequently mutated in colorectal cancer. COX-2 plays an important role in the pathogenesis of cancers progression. Previous studies have shown that prostaglandin E2 (PGE2) receptor is involved in intestinal carcinogenesis and activation of downstream pathways. However, the molecular mechanisms that link to K-ras, COX-2 and PGE2 receptor are currently unclear. In this study, we transfer pcDNA3.1 and pcDNA-K-ras in to HT29 colorectal cancer cells. Then were analyzed cell proliferation by MTT assay, protein expression by Western blot, transcription factor activity by EMSA (Electrophoretic mobility shift assay) and cell cycle by flow cytometry. In our data showed K-ras induce HT29 cells proliferation, but it did not affect other small G protein family expression. In addition to K-ras induce COX-2 and EP1/EP4 expression. K-ras inhibits GSK3β activity through pAkt. So we suppose over-expression Ras protein led to cell proliferation with activation of the phosphatidylinosotol-3 Kinase (PI3K)/Akt pathway, an effect likely to be due to inhibits GSK3β (Glycogen synthase kinase 3β)activity. Inhibition of GSK3 stabilizes β-catenin (decrease its degradation) and promotes β-catenin nuclear translocation and transcriptional activation of TCF-regulated gene further induction of COX-2 and activation EP4. We also used COX-2 inhibitor (NS398) or PI3K inhibitor (wortmannin) to confirm this pathway. The cell cycle result attestation K-ras mutation prolong S phase and increase G2/M phase ratio. At the same time, we collected colorectal tumor tissues as well as to confirm tumor tissues were overexpression Ras, pAkt and EP4 protein. Thus our data provided one newly thinks in the treatment which early colorectal cancer causes K-ras mutation may be consider.

2.
Base on the data of literatures and epidemiological statistics, abundant lipid accumulation usually appears obesity. In normal liver tissue, little lipid such as triglyceride, phospholipid, glycolipid and cholesterol existing in body is equal to 4-5 % of whole liver weight. Since lipid content up to 5 % of liver weight or sterosis up to 10 % of liver in biopsy diagnosis is so-called fatty liver. Although fatty liver without dominate symptoms, the progression would further cause fatty hepatitis, liver fibrosis, liver cirrhosis or promoting hepatoma formation. In this study, we investigated the anti-obesity effect of a flavonoid-enriched extract from Nelumbo nucifera leaf (NLFE) in vivo. C57BL/6 mice were fed with a high-fat diet (HFD) to induce obesity. NLFE reduced the body weight, body lipid accumulation, and activities of fatty acid synthase (FAS), glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase. NLFE also suppressed the expression of FAS, acetyl-CoA carboxylase, and HMGCoA reductase, and increased the phosphorylation of AMP-activated protein kinase in the liver. Taken together, we demonstrated that NLFE targets lipid-regulated enzymes, and may be effective in attenuating body lipid accumulation and preventing obesity.

並列關鍵字

colorectal cancer ； K-ras ； PGE2 (prostaglandin E2) receptor EP4 ； GSK3β (Glycogen synthase kinase 3β)

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