1.EP4 Upregulation of Ras Signaling and Feedback Regulation of Ras in Human Colon Tissue and Cancer cell2.Improvement in High-fat Diet-induced Obesity and Liver Fat Accumulation by a Nelumbo nucifera Leaf Flavonoid-rich Extract in Mice

• Main Authors: Cheng-Hsun, 吳政訓
• Other Authors: 王朝鐘
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/69424464540085709822

博士 === 中山醫學大學 === 生化暨生物科技研究所 === 98 === 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.