| Abstract: | 近幾年來腦血管與心臟疾病位居台灣地區十大死因之第二及第三名，僅次於惡性腫瘤之後，顯示癌症仍為十大死因之首，因此心血管疾病與癌症的預防與治療，是急待解決與重視的課題。如何治療癌症病人，尋找更有效的抗癌藥物，也是一個迫切的問題。長期以來，中草藥被應用於傳統醫學及民俗療法中的各種治療，已有多種中草藥被確認其具有抗氧化、抗發炎、抗過敏及抗癌等活性，但是到目前為止，對於中草藥預防心血管疾病、抑制癌細胞轉移、誘導癌細胞凋亡以及其中之詳細機轉的都還不是很清楚。在初步試驗結果中，發現覆盆子、七葉膽與番石榴葉之50%酒精萃取物具有顯著防止ApoB片段化及LDL氧化的效果；覆盆子、七葉膽與番石榴葉也能有效保護內皮細胞不受oxLDL之傷害與foam cells 的形成。此外，我們利用覆盆子、七葉膽與番石榴葉之酒精萃取物觀察其抑制肺癌(A549)轉移及誘導骨癌(143-B 與MG-63)、肝癌細胞(SK-Hep-1)與腎臟癌細胞(786-O) 凋亡之功效。經本實驗室初步分析，發現覆盆子、七葉膽與番石榴葉萃取物能有效誘導人類骨癌、肝癌與腎臟癌細胞走向死亡的現象。而覆盆子萃取物則可明顯抑制人類肺癌細胞之細胞侵襲及MMP-2蛋白分解?的表現，此外覆盆子亦可有效抑制癌症促進因子PMA所誘導之細胞癌化能力，並顯著促進細胞骨架蛋白E-cadherin的表達。綜合上述萃取物，在預防動脈粥狀硬化、抑制癌瘤細胞侵襲轉移能力研究與促進癌細胞走向凋亡方面應該會有所貢獻。因此，本研究將分三年來完成，第一年(1)探討覆盆子、七葉膽與番石榴葉抗氧化之效果。利用體外培養人類臍帶靜脈內皮細胞、平滑肌細胞與巨噬細胞株，以氧化型低密度脂蛋白誘導血管內皮細胞與巨噬細胞之細胞凋亡及巨噬細胞所誘導之發炎反應，來模擬人體內動脈粥狀硬化的形成；(2)探討覆盆子抑制多種不同癌細胞轉移、貼附之能力，並分析覆盆子對肺癌細胞侵襲相關基因之蛋白層次與mRNA層次的影響；(3)與其是否會影響多種細胞骨架蛋白的表達；(4)探討覆盆子、七葉膽與番石榴葉抑制不同癌細胞增生並誘導癌細胞凋亡之能力。第二年(1)深入探討覆盆子、七葉膽與番石榴葉這些天然萃取物作用在血管內皮細胞、平滑肌細胞與巨噬細胞之保護作用，並釐清其訊息傳遞路徑與作用機轉，同時研究在oxLDL刺激下，覆盆子、七葉膽與番石榴葉對於內皮細胞與單核球細胞的黏著作用之影響；(2)深入探討覆盆子抑制由PMA與IL1-β所誘導肺癌細胞轉移；和(3)細胞骨架重整與；(4)覆盆子、七葉膽與番石榴葉抑制誘導癌細胞凋亡之能力其中的詳細機轉與訊息傳遞路徑。第三年(1)利用餵食高膽固醇之動物模式(紐西蘭大白兔與倉鼠)，進一步研究其在活體內抑制動脈粥狀硬化之功效。並且針對具有保護效果之天然萃取物以LC-MS進行有效成分的分離純化及鑑定；(2)購買文獻已知覆盆子、七葉膽與番石榴葉之單離成分，做有效成分之鑑定分析; (3)以免疫缺陷的小鼠(BALB/c nu/nu mice)由尾靜脈接種肺癌細胞，與LLC-bearing mice model探討覆盆子抑制肺癌細胞轉移之能力；(4)以免疫缺陷的小鼠(BALB/c nu/nu mice)皮下接種各種不同之人類癌瘤細胞，觀察覆盆子、七葉膽與番石榴葉抑制腫瘤生長的能力。
The cerebrovascular and heart diseases are the second and third among the top ten of death in Taiwan. Among this, cancer is the biggest death rate among the top ten causes in Taiwan. So far, the most important thing for finding new cancer and cerebrovascular drug is urgent. A recent report showed that many kinds of Chinese medicinal herbs have the antioxidant, anti-inflammation, anti-allergic and anti-tumor activeness. However, the effects and detailed mechanisms of Chinese medicinal herbs on preventive cerebrovascular diseases, cancer cell metastasis, and inducing cell apoptosis were still unclear. In our preliminary study, we determined the effect of the extracts of Rubus idaeus L. (RIE), Gynostemma pentaphyllum (GPE), and Psoralea guajava L. (PGE) on inhibitory of LDL oxidation and ApoB fragmentation. RIE, GPE, and PGE showed great protection on oxLDL-induced cytotoxicity in endothelial cell and foam cell formation. Moreover, anti-metastasis and anti-tumor agents were investigated using 50% alcohol extracts of RIE, GPE, and PGE, on various lung cancer call lines (A549), osteosarcoma cell lines (143-B and MG-63), renal cancer cell lines (786-O) and liver cancer cell lines (SK-Hep-1). In our preliminary study, we demonstrated that RIE induced cell death on 143-B cells and PGE induced cell death on 786-O and SK-hep1 cells. RIE inhibited cell invasion and the secretion of MMP-2, and was sufficient to inhibit PMA-induced MMP-9 expression in A549 human lung cancer cells. RIE also could increase E-cadherin expression in A549 cells. Therefore, this three years grant proposal aims to study whether treatment of RIE, GPE, and PGE would protect against cardiovascular disease, inhibit cancer metastasis, and induce cancer cell death. Therefore, in the first year study, (1) we will focus on studying whether these extracts could inhibit LDL oxidation, rescue human umbilical vein endothelial cells and macrophage from oxLDL-mediated dysfunction and apoptosis, and inhibit cytokine-induced smooth muscle cell migration and proliferation. (2) investigate the effect of RIE on invasion of various cancer cells and analyze the protein and mRNA levels of invasion relevance genes, (3) investigate the effect of cytoskeleton conformation by RIE in human lung cancer cells, (3) analyze the effects for the down-regulation of cell viability and induced apoptosis by RIE, GPE, and PGE in various human cancer cell lines. In the second year of this study, (1) we will aim to explore the underlying mechanism involved in the preventive effects of herb extracts against oxLDL-indeced apoptosis in endothelial cell and macrophage and the inhibitive of these extracts on cytokine-induced smooth muscle cell migration and proliferation; to explore the underlying mechanism involved in (2) anti-invasiveness, (3) induced-apoptosis, and (4) the influences of the cytophysiological changes by RIE, GPE, and PGE. In the third year of this study, we will propose to (1) isolate and identify the active component of RIE, GPE, and PGE. Finally, (2) analyze the anti-atherosclerotic effect of these herb extracts via an animal model. (3) via an LLC-bearing mice model and BALB/c nude mice model determine the effect of RIE on cancer metastasis, (4) finally investigate tumor growth of different cancer cell lines in vivo via cancer cell xenografted nude mice model and BALB/c-bearing mice model. |
