荷葉多酚活化p38 MAPK / Fas L致人類乳癌細胞MCF-7凋亡之作用

Nuciferine Leaf Polyphenol rich-extract induced human breast cancer cell apoptosis involving p38 MAPK / Fas L pathway

鄭雅方(Ya-Fung Jeng)

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/碩士(2006年)

摘要

癌症，在近十年來由衛生署所做的台灣十大死亡原因調查，仍舊是高居榜首，為十大死因之第一名，所以如何預防及延緩癌症形成是目前研究的熱門主題。而目前廣泛研究趨向於以天然物質來抑制癌細胞之增生及惡化，有許多研究證實植物中多酚(polyphenols)其具有抗氧化以及抗腫瘤促進作用之功效，如綠茶、洛神花、紅酒和蘋果中的多酚。本研究室先從荷葉中萃取出多酚，並加以作定性及定量。在定量方面，計算萃取之荷葉多酚含量約有85-95％。而定性方面，利用HPLC分析荷葉中所含之多酚成份。然後利用所萃取之荷葉多酚(NPRE)針對：肝癌(Hep G2、Hep 3B)、口腔上皮癌(KB)、大腸癌(Caco-2)、血癌(HL-60)、胃癌(AGS)、老鼠正常纖維母細胞( NIH 3T3 )，乳癌(MCF-7、MDA-231) 九種不同的癌細胞株進行篩選。結果顯示：人類乳癌細胞(MCF-7 )對於NPRE抑制細胞存活的敏感度最高(IC50 = 0.63 mg/ml)，而且呈現dose-dpendent。接著以 MCF-7 細胞為實驗模式，處理在不同劑量NLE、NPRE下，觀察細胞凋亡( apoptosis )的現象。我們發現隨著NLE、NPRE濃度的增加對於造成細胞凋亡的現象也愈顯著：包括細胞型態的改變，以及 DNA fragmentation。利用流式細胞儀分析發現在相同劑量下，NLE會對MCF-7乳癌細胞造成少許細胞凋亡(apoptotic cell)和其細胞週期停滯在G0/G1 phase的現象；而NPRE，則會造成MCF-7乳癌細胞的大量死亡，而這樣的現象則可被抑制劑SB203580所阻擋，使NPRE所誘導MCF-7乳癌細胞凋亡現象，從44.44％降到17％，回復率約60％。於是，我們推測：NPRE所誘導MCF-7乳癌細胞走向凋亡的路徑中，p38蛋白可能扮演一個相當重要的角色。相同的結果也出現在Western Blots的分析當中，發現p38、Jun以及p53蛋白有被NPRE活化，也同樣受到p38抑制劑所調控，因而抑制了細胞死亡相關蛋白的表現。

關鍵字

荷葉 ； 多酚 ； 細胞凋亡

並列摘要

According to the research from the Department of Health of Executive Yuan, cancer has been the first reason of death in Taiwan. Therefore, it is a popular topic of discussing how to prevent and postpone the formation of cancer. Nowadays, most researches tend to use natural materials to control the growing and aggravation of the cancer cell. Many studies demonstrate that Polyphenols in plants have the effect of antioxidation and anticarcinogenesis, such as the Polyphenols in the Hibiscus sabdanffa L. ,green tea ,red wine and apples.

This study first extracted the polyphenols from the Nelumbo nucifera leaves to use as qualitative and quantitative。In the qualitative part, the calculated amount of polyphenols extraction was about 85-95%. As for the quantitative part, the polyphenols ingredient in Nelumbo nucifera leaves was analyzed with HPLC. And then the NPRE was sieved using nine different cancer cell line, including Hep G2, Hep 3B, KB, Caco-2, HL-60, AGS, NIH 3T3, MCF-7 and MDA-231. The results indicated that MCF-7 was the most sensitive to inhibit the survival ratio of the cell (IC50 = 0.63 mg/ml) in a dose-dpendent manner. Next, the MCF-7 cell was used as the experimental model to observe the apoptosis phenomena under the circumstances of different concentrations of NLE and NPRE.

It is discovered that the more NLE and NPRE consistency increased, the more conspicuous apoptosis became, including the changes of the morphology and the DNA fragmentation. Analyzing with Flow cytometric, we found that under the same amount, the NLE caused apoptotic cell of MCF-7 and the cell cycle arrest in the G0/G1 phase while the NPRE caused the death of MCF-7. But the results could be blocked by the inhibitors SB203580 which made the declining of MCF-7 induced by NPRE slow from 44.44% down to 17%. The recovery rate is about 60%. Therefore, it is estimated that the p38 protein may play an important role in the declining pace of MCF-7 which induced by NPRE. The same result to occuring in the Western Blots analysis, we found that the p38、Jun and p53 protein were activation by NPRE , which means they also could be blocked by the p38 inhibitor and decline in p38、Jun and p53 protein.

並列關鍵字

Nelumbo nucifera leaves ； polyphenols ； apoptosis ； Nelumbo nucifera leaf polyphenol rich-extract (NPRE)

參考文獻

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