| 摘要: | 人參皂?Rh2抑制肺腺癌細胞生長及造成凋亡之分子作用機轉研究 中國醫藥大學中國醫學研究所 研究生：楊淑媚 指導教授：陳榮洲博士 Rh2是從紅參中萃取出來的一種二醇類人參皂?，在過去的研究中發現其對於某些種類的癌細胞具有抑制癌細胞增殖 (antiproliferative effect)的作用，但其抗癌的分子作用機轉尚有未清楚之處，本文以肺腺癌 (A549)細胞作為研究，探討Rh2是否會對其生長造成影響，並進一步探討其分子作用機轉。肺腺癌 (A549)細胞以30 μg/ml Rh2濃度處理，發現有癌細胞生長停滯及細胞凋亡的情形。以流式細胞分析儀 (flow cytometry)分析發現細胞週期停滯在G1期，我們進一步分析調控細胞週期的相關分子表現，蛋白質西方墨點法分析 (western blot)發現Cyciln-D1, -E及Cdk-6表現量下降，以及pRb2含量的上升。Rh2處理後的肺腺癌細胞不論是在細胞形態上或是TUNEL assay 及DNA fragmentation analysis都發現Rh2會造成肺腺癌細胞的凋亡，我們進一步探討其凋亡路徑，分析了Bcl-2家族相關分子，發現其表現量無多大的變化，其凋亡路徑與粒腺體凋亡路徑的Bcl-2家族無關。但分析了細胞膜上的死亡受體路徑，發現TRAIL-R1 (DR4)的表現量增加，所以其凋亡路徑可能走的是細胞膜上的死亡受體路徑。在劊蛋白? (caspase)方面，caspase-2, -3及-8有被活化的現象，若加入caspase-8的抑制劑則會影響caspase-2及-3的活化，所以可能是先活化了caspase-8，之後再活化caspase-2及-3。 總結人參皂?Rh2的作用：Rh2會造成肺腺癌細胞週期停滯於G1期並誘發細胞凋亡。Rh2對細胞週期的作用為使Cyciln-D1, -E及Cdk-6表現量下降，以及pRb2含量的上升，促使細胞週期停滯在G1期。Rh2另一方面亦會造成肺腺癌細胞的凋亡現象，其所誘發的凋亡路徑並非粒腺體凋亡路徑而是細胞膜上的死亡受體路徑，Rh2會使TRAIL-R1 (DR4)的表現量增加，先誘發caspase-8活化，再誘導caspase-2, -3的活化，進而造成DNA被裂解，促使肺癌細胞凋亡。 關鍵詞：人參皂?Rh2，肺腺癌 (A549)細胞，細胞週期，凋亡路徑; Molecular mechanisms of Ginsenoside Rh2 mediated G1 growth arrest and apoptosis in human lung adenocarcinoma A549 cells Shu-Mei Yang Major Professor: Jung-Chou Chen Institute of Chinese Medical Science, China Medical University Ginsenoside Rh2 (Rh2), a purified ginseng saponin, has been shown to have antiproliferative effects in certain cancer cell types. However, the molecular mechanisms of Rh2 on cell growth and death have not been fully clarified. In this study, the anti-proliferative effect of Rh2 in human lung adenocarcinoma A549 cells was investigated. Treatment of A549 cells with 30 μg/ml Rh2 resulted in a G1-phase arrest, following progressed to apoptosis. This Rh2-mediated G1 arrest was accompanied by the down-regulation of the protein levels and kinase activities of cyclin-D1, cyclin-E, and Cdk6, and the up-regulation of pRb2/p130. In addition, Rh2-induced apoptosis was confirmed by TUNEL assay and DNA fragmentation analysis. Administration of Rh2 caused an increase in the expression levels of TRAIL-R1 (DR4) death receptor but did not alter the levels of other death receptors and Bcl-2 family molecules. Furthermore, the Rh2-induced apoptosis was significantly inhibited by DR4:Fc fusion protein, which inhibits TRAIL-DR4-mediated apoptosis. In addition, the caspase-2, —3 and -8 were drastically activated upon Rh2 treatment. The inhibitors of caspase-2, -3, and caspase-8 markedly prevented the cell death induced by Rh2. The inhibitor of caspase-8 significantly inhibited the activation of caspase-2, -3 and —8. These observations indicate that multiple G1-related cell cycle regulatory proteins were regulated by Rh2 and contributed to Rh2-induced G1 growth arrest. The increase in the expression level of DR4-death receptor may play a critical role in the initiation of Rh2-triggered apoptosis, and the activation of caspase-8/caspase-3 cascade acts as the executioner of the Rh2-induced death process. Key words: Caspase, Cyclin, Ginsenoside Rh2, pRb2/p130, TRAIL-R1 |
