摘要

1. Magnolol抑制人類肝癌細胞Hep-3B增生的分子機轉Molecular Mechanisms of Magnolol-Induced Antiproliferation effect in Hep-3B cells • 本篇研究計畫主要在探討中藥厚朴的酚類萃取物magnolol（厚朴中分離出含有hydroxylated biphenyl 的化合物）對人類肝癌細胞株（Hep-3B）的生長抑制作用及其作用的分子機轉。先前本實驗室發現magnolol可藉由增加p21蛋白的表現來抑制大腸癌細胞株（Colo-205及HT-29）及肝癌細胞株（Hep-G2及Hep-3B）的生長。一般認為p21的表現是受其上游基因p53的調控，然而這些對於magnolol敏感的細胞中有的p53基因是具功能性（Colo-205及Hep-G2）；而有的p53基因則不具功能，或喪失部分功能（Hep-3B及HT-29）。因此本研究的目的是想利用Hep-3B肝癌細胞株（p53基因不具功能）來進一步探討magnolol的抗癌作用機轉。本實驗發現，以低濃度的magnolol（10 M~50 M）處理，Hep-3B的生長有被抑制的情形；以高濃度的magnolol（100 M）處理下，則觀察到Hep-3B細胞進行程式凋亡的現象。利用[3H] thymidine incorporation 和 flow cytometry的方法，我們發現magnolol會使得Hep-3B 細胞週期停滯在G0/G1-phase，進而抑制DNA synthesis 。另外我們藉由Western blot analysis的方式觀察細胞週期調控蛋白及程式凋亡特定蛋白表現時，發現當magnolol抑制Hep-3B細胞生長時除了p21的表現有增高的現象外，其餘之細胞週期調控蛋白的表現則不受影響；而當magnolol造成Hep-3B程式凋亡時，p21的表現量反而降低。此外，我們也觀察到與細胞程式凋亡有關的一些蛋白如bax、caspase3、caspase8及caspase9皆有被活化的現象。綜合上述的研究結果，我們推測magnolol的抑癌作用主要是藉由增加p21蛋白的表現來抑制的CDK2酵素活性，進而造成細胞週期停滯於G0/G1期，進而抑制癌細胞的生長；同時magnolol亦可經由活化caspase的途徑來造成癌細胞的凋亡。

2. The aim of this study is to investigate the antiproliferation effect of magnolol on the human hepatoma cell line Hep-3B (p53 null) and its underlying molecular mechanisms. Magnolol is one of the phenolic pure compounds isolated from the bark of Hou pu’ (Cortex Magnolia officina1is), which has been commonly used in Chinese herb medicine in the treatment of fever, headache, anxiety, diarrhea and stroke.In vitro studies, we showed that magnolol at low concentrations (10~50 M) induced a dose-dependent inhibition of cell growth in Hep-3B. However, magnolol at a higher concentration (100 M) induced the occurrence of apoptosis. To study further the molecular mechanisms of anti-liver cancer effect of magnolol, we analyzed the cell population during cell cycle by [3H]thymidine incorporation and flow cytometry analyses, and detected the expression levels of cell cycle regulatory proteins by using western blot analysis. Treatment of Hep-3B cells with magnolol (10-50 M) induced a decrease in DNA systhesis and retardation of cell cycle at G0/G1 phase of cell cycle. The protein levels of cyclins and CDKs were not changed significantly in the magnolol-treated Hep-3B cells as compared with the control cells. However, the p21 protein was upregulated during magnolol-mediated antiproliferation, and downregulated during magnolol-induced apoptosis. Moreover, the CDK2, but not CDK4, kinase activity was downregulated in the magnolol-treated Hep-3B in a dose-dependent manner. For the molecular mechanisms involved in the magnolol-induced apoptosis in Hep-3B, we observed that intracellular events included (1) degradation of bax and (2) activation of caspases 3, 8, and 9.