- 學位論文
欣脂清藉由促進GSK3β及下調Cdk4/Cyclin D1、Cdk2/Cyclin E1表現量誘導國人結腸直腸癌細胞株細胞週期G1期停滯。
Simvastatin induced cell cycle G1 arrest in compatriot primary colorectal cancer cell lines by promoting GSK3β and down-regulating Cdk4/Cyclin D1, Cdk2/Cyclin E1 expression.
摘要
背景：欣脂清(simvastatin)同為HMG-CoA還原酶小分子抑制劑，廣泛作為降低膽固醇的藥物。欣脂清在許多體外和活體內研究顯示也具有抗癌的作用。 目的：本研究旨在於探討欣脂清對於國人結腸癌細胞株(CPs)的分子作用機轉。 方法：對於欣脂清抑制CPs的細胞生長，我們使用MTT試驗法進行檢測。利用流式細胞儀觀測細胞週期及細胞凋亡的實驗。西方墨點法則用於探討細胞週期G1期的相關蛋白質表現量，包含Cdk4/Cyclin D1, Cdk2/Cyclin E1和GSK3β。在基因表現的檢測上則有賴於RT-PCR及RealTime-PCR。 結果：CPs經欣脂清作用24至72小時後，呈現劑量與時間上的依賴性。經由流式細胞儀並配合PI單一染色與PI/Annexin V雙重染色，用以確定細胞存活率的下降是否歸因於細胞週期停滯。從CPs經由欣脂清作用24小時候顯示出，G1期的DNA含量有所增加且並未造成細胞凋亡或壞死的現象。此外在西方墨點法的試驗中，欣脂清也促成GSK3β蛋白質的表現量上升及下調Cdk4/Cyclin D1, Cdk2/Cyclin E1的蛋白質表現。RT-PCR的結果顯現出，Cdk4/Cyclin D1, Cdk2/Cyclin E1和GSK3β的基因表現，並未因欣脂清的作用而有所改變。然而，RealTime-PCR的結果顯示，GSK3β和p27經新脂清作用後表現量上升，而Cdk4/Cyclin D1, Cdk2/Cyclin E1的基因表現量則是受到抑制。 結論：國人結腸直腸癌細胞株(CPs)經藥物作用後，會促進GSK3β及下調Cdk4/Cyclin D1, Cdk2/Cyclin E1的蛋白質表現量，使細胞週期停滯在G1，因此抑制細胞的生長。
並列摘要
BACKGROUND: Simvastatins are similar to HMG-CoA reductase small molecule inhibitors, which are widely administered for the reduction of cholesterol levels. Simvastatins have also demonstrated anti-cancer effects in vitro and in vivo. OBJECTIVES: This study sought to reveal the molecular mechanisms underlying the effects of simvastatin on primary colorectal cancer cell lines (CPs). METHODS: MTT assay was performed to evaluate simvastatin-inhibited CPs proliferation. Cell cycle and apoptosis were investigated by flow cytometry. Western blotting was used for the detection of Cdk4/Cyclin D1, Cdk2/Cyclin E1 and GSK3β as well as the expression of G1 phase-related proteins. Genes expression was analyzed using RT-PCR and RealTime-PCR. RESULTS: Treated of CPs with simvastatin for 24 to 72 hours in dose- and time- dependent manner. Flow cytometry with PI staining and PI/Annexin V double staining were used to determine whether the reduction in CP cell viability following simvastatin treatment could be attributed to cell cycle arrest. Simvastatin treatment for 24 hours was shown to increase the content of DNA in G1 phase without inducing cell apoptosis or necrosis. In addition, this treatment was shown to promote the expression of GSK3β and down-regulate the expression of Cdk4/Cyclin D1, Cdk2/Cyclin E1 proteins. RT-PCR showed that treating CPs with simvastatin did not affect gene expression, including Cdk4/Cyclin D1, Cdk2/Cyclin E1, or GSK3β. However, results from RealTime-PCR showed that GSK3β and p27 were increased and Cdk4/Cyclin D1, Cdk2/Cyclin E1 were suppressed by simvastatin in CPs. CONCLUSIONS: The treatment of primary colorectal cancer cell lines (CPs) with simvastatin was shown to induce cell cycle arrest in G1 phase by promoting GSK3β and down-regulating Cdk4/Cyclin D1, Cdk2/Cyclin E1 protein expression, thereby inhibiting the cell proliferation.
參考文獻
被引用紀錄
延伸閱讀
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