| 摘要: | 薑黄素引起人類大腸癌細胞(colo 205)的凋亡和抑制 基質金屬蛋白酶表現的分子機轉 研究生：蘇進成 指導教授：陳光偉 中國醫藥大學 中國醫學研究所 薑黃素(Curcumin, diferuloylmethane, C21H20O6)是從薑黃根莖中提取的一種黃色多酚類化合物，有消炎、抗氧化和抗腫瘤發生等功能，也能夠抑制腫瘤細胞的生長，使細胞停滯在不同的細胞週期而導致凋亡，並且有減弱癌細胞侵襲的能力。在亞洲地區和傳統醫學中被廣泛使用。但確實的分子機轉還是沒有被完全明瞭。本研究將探討薑黄素作用於人類大腸癌細胞株(colo 205)的分子機轉，以提供更多訊息。利用PARP抗體、DAPI染色和Comet assay檢測，確認薑黃素會誘導colo 205細胞走向凋亡(apoptosis)。利用流式細胞儀(Flow cytometry)檢測，結果顯示薑黃素對colo 205的增殖抑制和促細胞凋亡作用有劑量和時間依存效應。也發現薑黃素作用於colo 205細胞鈣離子濃度和ROS的量快速上升，粒腺體的膜電位下降，Caspase-3活性大幅上升，引發細胞凋亡。利用西方墨點法(Western blot)檢測，結果發現薑黃素可使colo 205細胞凋亡相關信號傳遞的蛋白p53、p21 和Bcl-2的表現量下降，Bax、Cytochrome c的表現量上升。結果也顯示可使Cyclin B1、Cdk1和Cdc25c的表現量下降，而Wee1的表現量上升。另外利用生物晶片檢測，也顯示薑黃素使colo 205 細胞 Wee1的基因表達上調，Cdc25c、Cyclin B1和Cdk1的基因表達下調，和蛋白的表現結果一致。表示薑黃素可透過下調Cdc25c，Cyclin B1 和 Cdk1基因表達和上調Wee1基因表達，使colo 205細胞停滯在G2/M 期。利用西方墨點法檢測，結果發現薑黃素可下調colo 205 細胞中的NF-κB p65，Cox-2及MMP-2蛋白的表現量，而利用生物晶片檢測，也確認可下調colo 205 細胞NF-κB p65、Cox-2和MMP-2基因的表達。另外由侵襲轉移能力檢測，顯示薑黃素可減少colo 205細胞侵襲的能力。以上的結果顯示薑黃素可促使colo 205 細胞發生凋亡，可能透過下調p53、p21、Bcl-2、Cyclin B1、Cdk1和Cdc25c；上調Bax、Cytochrome c 和Wee1的蛋白表現量和基因的表達。可能透過下調NF-κB p65、Cox-2和MMP-2的蛋白表現量和基因的表達，減低侵襲能力。薑黃素口服耐受性高，在未來可能成為預防和治療癌症的口服藥物。; The Molecular Mechanism of Curcumin Induced Apoptosis and Inhibited Matrix Metalloproteinase Expression in Human Colon Cancer Cells (colo 205) Chin-Cheng Su Major professor: Guang-Wei Chen Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University Curcumin (diferuloylmethane, C21H20O6), is a natural yellow polyphenolic pigment, derived from the rhizomes of the plant Curcuma longa (Linn), existing anti-inflammatory, antioxidant, and Anticarcinogenic properties. It is used extensively in Asian countries and in traditional medicine. Curcumin is known to inhibit proliferation of cancer cells by arresting them at various phases of the cell cycle and is known to induce apoptosis and inhibit invasion in tumor cells. However, the exact mechanisms of curcumin induce apoptosis and cell cycle arrest and inhibit invasion of cancer cells are unclear. This study will provide increased insight into the mechanism of action of curcumin in colon cancer colo 205 cells.In the present, we investigated the induction of apoptosis from colon cancer colo 205 cells to underscore the need for further understanding of the multiple mechanisms of cell death unleashed by curcumin. We examined cytotoxicity and apoptosis in curcumin treated colon cancer colo 205 cells by using flow cytometry and the results demonstrated that curcumin induced cytotoxicity and apoptosis in these examined cells and that these effects are dose-and time-dependent. We used PARP and DAPI staining to confirm curcumin induced apoptosis. We also used Comet assay to show curcumin induced DNA damage in these examined cells. We also used flow cytometry to show curcumin induced reactive oxygen species (ROS) and Ca2+ productions and decreased the levels of mitochondria membrane potential and induced caspase-3 activity. We also used Western blotting to analyze the levels of Bax, Bcl-2, cytochrome C, p53 and p21 in curcumin treated cells and the results demonstrated that curcumin promoted the expression of Bax and cytochrome C but inhibited the expressions of p53, p21 and Bcl-2. We used flow cytometry to analyze the cell cycle after colo 205 cells were treated with various concentrations of curcumin. The results demonstrated that curcumin induced G2/M arrest in these examined cells and that those effects are dose-and time-dependent. In order to further understand the mechanism of curcumin induced G2/M arrest, the checkpoint associated with enzymes of the cell cycle were also investigated by Western blotting methods. The results demonstrated that curcumin induced Wee1 expression and decreased the Cdc25c, cycling B1 and CDK1 expressions. The cDNA microarray assay also employed to confirm gene expressions. The results demonstrated that curcumin promoted the gene expression of Wee1 and inhibited the gene |
