| 摘要: | 從古自今蔓荊子黃素(Casticin)廣泛當做抗發炎劑使用，過去研究顯示可促使癌症細胞的凋亡。本研究中我們探討 Casticin誘導人類黑色素瘤A375.S2細胞株細胞凋亡的機制，首先我們利用MTT試驗測定A375.S2細胞存活率和觀察細胞形態變化，藉由流式細胞儀偵測A375.S2細胞株的細胞週期變化、粒線體膜電位改變、活性氧基群的變化和Caspase-3活性表現。由DAPI試驗，蔧星試驗和DNA凝焦電泳試驗也可發現DNA損傷、斷裂情形。並藉由西方墨點法檢測凋亡相關蛋白和細胞週期調控蛋白的表現量。最後利用EMSA觀察NF-kB與DNA結合能力。在我們的研究結果顯示，Casticin誘導A375.S2細胞存活率呈現劑量依賴性而下降並能夠引起A375.S2細胞的細胞週期停滯於G2/M期和DNA受損。Casticin也造成A375.S2細胞內活性氧基群的增加和粒線體膜電位的減少。西方墨點法證實凋亡相關蛋白Cytochrome c, Bax, Bak, AIF, Edno G, Caspase-3蛋白表現量增加和抗凋亡蛋白Bcl-2, Bcl-xL, x-IAP, Mcl-1蛋白表現量下降。另外西方墨點法也證明細胞週期G2/M調控蛋白CHK-2, p21蛋白表現量增加和Cyclin B, Cyclin A, CDK-1和Cdc25c蛋白表現量減少。此外利用免疫螢光染色法觀察AIF與Endo G被釋放到細胞質誘導下游路徑的活化並造成細胞凋亡。然而Casticin也抑制NF-kB與核內DNA結合的活性。基於上述結果，我們證實Casticin誘導人黑色素瘤A375.S2細胞週期停滯在G2 / M期並藉由粒線體內凋亡路徑造成細胞凋亡。
Casticin have long been widely used as an anti-inflammatory agent, has been shown to apoptosis promoting activite in cancer cell lines. In this study, we explored the mechanism of casticin- induced cell apoptosis in human melanoma A375.S2 cells. First, we determined cell viability by MTT assay and the impact of cell morphology by phase-contact microscope were observed in A375.S2 cells. Flow cytometry analysis for the changes of the cell cycle distribution, reactive oxygen species (ROS) production, mitochondrial membrane potential (???卌), intracellular release of caspase-3 activity in A375.S2 cells were examined. DNA damage and DNA breakage were examined by DAPI staining and Comet assay. Apoptosis associated protein and cell cycle protein expressions were examined by Western blotting. Finally we also used EMSA to measure the DNA binding ability in A375.S2 cells.
Our results showed that casticin induced the decrease of cell viability in a dose-dependent manner. Casticin induced cell cycle arrest at G2/ M phase and induced DNA damage in A375.S2 cells. Castacin caused intracellular ROS increased and loss of mitochondrial membrane potential (???卌). Caspase-3 activity was significantly increased after treatment with casticin at different time. Western blot analytics proved the increase of pro-apoptotic proteins such as Bax,Bak, AIF, Endo G, cytochrome c, caspase-3, and declined the anti-apoptotic proteins Bcl-2, BCl-xL, xIAP, Mcl-1.
In addition, Western blotting showed an increase in the levels of CHK-2, p21 and a decrease in the levels of Cdc25c, Cyclin B, Cyclin A, and CDK-1. The intracytosolic release of AIF and Endo G contributing to the occurrence of apoptosis were demonstrated by confocal microscopy. The translocation and DNA binding ability of NF-kB were decreased in A375.S2 cell line of the exposed to casicin. Based on the above results, we confirm that casticin inhibit the proliferation of human melanoma A375.S2 cells through the cell cycle arrest at G2/ M phase and induction of apoptosis through mitochondrial apoptosis pathway. |
