Casticin induces Cell Death through G2/ M PhaseArrested and Apoptosis in Human MelanomaCancer A375.S2 Cells
| Autor: | Yin-Wen Shiue, 薛茚文 |
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| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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