Mechanism of the Baicalein - Induced Cell Death In B16F10 Mouse Melanoma Cells
| Autor: | Yu-Kai Hsiao, 蕭宇凱 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 In our previous study, we demonstrated that baicalein induces the formation of superoxide and hydroxyl radicals via 12-lipoxygenase (12-LOX) in B16F10 mouse melanoma cell line; simultaneously we also found that baicalein caused a reduction in cellular viability and induce cell apoptosis. At present, whether baicalein-induced cell death involves the 12-lipoxygenase (12-LOX) suppression or ROS generation is still unclear. In the present investigation, we utilized the extracellular ROS scavengers namely mannitol and catalase, and the intracellular ROS scavengers such as DMPO and CMH to assess their scavenging ability on ROS produced by baicalein. In addition, we also found that the ROS scavengers had no effect on cell growth differentiation, but in the cellular viability (MTT) assay they could effectively reverse the cell viability reduction induced by baicalein. Moreover western blot analysis revealed that the ROS scavengers didn’t respond the cell apoptosis protein (active caspase-3) and the intrinsic apoptosis pathway protein (BAX). In the 12-LOX aspect, we use the 12-LOX downstream product (12-HETE) to counterbalance the 12-LOX inhibitory action of baicalein. We found that the 12-HETE had no difference in cell growth differentiation, but it could reverse the reduction of cellular viability caused by baicalein in B16F10 mouse melanoma cell line effectively, this results are as similar as in ROS scavengers. The 12-HETE also possesses an inhibitory effect on the increase in expression of active caspase-3 caused by baicalein. Finally we pretreated the ROS scavengers and 12-HETE at the same time, the results showed that both drugs minimize the damage caused by baicalein. We inferred that the B16F10 mouse melanoma cell death caused by baicalein is related to both of 12-LOX suppression and ROS generation, but the apoptosis is only because of the 12-LOX suppression by baicalein. In addition, we also used siRNA technology to reduce the performance of 12-LOX; we found the cellular viability reduction of Electro control group is more pronounced than the 12-LOX siRNA group. Therefore, we inferred the cell death caused by baicalein through the 12-LOX; the 12-LOX siRNA and 12-LOX inhibition by baicalein are in different mechanisms. According to our previous study, the 12-LOX siRNA group would decrease the ROS generation caused by baicalein, so we assumed that the main reason of cell death caused by baicalein is the ROS generation and could lead to cell necrosis. In order to confirm the above arguments, we also use the cytometric analysis to examine the cell death caused by baicalein in which we found that the cell necrosis is the major part of cell death, and the apoptosis play a minimum role. The data generated from this study, we can conclude that the B16F10 mouse melanoma cell death caused by baicalein is mainly because of the ROS generation through the 12-LOX. Therefore, the majority of cell death may be due to cell necrosis. The cell apoptosis caused by 12-LOX suppression of baicalein is only a small part. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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