白藜芦醇对恶性黑素瘤细胞株增殖的影响

Effect of resveratrol on the proliferation of malignant melanoma cell lines in vitro

目的研究白藜芦醇对恶性黑素瘤体外抗癌作用，探讨其抗癌作用的分子机制。方法采用MTT法检测白藜芦醇对人恶性黑素瘤细胞系A375及小鼠恶性黑素瘤细胞系B16-F1增殖的影响；Annexin—V／PI双标流式细胞术检测白藜芦醇对A375、B16-F1细胞凋亡的影响，PI单标流式细胞术测定白藜芦醇对A375、B16-F1细胞周期的影响；Western印迹法检测白藜芦醇对A375、B16-F1细胞cel-2、Bax蛋白表达的影响。结果白藜芦醇对A375、B16-F1细胞增殖具有明显的抑制作用，且呈量效及时效关系。25μmol／L白藜芦醇作用24h即可诱导A375细胞凋亡，其细胞凋亡率为16．7％±2．1％。100μmol／L白藜芦醇作用24h时B16-F1细胞凋亡率可达39．6％±3．3％。100μmol／L白藜芦醇作用12h时A375细胞凋亡率为17．2％±1．7％，作用72h时达52．3％±4．1％；相同浓度白藜芦醇作用12h时B16-F1细胞凋亡率为18．4％±1．6％，作用72h时达56．7％±4．5％。白藜芦醇处理组A375及B16-F1细胞周期均发生变化，细胞周期被阻滞于G1期，此阻滞效应随白藜芦醇浓度的增加而增加，25μmol／L、100μmol／L白藜芦醇作用24h时，A375G1期比例分别为40．51％±3．97％和55．64％±4．95％，B16-F1细胞分别为41．34％±3．12％和53．93％±5．12％。白藜芦醇明显下调A375及B16-F1细胞抗凋亡蛋白Bcl-2的表达，同时上调促凋亡蛋白Bax的表达水平。结论白藜芦醇可通过调控细胞周期进程、诱导细胞凋亡而有效抑制A375及B16-F1的增殖，其机制与调节Bcl-2、Bax的表达有关。

Objective To investigate the anticancer activities of resveratrol on malignant melanoma cells in vitro and involved mechanisms. Methods A375 human malignant melanoma cells and B16-F1 mouse malignant melanoma cells were cultured and treated with various concentrations of resveratrol for different durations. The cell proliferation, apoptosis and cycle of both B16-F1 and A375 cells were detected with MTT assay, Annexin V-FITC/propidium iodide （PI） double staining flow cytometry and propidium iodide flow cytometry, respectively. Western blot analysis was performed to measure the expression of Bcl-2 and Bax protein in both cells. Results Resveratrol inhibited the proliferation of A375 and B16-F1 cells in a time- and dose- dependent manner. The apoptosis rate of A375 cells was （ 16.7 ±2.1 ）%, （ 17.2 ±1.7）% and （52.3 ± 4.1 ）% after treatment with resveratrol of 25 ixmol/L for 24 hours, resveratrol of 100 μmol/L for 12 and 72 hours, respectively; and resveratrol of 100 μmol/L induced the apoptosis of B16-Fl at a rate of （ 18.4 ± 1.6）%, （39.6±3.3）% and （56.7±4.5）% at 12, 24 and 72 hours, respectively. Flow cytometry showed that A375 and B16-F1 cells treated with resveratrol were arrested in the G1 phase of cell cycle, and the blocking effect increased in a dose-dependent manner. The percentage of A375 and B16-F1 cells in G1 phase was （40.51 ± 3.97）% and （41.34 ±3.12）%, respectively, after 24-hour treatment with resveratrol of 25 μmol/L, （55.64 ± 4.95）% and （53.93 ± 5.12）%, respectively with resveratrol of 100 μmol/L for the same duration. The expression of Bcl-2 protein was decreased in malignant melanoma cells treated with resveratrol, while that of Bax protein increased. Conclusions Resveratrol can effectively inhibit the proliferation of malignant melanoma cells by regulating the cell cycle and inducing cell apoptosis, which seems to be associated with the regulation of Bcl-2 and Bax expressions.