酪氨酸激酶抑制剂达沙替尼抗黑色素瘤的体外研究

Effect of tyrosine kinase inhibitor dasatinib on melanoma cells

目的探讨达沙替尼对小鼠黑色素瘤B16F10细胞株体外增殖、迁移和凋亡的影响。方法采用MTT法检测不同浓度(3.125、6.250、12.500、25.000、50.000μg/m L)达沙替尼在24、48 h对B16F10细胞增殖能力的影响;采用细胞划痕实验和Transwell体外迁移实验检测达沙替尼对B16F10细胞迁移能力的影响;DAPI染色法观察给药后细胞核形态的变化;流式细胞仪检测达沙替尼对B16F10细胞凋亡率和细胞周期的影响;荧光显微镜观察达沙替尼对B16F10细胞线粒体膜电位的影响,并用荧光分光光度计检测Caspase 3和Caspase 9的活化程度。结果达沙替尼对B16F10细胞的增殖有抑制作用,并呈浓度和时间依赖性。细胞划痕实验及Transwell体外迁移实验结果表明:达沙替尼能够有效地抑制B16F10细胞的迁移。分别以低、中、高3个浓度(6.250、12.500、25.000μg/m L)的达沙替尼作用于B16F10细胞24 h后,细胞形态发生明显改变,核裂解,形成多个凋亡小体,凋亡率分别为(34.06±0.83)%、(50.24±1.66)%和(88.91±0.96)%,与对照组比较差异均有统计学意义(P<0.05)。达沙替尼对B16F10细胞周期的影响较小,其中对G1期有微弱的阻滞作用。中浓度(12.500μg/m L)达沙替尼组能够引起B16F10细胞线粒体膜电位的降低,Caspase 3和Caspase 9活性的增加,表明达沙替尼可能是通过线粒体介导的Caspase通路引起细胞凋亡。结论达沙替尼能有效地抑制小鼠黑色素瘤B16F10细胞株体外增殖与迁移,诱导细胞凋亡,有望成为一种有效的抗黑色素瘤药物。

Objective To determine the effect of dasatinib on the proliferation, migration and apoptosis of murine melanoma cell line B16F10. Methods MTT assay was used to detect the proliferation of B16F10 cells after the treatment of dasatinib at different doses （3.125, 6.250, 12.500, 25.000 and 50.000 μg/mL） for different time periods （24 and 48 h）. Wound healing assay and Transwell chamber assay were used to assess the effect of dasatinib treatment on the migration of B16F10 cells. DAPI staining was used to observe the cell morphology before and after the treatment. Flow cytometry was employed to analyze the effect of dasatinib on the apoptotic rate and cell cycle of B16F10 cells. Fluorescence microscopy was performed to measure the effects of dasatinib on the mitochondrial membrane potential of B16F10 cells. Fluorescence spectrophotometery was used to measure the activities of Caspase 3 and Caspase 9. Results Dasatinib markedly inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. Wound healing assay and Transwell chamber assay showed that dasatinib effectively suppressed the migration of B16F10 cells. After the cells were treated by 6.250, 12.500 and 25.000 μg/mL dasatinib for 24 h, obvious morphological changes including nuclear fragments and apoptotic bodies were observed, and the apoptotic rate were （34.06±0.83）%, （50.24±1.66）% and （88.91±0.96）%, respectively, which were significant different with that of the control group （P〈0.05）. Dasatinib had little effect on cell cycle and showed mild arrest on the cells in G1 phase. Dasatinib of 12.500 μg/mL resulted in the reduce of mitochondrial membrane potential and enhanced activities of Caspase 3 and Caspase 9, which indicating that dasatinib may induce cell apoptosis through mitochondria mediated Caspase signal pathway. Conclusion Dasatinib effectively inhibits the proliferation and migration, induces the apoptosis in murine melanoma B16F10 cells. It might be used as an effectively therapeutic agent against human melanoma.