中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (6): 404-407.

• 论著 • 上一篇    下一篇

甲磺酸伊马替尼对黑素瘤细胞Hs294T生物学活性的影响

刘启方1,周晓燕2,徐玉荣2,廖文俊3,李承新3,高天文4   

  1. 1. 66347部队卫生队
    2. 第四军医大学西京医院皮肤科
    3. 第四军医大学西京医院全军皮肤病研究所
    4. 西安第四军医大学西京医院皮肤科
  • 收稿日期:2011-07-21 修回日期:2011-10-11 出版日期:2012-06-15 发布日期:2012-05-31
  • 通讯作者: 廖文俊 E-mail:liaowj@fmmu.edu.cn

Effects of imatinib mesylate on the biological activity of Hs294T melanoma cells

  • Received:2011-07-21 Revised:2011-10-11 Online:2012-06-15 Published:2012-05-31

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摘要：

【摘要】 目的 探讨酪氨酸激酶抑制剂甲磺酸伊马替尼对黑素瘤细胞Hs294T生物学活性及其Wnt/β-连环蛋白通路的的影响。 方法 用甲磺酸伊马替尼作用黑素瘤细胞Hs294T后，用MTT实验、Annexin V-PI双染色法、基质胶侵袭实验分别观察细胞增殖活性、凋亡和侵袭能力的变化。用激光共聚焦显微镜观察细胞中Wnt通路关键性蛋白分子β-连环蛋白定位的改变。用Western印迹和荧光定量PCR分别检测β-连环蛋白，靶基因细胞周期蛋白D1蛋白的表达变化，及通路转录因子LEF1、靶基因C-myc 的mRNA表达的变化。结果 4 ～ 10 μmol/L浓度药物能使细胞增殖活性呈梯度下降（P ＜ 0.05）；用10 ?滋mol/L药物可使细胞增殖能力呈时间梯度下降（P ＜ 0.01）；与对照组相比，处理组细胞早期及晚期凋亡比例均明显增加，细胞侵袭能力显著降低约48%（P ＜ 0.05），同时β-连环蛋白蛋白表达未见明显变化，但其胞核定位相对减少，LEF1、C-myc、细胞周期蛋白D1的表达均下调。结论 甲磺酸伊马替尼可能通过下调Wnt/β-连环蛋白通路而抑制黑素瘤细胞增殖、促进凋亡并降低侵袭力。

关键词: 黑素瘤

Abstract:

【Abstract】 Objective To investigate the effects of imatinib mesylate as a tyrosine kinase inhibitor on the biological activity of and Wnt/β-catenin pathway in Hs294T melanoma cells. Methods After Hs294T cells were incubated with imatinib mesylate at various concentrations （4, 8, 10, 16, 20 and 24 μmol/L） for 24 hours or imatinib mesylate at 10 μmol/L for 24, 48 and 72 hours, methyl thiazolyl tetrazolium （MTT） assay was performed to estimate the proliferation of cells and to determine the effects of imatinib mesylate on the proliferation of Hs294T cells. Then, Hs294T cells were treated with imatinib mesylate at 10 μmol/L or dimethyl sulfoxide （DMSO） for different durations, followed by the detection of cell apoptosis with flow cytometry, localization of β-catenin with annexin Ⅴ/propidium iodide-double staining and laser confocal microscopy, quantification of β-catenin and cyclin D1 protein with Western blot, and measurement of LEF1 and C-myc mRNA expression with real time fluorescence-based quantitative PCR. Matrigel invasion assay was performed to evaluate the invasiveness of Hs294T cells after treatment with imatinib mesylate at 5 μmol/L or DMSO for 24 hours. Results Imatinib mesylate at 4－10 μmol/L elicited a dose-dependent decline in the proliferation of Hs294T cells （F = 125.3, P ＜ 0.05）, and imatinib mesylate at 10 μmol/L induced a time-dependent decrease from 24 to 72 hours （F = 714.6, P < 0.01）. The percentage of early and late apoptotic cells was markedly increased, while the invasiveness was decreased by about 48% （P < 0.01）, together with a downregulation in the expression of LEF1, C-myc and Cyclin D1 in imatinib mesylate-treated Hs294T cells compared with the DMSO-treated cells. No obvious changes were observed in the protein expression of β-catenin, but a decline in the nuclear localization of β-catenin was noted in Hs294T cells after being treated with imatinib mesylate. Conclusion Imatinib mesylate may suppress the proliferation and invasion of, but promote the apoptosis in, melanoma cells, by downregulating the Wnt/β-catenin pathway.

Key words: melanoma