中华皮肤科杂志 ›› 2008, Vol. 41 ›› Issue (6): 387-390.

• 论著 • 上一篇    下一篇

KAAD-环巴胺抑制Hedgehog信号通路对人鳞状细胞癌细胞株A431生长及凋亡的影响

刘海燕 周扬 白育萍 刘玉峰 李承新   

  1. 第四军医大学西京医院全军皮肤病研究所；兰州军区兰州总医院皮肤科 西安第四军医大学西京医院皮肤科 第四军医大学西京医院全军皮肤病研究所
  • 收稿日期:2007-10-22 修回日期:2007-11-22 发布日期:2008-06-15
  • 通讯作者: 刘海燕 E-mail:lhyxyj@fmmu.edu.cn

Effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431

Liu Hai-Yan Cheng-xin LI   

  • Received:2007-10-22 Revised:2007-11-22 Published:2008-06-15
  • Contact: Liu Hai-Yan E-mail:lhyxyj@fmmu.edu.cn

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摘要： 目的 探讨Hedgehog信号转导通路的特异性抑制剂KAAD-环巴胺对人鳞状细胞癌（squamous cell carcinoma, SCC）细胞增殖及凋亡的影响。方法 采用 MTT 法测定KAAD-环巴胺对A431细胞的增殖抑制情况，光镜下观察细胞形态，流式细胞仪检测KAAD-环巴胺对A431细胞周期的影响，用 Annexin-V-FITC/PI双染色法检测细胞凋亡率的变化。结果 MTT 结果显示0.5，1，2，5 μmol/L KAAD-环巴胺对A431细胞的生长抑制率依次为（7.0 ± 2.3）%，（20.6 ± 2.8）%，（48.3 ± 3.4）%和（61.6 ± 3.3）%，F = 49.92，P ＜ 0.01；5 μmol/L KAAD-环巴胺作用1 ～ 5 d 的生长抑制率分别为（18.3 ± 2.6）%，（56.1 ± 3.7）%，（65.4 ± 2.8）%，（71.2 ± 1.9）%，（75.9 ± 3.0）%，F = 16.32，P ＜ 0.01，表明KAAD-环巴胺可显著抑制A431细胞增殖，且呈浓度和时间依赖性：r浓度 = 0.91，P ＜ 0.01；r时间 = 0.86，P ＜ 0.05。光镜下细胞形态明显受损。细胞周期结果显示KAAD-环巴胺作用组G1期细胞数为（76.2 ± 1.8）%，较空白对照（51.8 ± 2.9）%明显增加，F = 26.34，P ＜ 0.01，G1期前亚二倍体峰由空白对照组（1.7 ± 0.3）%增至（8.7 ± 0.2）%，F = 6.32，P ＜ 0.05，表明 KAAD-环巴胺可以将A431阻滞于G1期。凋亡检测结果显示KAAD-环巴胺可以明显诱导A431细胞发生凋亡，凋亡率由空白对照（18.5 ± 3.1）% 增至（46.2 ± 2.8）%，F = 32.01，P ＜ 0.01。阴性对照组实验结果与空白对照组比较差异均无统计学意义（P ＞ 0.05）。结论 Hedgehog信号通路的特异性抑制剂KAAD-环巴胺能够显著抑制A431细胞增殖并诱导其凋亡。

关键词: Hedgehog信号通路, KAAD-cyclopamine, A431 细胞, 凋亡

Abstract: Objective To investigate the in vitro effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431. Methods A431 cells were cultured and treated with KAAD-cyclopamine （0.5, 1, 2, 5 μmol/L）. Then, MTT assay was used to detect the proliferation of A431 cells, and light microscopy to observe cell morphology at different time points with a 24-hour interval. Flow cytometry was used to assess cell cycle, and annexin-V/propidium iodide double staining to evaluate the apoptosis in these cells after 48 hours of treatment with KAAD-cyclopamine. Results KAAD-cyclopamine of 0.5, 1, 2 and 5 μmol/L inhibited the proliferation of A431 cells by （7.0 ± 2.3）%, （20.6 ± 2.8）%, （48.3 ± 3.4）% and （61.6 ± 3.3）%, respectively （F = 49.92, P < 0.01）. Furthermore, in the presence of KAAD-cyclopamine of 5 μmol/L, on day 1, 2, 3, 4, and 5 the proliferation of A431 cells was suppressed by （18.5 ± 2.6）%, （56.1 ± 3.7）%, （65.4 ± 2.8）%, （71.2 ± 1.9）% and （75.9 ± 3.0）%, respectively, the difference was significant among these time points（F = 16.32, P < 0.01）. Statistical analysis showed that KAAD-cyclopamine downregulated the growth of A431 cells in a dose- and time-dependent manner （r = 0.91, 0.86, P < 0.01 and 0.05, respectively）. Light microscopy revealed typical morphological changes of cell damage in A431 cells. KAAD-cyclopamine increased the percentage of cells in G1 phase from （51.8 ± 2.9）% to（76.2 ± 1.8）% （F = 26.34, P < 0.01）, the proportion of hypoploid cells from （1.7 ± 0.3）% to （8.7 ± 0.2）% （F = 6.32, P < 0.05）, which suggested that KAAD-cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cyclopamine-treated cells was significantly higher than that in the untreated control ［（46.2 ± 2.8）% vs （18.5 ± 3.1）%, F = 32.01, P < 0.01］. Tomatidine treatment did not affect the proliferation or apoptosis of A431 cells（both P > 0.05）. Conclusion KAAD-cyclopamine can markedly suppress the proliferation and induce apoptosis of A431 cells.

Key words: Hedgehog signaling pathway, KAAD-cyclopamine, A431 cell, Apoptosis