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

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

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—FTTC／PI双染色法检测细胞凋亡率的变化。结果MTT结果显示0．5，1，2，5μmol／LKAAD-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；5μmol／LKAAD-环巴胺作用1～5d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。

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 KAA -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 μ mol/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 5μmol/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 in creased 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 KAA -cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cy- clopamine-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.