曲古菌素A对A431细胞生长停滞与DNA损伤诱导蛋白45α表达及对细胞增殖凋亡的影响

Effects of trichostatin A on the expression of growth arrest and DNA damage-inducible protein 45 alpha in, proliferation of and apoptosis in a human epithelial carcinoma cell line A431

目的探讨不同浓度曲古菌素A对A431细胞生长停滞与DNA损伤诱导蛋白45α（Gadd45α）表达及对细胞增殖、凋亡的影响。方法0．05、0．1、0．25、0．5、μmol／L曲古菌素A处理A431细胞，采用CCK8法检测曲古菌素A对A431细胞增殖的影响；流式细胞仪检测药物处理后A431细胞周期、凋亡率的变化；RT—PCR及Western印迹法检测Gadd450tmRNA及蛋白的表达。结果不同浓度曲古菌素A均能抑制A431细胞的增殖，在一定时间范围内（6、12、24、48h），随药物浓度增加，细胞存活率下降，具有明显剂量依赖性；同一药物浓度下，细胞存活率随时间延长而下降，有时间依赖性。0．1、0．25、0．5μmol／L曲古菌素A作用A431细胞24h后，G1期细胞比例依次为26．910％±0．799％、30．406％±0．625％、32．896％±0．599％，较对照组（21．633％±1．144％）显著增高（F=105．93，P〈0．05）；A431细胞早期凋亡率、晚期凋亡率均有递增的趋势；A431细胞Gadd45ctmRNA表达随浓度增加而表达增强；Western印迹结果显示，A431细胞Gadd450L蛋白A值分别为0．6536±0．2193、0．6990±0．0110、0．9040±0．1971，较对照组（0．3766±0．0241）增高（F=332．88，P〈0．05）。结论曲古菌素A在体外可诱导A431细胞上调Gadd45α表达；Gadd45α可能参与了曲古菌素A处理后A431细胞的抑制增殖、诱导凋亡过程。

Objective To investigate the effect of trichostatin A on the expression of growth arrest and DNA damage-inducible protein 45 alpha （Gadd45a in, proliferation of and apoptosis in a human epithelial carcinoma cell line A431. Methods Cultured A431 cells were treated with different concentrations （0.05, 0.1, 0.25, 0.5, μmol/L） of trichostatin A for various durations （6, 12, 24, 48 hours）. Subsequently, cell proliferation, cycle and apoptosis were detected by cell counting kit-8 （CCK8） and flow cytometry respectively. The expression of Gadd45a mRNA and protein in cultured A431 cells was detected by reverse transcription-PCR and Western blot respectively. Results Trichostatin A inhibited the proliferation of A431 cells in a dose （0.05 - 1.0 μmol/L ）- dependent manner at all the 4 time points （F = 3554.71, P 〈 0.05）, as well as in a time （6 - 48 hours）-dependent manner at these tested concentrations （F = 1685.18, P 〈 0.05）. A statistical increase was induced in the early apoptosis rate, late apoptosis rate and Gadd45a mRNA expression in A431 cells by the 24 hour-treatment with trichostatin A of 0.1 to 0.5 μmol/L. Elevated percentage of cells at G1 phase （26.910% ± 0.799%, 30.406% ± 0.625%, 32.896% ± 0.599% vs. 21.633% ± 1.144%, F = 105.93, P 〈 0.05） and expression of Gadd45a protein （0.6536 ± 0.2193, 0.6990 ± 0.0110, 0.9040 ± 0.1971 vs. 0.3766 ± 0.0241, F = 332.88, P 〈 0.01） were observed in A431 cells treated with trichostatin A of 0.1, 0.25 and 0.5 μmol/L for 24 hours compared with untreated A431 cells. Conclusions Trichostatin A can enhance the mRNA and protein expression of Gadd45a in A431 cells, which may be involved in the suppression of cell proliferation as well as acceleration of apoptosis of A431 cells by trichostatin A.