4-氨基吡啶与多西紫杉醇抗人乳腺癌细胞MCF-7 作用及相互关系探讨*

目的:通过研究钾离子通道阻断剂4- 氨基吡啶（4-AP ）对肿瘤化疗药物多西紫杉醇（docetaxel ，DOC ）的抗人乳腺癌细胞MCF-7 作用的影响，探讨4-AP 能否增强DOC 的作用。方法:用MTT 比色法分析DOC 、4-AP 以及两药联合应用对人乳腺癌细胞MCF-7 增殖的影响；用流式细胞术PI 单染法及Annexin V-FITC/PI双染法检测上述两种药物对人乳腺癌细胞MCF-7 的细胞周期及早期凋亡的影响。结果:DOC 能明显抑制人乳腺癌细胞株MCF-7的增殖，并呈剂量和时间依赖性。4-AP 对MCF-7 细胞增殖亦具有一定的抑制作用，在用药后24h、48h 及72h 的抑制率分别为11.9%±1.7% 、42.1%±3.2% 、44.2%±1.6% 。且5mmol/L 4-AP 可使DOC 的作用增强，使25μ mol/L DOC 对人乳腺癌细胞株MCF-7 最大杀伤作用时间从72h 提前至24h。5 μ mol/L DOC 能够使MCF-7 G2/M期细胞比率明显增加（53.58%±1.44% 与对照组8.83%±0.44% 相比，P<0.01），使G0/G1 期细胞减少（11.48%±0.14% 与对照组63.89%±0.98% 相比，P<0.01），说明DOC 主要在G2/M期阻滞MCF-7 细胞的增殖。5mmol/L 4-AP 作用于MCF-7 使其G0/G1 期细胞比率增加，G2/M期细胞明显减少，甚至消失（0.42%±0.17% 与对照组8.83%±0.44% 相比，P<0.05）。 而两药联用可见4-AP 使MCF-7 G2/M期细胞比率有所减少，而G0/G1 期细胞比率有所增加。DOC 单独作用于人乳腺癌细胞株MCF-7 细胞24h 后，能明显增加晚期凋亡和死亡率（由6.97%±0.75% 增加到20.77%±0.75% ，P<0.05）；而两药联合时，与对照组相比，早期凋亡比例由4.60%±0.91% 增加到12.20%±0.82%（P<0.05），晚期凋亡和死亡细胞比例由6.97%±0.75% 增加到58.42%±0.31%（P<0.05），提示4-AP（5mmol/L ）能明显增加DOC 诱导的MCF-7 早期凋亡。结论:DOC 和4-AP 分别在G2/M期和G0/G1 期抑制MCF-7 细胞增殖，4-AP 通过促进DOC 诱导细胞凋亡发挥抑制MCF-7 细胞增殖的作用。 

The Effect of Docetaxel Combined with 4-AP on Human Breast Cancer MCF-7 Cells

Objective: To study the effect of docetaxet (DOC) combined with 4-AP on human breast can-cer MCF-7 cells and to explore whether 4-AP could strengthen the effect of docetaxel. Methods: MTT assays were performed to investigate the effect of docetaxel, 4-AP and the combination of them on the proliferation of MCF-7 cells. Flow cytometry was employed to detect cell cycles and cell apoptosis after the cells were stained by PI alone or by Annexin-V and PI. Results: Docetaxel could significantly inhibit the proliferation of MCF-7 cells in a dose- and time- dependent manner. 4-AP could inhibit the proliferation of MCF-7 cells and the inhibitory rates were 11.9%±1.7%, 42.1%±3.2%, and 44.2%±1.6% at 24h, 48h and 72h after adding 4-AP. Moreover 4-AP (5mmol/L) could strengthen the effect of docetaxel. 4-AP (25μmol/L) could increase the effect of Docetaxel. Docetaxel at 5μmol/L could significantly increase the percentage of cells at G_2/M (53.58%± 1.44% vs. 8.83%±0.44%, P<0.01) and decrease the percentage of cells at G_0/G_1 (11.48%±0.14% vs. 63.89%±0.98%, P<0.01), indicating that docetaxel blocked MCF-7 cells at G_2/M phase. 4-AP at 5mmol/L could in-crease the percentage of MCF-7 cells at G_0/G_1 and decrease the percentage of cells at G_2/M (0.42%±0.17% vs. 8.83%±0.44%, P<0.05). Docetaxel could significantly increase late apoptosis and death of MCF-7 cells af-ter treatment over 24h (from 6.97%±0.75% to 20.77%±0.75%, P<0.05). Docetaxel combined with 4-AP could increase early apoptosis rate from 4.60%±0.91% to 12.20%±0.82% (P<0.05) and could increase late apopto-sis rate and death rate from 4.60%±0.91% to 12.20%±0.82% (P<0.05). Conclusion: Both docetaxel and 4-AP can inhibit the proliferation of MCF-7 cells. Docetaxel can increase the percentage of cells at G_2/M phase and 4-AP can increase the percentage of cells at G_0/G_1 phase. 4-AP could strengthen the inhibitory effect of docetaxel on the proliferation of MCF-7 cells through inducing cell apoptosis.