抗癌生物活性肽对人乳腺癌nm231细胞的增殖抑制作用

 目的 探讨抗癌生物活性肽（ACBP）对人乳腺癌 nm231细胞的作用及其机制。 方法 nm231 细胞经不同浓度（0.05、0.10、0.20、 0.25 µg/ml）ACBP 作用 72 h，以噻唑蓝（MTT）法测定细胞增殖活性。以 0.25 µg/ml 的 ACBP 分别作用于 nm231 细胞 4、6、24、48、72 h，行光镜和透射电镜（作用 48 h 细胞）观察。应用 DNA 凝胶电泳和磷脂结合蛋白 V（Annexin V，AV）/碘化丙啶（PI）双标记染色流式细胞术等方法，观察 0.25 µg/ml 的 ACBP 作用不同时间对nm231 细胞凋亡发生及作用 48 h 对细胞周期的影响。以上各项检测均设以 RPMI 1640 培养液取代 ACBP 的对照组。 结果 浓度为 0.1 µg/ml 的 ACBP 即对 nm231 细胞的增殖有明显抑制作用，抑制率为 10.3%，抑制作用具有浓度依赖性，ACBP 浓度为 0.25 µg/ml 时抑制率达 35.1%。光镜观察显示，经 0.25 µg/ml 的 ACBP 作用 24 h，细胞出现凋亡特征；作用 48 h，光镜及电镜下均可见大量的典型凋亡细胞。DNA 凝胶电泳显示，经 0.25 µg/ml 的ACBP 作用 24 h 的细胞出现 DNA 断裂；作用 48 h 的细胞出现典型的梯形电泳图谱。流式细胞术分析显示，ACBP 作用后AV（+）/ PI（－）细胞和 AV（+）/ PI（+）细胞比例均随培养时间延长而增大；作用 48 h 的 nm231细胞 G0/1 期细胞比例高于对照组（P < 0.01），而细胞增殖指数低于对照组（P < 0.01）。 结论 ACBP 可抑制 nm231 细胞的增殖，其机制与抑制nm231 细胞的 DNA 合成和诱导细胞凋亡相关。

Inhibitory effect of anti-cancer bioactive peptide on the proliferation of human breast cancer cell line nm231

Objective To investigate the effect of anti-cancer bioactive peptide (ACBP) on human breast cancer cell line nm231 and its mechanisms. Methods After being treated with different concentrations of ACBP (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, and 0.25 μg/ml respectively) for 72 hours, the proliferative activity of the nm231 cells were detected by MTT. And the nm231 cells that treated with 0.25 μg/ml ACBP was observed under a light microscope at 4, 6, 24, 48, and 72 hours respectively, and examined by transmission electron microscopy at 48 hours. Gel electrophoresis of DNA and flow cytometry using AV/PI double staining were used to detect the effect of ACBP (0.25 μg/ml) on the cell apoptosis at different time points, and its effect on the cell cycle at 48 hours. In all the experiments, the nm231 cells in the control group were cultured in RPMI 1640 medium instead of ACBP. Results The proliferation of nm231 cells was markedly inhibited by 0.1 µg/ml ACBP with an inhibitory rate of 10.3%. The rate reached 35.1% in the cells treated with 0.25 µg/ml ACBP. The inhibitory effect showed a concentration-dependent manner. After being treated with 0.25 µg/ml ACBP for 24 hours, apoptotic features could be detected in the nm231 cells under a light microscope. Then, at 48 hours, a large number of typical apoptotic cells could be observed by both light microscopy and transmission electron microscopy. DNA gel electrophoresis showed DNA fragmentation in the cells treated with 0.25 µg/ml ACBP for 24 hours, and typical DNA ladder in those treated for 48 hours. Flow cytometry found that the proportions of AV+/PI- and AV+/PI+ cells increased with culture time. In the nm231 cells treated with ACBP for 48 hours, the proportion of the cells in G0/1 phase was significantly higher than that in the control, while the proliferation index was significantly lower than in the control. (both P < 0.01). Conclusion ACBP can inhibit the proliferation of nm231 cells by inhibiting the DNA synthesis and inducing cell apoptosis.