Effect of 3-Methyladenine on Apigenin-Induced Autophagy and Apoptosis in the Breast Cancer T47D Cell Line
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摘要: 研究自噬抑制剂3-甲基腺苷（3-methyladenine，3-MA）对芹菜素诱导乳腺癌T47D细胞系自噬和凋亡的影响。方法：常规培养人乳腺癌T47D细胞，分为对照组、3-MA组、芹菜素组、3-MA+芹菜素组。MTT法检测各组的细胞增殖抑制率；GFP-LC3质粒转染观察各组细胞自噬情况；Hochest/Mito-Red/YO-PRO-1染色法观察各组细胞凋亡形态；AnnexinV/PI双染法流式细胞仪检测各组细胞凋亡率；Western blot法检测LC3及PARP的变化。结果：MTT示3-MA+芹菜素组的增殖抑制率明显高于其他各组（P<0.05）；GFP-LC3质粒转染结果显示：对照组，3-MA组及3-MA+芹菜素组自噬不明显，而芹菜素组与其他各组相比自噬明显增多（P<0.05）；3-MA+芹菜素组的凋亡细胞增多，各组凋亡率分别为（12.73±0.05）%，（18.46±0.03）%，（23.27±0.07）%，（34.14±0.05）%，与对照组相比有统计学意义（P<0.05）；Western blot结果显示，芹菜素组LC3-Ⅱ增高，而3-MA组及3-MA+芹菜素组的LC3-Ⅱ显著减少，3-MA+芹菜素组PARP的剪切带相比其他各组明显增加。结论：自噬抑制剂3-MA抑制细胞自噬后，能够明显增强芹菜素对乳腺癌T47D细胞系的凋亡诱导效应。Abstract: To study the effect of 3-methyladenine ( 3-MA ) on apigenin-induced autophagy and apoptosis in the breast cancer T47D cell line. Methods: T47D cells cultured in vitro were divided into control, 3-MA, apigenin, and 3-MA+apigenin groups. MTT assay was used to detect the inhibition rate of each group. GFP-LC3 plasmid transfection was used to observe the autophagy of each group by fluorescence microscopy. Hoechst/Mito-Red/YO-PRO-1 staining was used to observe the cell apoptosis of each group using fluorescence microscopy. AnnexinV/PI staining was used to detect the apoptotic rate by flow cytometry. Western blot analysis was performed to detect changes in LC3 and PARP. Results: MTT assay showed that the inhibitory rate of the 3-MA+apigenin group was significantly higher than those of the other groups ( P < 0.05 ). GFP-LC3 plasmid transfection showed that autophagy was not obvious in the control, 3-MA, and 3-MA+apigenin groups but was significantly increased in the apigenin group ( P < 0.05 ). Apoptosis increased in the 3-MA+apigenin group, and the apoptotic rates of the four groups were ( 12.73 ± 0.05 ) %, ( 18.46 ± 0.03 ) %, (23.27 ± 0.07 ) %, and ( 34.14 ± 0.05 ) %, respectively ( P < 0.05 ). Western blot analysis showed that LC3-II levels increased in the apigenin group but significantly decreased in the 3-MA and 3-MA+apigenin groups. Cleaved PARP significantly increased in the 3-MA+apigenin group compared with the other groups. Conclusion: The inhibition of autophagy by 3-MA could significantly increase apigenin-induced apoptosis in the breast cancer T47D cell line.
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Keywords:
- Apigenin /
- 3-Methyladenine /
- Breast cancer /
- Apoptosis
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