褪黑素通过激活自噬抑制MDA-MB-231乳腺癌细胞的生长和转移

目的 探讨褪黑素对乳腺癌细胞MDA-MB-231生长和转移的作用及其机制。方法 将MDA-MB-231细胞设置对照组以及1、3、5 mmol/L褪黑素处理组，Western blot检测自噬对细胞生长、转移的影响时，添加3-甲基腺嘌呤（3-MA）组、3-MA联合褪黑素组。使用不同浓度褪黑素处理MDA-MB-231乳腺癌细胞24、48 h，并加入0.1%无水乙醇的细胞作为对照，CCK-8法检测细胞增殖活性确定最佳处理浓度和时间，筛选出3 mmol/L褪黑素用于后续实验。集落形成实验及划痕实验检测不同浓度褪黑素对乳腺癌细胞集落形成能力和迁移能力的影响；流式细胞术、免疫荧光检测3 mmol/L褪黑素对MDA-MB-231细胞凋亡率和自噬蛋白阳性着色情况；Western blot检测自噬相关蛋白LC3、P62、Beclin1，凋亡相关蛋白Bcl2、Bax，上皮-间质转化（EMT）相关蛋白E-cadherin、Snai1的水平。结果 CCK-8结果显示，与对照组相比，褪黑素呈浓度及时间依赖性抑制乳腺癌细胞的增殖（P<0.05）；集落形成实验显示，3个浓度褪黑素处理组的集落数低于对照组；褪黑素作用24 h后明显抑制乳腺癌细胞划痕愈合速度（P<0.01），并诱导细胞凋亡率增加（P<0.01）；且与对照组相比，褪黑素组的促凋亡蛋白Bax表达增加（P<0.05），抗凋亡蛋白Bcl2表达下调（P<0.05），Bcl2/Bax的比值逐渐减低（P<0.01）；转录因子Snail减少，上皮细胞标志蛋白E-cadherin增加；单独使用褪黑素能够诱导细胞内自噬标记蛋白LC3-ΙΙ/LC3-Ι、Beclin1表达增加，P62表达减少（P<0.05）以及Beclin1蛋白阳性着色增强，P62蛋白阳性着色减弱；3-MA+褪黑素组中自噬相关蛋白Beclin1（P<0.001）、LC3-ΙΙ/LC3-（Ι P<0.05）以及凋亡蛋白Bax（P<0.01）、上皮细胞标志蛋白E-cadherin（P<0.001）明显低于褪黑素组，抗凋亡蛋白Bcl2（P<0.05）、转录因子Snail以及Bcl2/Bax的比值（P<0.01）高于褪黑素组。 结论 褪黑素可通过诱导MDA-MB-231乳腺癌细胞发生自噬，抑制乳腺癌细胞增殖、转移并促进其凋亡，而减少自噬，可减弱褪黑素对乳腺癌细胞生长和转移的抑制作用。

Melatonin inhibits growth and metastasis of MDA-MB-231 breast cancer cells by activating autophagy

Objective To investigate the effects of melatonin on the growth and metastasis of MDA-MB-231 breast cancer cells and explore the mechanism. Methods MDA-MB-231 cells were treated with 1, 3 or 5 mmol/L melatonin, and the changes in cell proliferation were examined using CCK-8 assay. Colony-forming assay and wound healing assay were used to assess the effects of melatonin treatmnent on colony-forming ability and migration of the cells. Flow cytometry and immunofluoresnce assay were employed to examine apoptosis and positive staining for autophagy-related proteins in the cells treated with 3 mmol/L melatonin. The effects of melatonin treatment alone or in combination with 3-methyladenine (3-MA) on the expressions of the proteins associated with autophagy (LC3, P62 and Beclin1), apoptosis (Bcl2 and Bax) and epithelial-mesenchymal transition (E-cadherin and Snail) were examined with Western blotting. Results Melatonin treatment significantly inhibited the proliferation of breast cancer cells in a concentration- and time-dependent manner (P<0.05), suppressed colony-forming ability and migration (P<0.01), and promoted apoptosis of the cells (P<0.01). Melatonin treatment alone significantly increased the expressions of Bax (P<0.05), E-cadherin, LC3-II/LC3-I, and Beclin1 and lowered the expressions of Bcl2 (P<0.05), Snail, P62 (P<0.05), and Bcl2/Bax ratio (P<0.01) in the cells, and caused enhanced positive staining of Beclin1 protein and attenuated staining of P62 protein. Compared with melatonin treatment alone, melatonin treatment combined with 3-MA significantly decreased the expressions of Beclin1 (P<0.001), LC3-II/LC3-I (P<0.05), Bax (P<0.01), and E-cadherin (P<0.001) and increased the expressions of Bcl2 (P<0.05), Snail, and Bcl2/Bax ratio (P<0.01). Conclusion Melatonin can induce autophagy of MDA-MB-231 breast cancer cells to inhibit cell proliferation and metastasis and promote cell apoptosis, and suppressing autophagy can weaken the inhibitory effect of melatonin on the growth and metastasis of breast cancer cells.