Inhibitory effect of Xiangbeisan on growth of human breast cancer cells MCF-7 and MDA-MB-231 by activating AMPK/mTOR signaling pathway
Objective To investigate the in vitro anti-breast cancer effects of the Chinese herbal prescription Xiangbeisan and its molecular mechanisms.Results Human breast cancer MCF-7 and MDA-MB-231 cells were treated with Xiangbeisan 1,2,3,4,or 5 mg·mL-1,with a control group not receiving any drug.MTT assay and clone formation assay were used to detect the effects of Xiangbeisan on the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells,and flow cytometry was used to detect the changes in apoptosis of human breast cancer MCF-7 and MDA-MB-231 cells after the administration of Xiangbeisan.Subsequently,the effects of Xiangbeisan on the migration and invasion ability of human breast cancer MCF-7 and MDA-MB-231 cells were detected using cell scratch and cell invasion assays.Through acridine orange staining,the effect of Xiangbeisan on autophagy in human breast cancer cells was explored.Finally,the transcriptome sequencing technique was used to explore the mechanism of Xiangbeisan-induced breast cancer cell death,and the western blotting experiment was used to detect the protein levels of AMPK,mTOR,LC3,and Beclin-1.Results Compared with the control group,Xiangbeisan significantly inhibited the viability of MCF-7 and MDA-MB-231 cells(P＜0.05,0.001),and the median inhibitory concentration(IC50)at 48 h were 2.344 and 1.961 mg·mL-1,respectively,with time and concentration correlation.The colony formation ability of MCF-7 and MDA-MB-231 cells was significantly inhibited.Apoptosis of MCF-7 and MDA-MB-231 cells was significantly induced(P＜0.001).Xiangbeisan obviously inhibited the migration and invasion ability of MCF-7 and MDA-MB-231 cells in vitro.The results of acridine orange staining indicated that Xiangbeisan induced autophagy in MCF-7 and MDA-MB-231 cells.Western blotting experiments showed that Xiangbeisan significantly up-regulated the expressions of Beclin-1 and LC3-Ⅱ,the key autophagy proteins,in MCF-7 and MDA-MB-231 cells(P＜0.01,0.001).Transcriptome sequencing assay revealed that the most significant pathways with differential gene changes included the mTOR signaling pathway,autophagy pathway,etc.Western blotting experiment results showed that Xiangbeisan significantly down-regulated the phosphorylation level of mTOR(P＜0.001),and significantly up-regulated the phosphorylation level of AMPK(P＜0.001).Conclusion Xiangbeisan significantly inhibited the proliferation,migration and invasion of human breast cancer cells,induced apoptosis and autophagy,and the activation of AMPK/mTOR signaling pathway may be an important mechanism.
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