The Mechanism Of ZSW-1-17 Inhibits Triple-negative Breast Cancer By Activating AMPK

Background: Triple-negative breast cancer(TNBC)is a subtype of breast cancer.Due to the lack of targeted drugs,easy metastasis and recurrence,the 5-year survival rate of patients is less than 30 %.Adenylateactivated protein kinase(AMPK)was initially identified as a cell energy sensor and played a key role in cell energy homeostasis.In the past decade,in-depth studies on the molecular mechanism of AMPK have shown that AMPK phosphorylation regulates the biological functions of multiple downstream signaling molecules,including cell growth and proliferation,autophagy,cycle and metabolism.The TCGA database of breast cancer showed that the expression levels of AMPK subunit(α,β and γ)and its isomers in breast cancer tissues were lower than those in normal tissues.1,4-naphthoquinone compound ZSW-1-17 has novel structure,good stability and solubility,and has certain inhibitory effect on breast cancer cells,but the specific mechanism is not clear.Methods: First,MTT method and plate clone formation experiment were used to detect the inhibitory effect of ZSW-1-17 on triple-negative breast cancer cells.Cell apoptosis and cell cycle were detected by flow cytometry.The inhibitory effect of ZSW-1-17 on triple-negative breast cancer cells in vivo was verified by establishing a nude mouse xenograft model.Then,the transcriptome changes of breast cancer cells after drug treatment were analyzed by transcriptomics to find the possible action pathway of drugs.The results of transcriptomics were verified by q RT-PCR and Western Blot experiments.The molecular mechanism of ZSW-1-17anti-tumor activity was explored by constructing cell lines with knockout key genes in this pathway and calcium fluorescent probes.Results: ZSW-1-17 significantly inhibited the proliferation of multiple triple negative breast cancer cells,promoted the apoptosis of triple negative breast cancer cells,and had no significant effect on cell cycle.The results of transcriptome analysis suggested that ZSW-1-17 might play an anti-tumor role through AMPK signaling pathway.Western Blot detected that ZSW-1-17 up-regulated AMPK phosphorylation level in a concentration-dependent manner,and CRISPR-Cas9 technology was used to knock out AMPKα in MDA-MB-231 cells.The inhibitory effect of ZSW-1-17 on AMPKα-KO cell lines was significantly reduced.Calcium fluorescent probe assay showed that ZSW-1-17 could increase intracellular calcium concentration.The mouse xenograft model proved that ZSW-1-17 had a significant inhibitory effect on xenografts in vivo,and the mechanism was basically consistent with the cell level.Conclusion: ZSW-1-17 inhibits the proliferation,colony formation and apoptosis of triple negative breast cancer cells,and exerts its antitumor effect by activating AMPK.