Therapeutic Effect And Mechanism Of PDE4 Inhibitor ZL-n-91 On Triple-negative Breast Cancer

Triple negative breast cancer（TNBC）is the most malignant subtype of breast cancer,and the development of effective treatment strategies is the focus of current research.Studies have shown that phosphodiesterase 4（PDE4）is closely related to the occurrence and development of TNBC and suggest that the specific inhibition of PDE4 activity may have a certain therapeutic potential for TNBC.Compared with other PDE4 inhibitors on the market,the novel phosphodiesterase inhibitor 4 ZL-n-91 is highly selective against PDE4 D and PDE4 B,which can avoid serious side effects such as nausea and vomiting.It has been demonstrated to have the characteristics of neuroprotective activity.Besides,it has been shown excellent application prospects in acute lung injury and chronic obstructive pulmonary disease.However,its therapeutic effect and molecular mechanism on TNBC are still need to be further studied.In this study,the highly selective PDE4 inhibitor ZL-n-91 was selected to explore its inhibitory effect and mechanism on the growth of TNBC through in vivo and in vitro experiments,which provided a theoretical basis for the treatment of TNBC by the phosphodiesterase 4 inhibitor ZL-n-91.Objective: To investigate the therapeutic effect and mechanism of a novel phosphodiesterase 4 inhibitor ZL-n-91 on TNBC cells.Methods:（1）The effect of different concentrations of ZL-n-91 on the proliferation of TNBC cells in vitro was investigated by CCK-8 proliferation assay.（2）The effects of different concentrations of ZL-n-91 on the clonogenesis ability of TNBC cells were investigated by single-cell clonogenesis experiment.（3）Flow cytometry and Western blot were used to detect the effects of different concentrations of ZL-n-91 on cell cycle and the expression of cyclin related protein in TNBC cells.（4）Flow cytometry was used to detect the effects of different concentrations of ZL-n-91 on apoptosis of TNBC cells.（5）Transcriptome sequencing and Western blot were used to explore the molecular mechanism of the therapeutic effect of different concentrations of ZL-n-91 on TNBC cells.（6）MDA-MB-231 cells were used to construct subcutaneous transplanted tumor model in nude mice,and the therapeutic effect of ZL-n-91 on TNBC was further explored by immunohistochemical staining and other techniques.Results:（1）ZL-n-91 inhibited the proliferation of MDA-MB-231 and BT-549 cells in a dose dependent manner.（2）ZL-n-91 inhibited the clonogenesis of MDA-MB-231 and BT-549 cells in a dose-dependent manner.（3）ZL-n-91 significantly arrested the cell cycle of MDA-MB-231 at G0/G1 phase.The expression of CDK2,CDK4,Cyclin D and PCNA were significantly down-regulated.In addition,ZL-n-91 significantly arrested the cell cycle of BT-549 in G2/M phase by regulating Cyclin B and CDC2.（4）ZL-n-91 induced apoptosis of MDA-MB-231 and BT-549 cells in a dose-dependent manner.（5）ZL-n-91 significantly inhibited the DNA repair and replication ability of MDA-MB-231 cells,and aggravated DNA damage of MDA-MB-231 and BT-549 cells.（6）ZL-n-91 significantly inhibited the growth of subcutaneous transplanted tumor of MDA-MB-231 cells,down-regulated the expression of Ki67 in tumor cells and induced DNA damage of tumor cells in vivo.Conclusion: The novel PDE4 inhibitor ZL-n-91 has a strong anti-tumor effect on TNBC cells,which can promote DNA damage of TNBC cells,block the cell cycle and promote the apoptosis of TNBC cells,and is expected to provide a new breakthrough for the targeted treatment of TNBC.