The Function And Mechanism Of DZ-514 Induces Methuosis In Triple Negative Breast Cancer

Breast cancer is the most common cancer among women,and its age of onset is gradually getting younger,which seriously endangers the physical and mental health of women around the world.Triple Negative Breast Cancer(TNBC)is currently a pathological classification with a poor prognosis in breast cancer treatment due to its high heterogeneity,strong aggressiveness and high metastatic.F inding new therapeutic targets or new effective small-molecule inhibitors is of great significance for improving the treatment of TNBC.Methuosis is a new type of non-apoptotic cell death characterized by the accumulation of cytoplasmic acuoles.The discovery of new small molecule inducers that induce methuosis could provide more options for the treatment of apoptosis-resistant TNBC.In this study,we synthesized a series of N-phenyl-4-pyrimidine diamine derivatives based on the pyrimidine skeleton to screen for specific BCL6 small molecule inhibitors that effectively kill TNBC.We performed SRB assay and found that DZ-514 is one of the compounds with the most killing potential.This study explored the way in which DZ-514 induces the death of TNBC cells and further elucidates its mechanismOur study found that the compound DZ-514 can reduce cell viability,blocking cells in the G2/M phase.The results of Annexin/PI double-stained labeled apoptosis experiments showed that DZ-514 did not induce significant apoptosis in cells.Neither the pan-Caspase inhibitor Z-VAD-FMK nor the necrosis inhibitor Nec-1 blocked DZ514-induced cell death.Our further research found that compound DZ-514 can induce vacuolarization of breast cancer cytoplasm and has a broad spectrum.We explored the properties of cytoplasmic vacuoles induced by DZ-514 using the extracellular fluid tracer Lucifer Yellow in combination with the macrocytosis inhibitor Baf A1 and the autophagy inhibitor HCQ.Our results show that DZ-514 promotes cell uptake of Lucifer Yellow;Baf A1 can block the induction of cavitation by DZ-514;HCQ cannot block the phenomenon of cytoplasmic vacuolation induced by DZ-514.In addition,we,performed immunofluorescence and Western blotting assay and found that DZ-514 promotes the expression of endosomes LAMP1 and Rab7.The above experimental results show that the death of DZ-514-induced cells is methuosis.In the HCC1806 xenograft mouse model,we found that DZ-514 significantly inhibited the growth of tumor through methuosis and showed good biosafety in vivo.Mechanistically,we found that DZ-514 did not depend on BCL6 to exert a cancer suppressor effect.The ERK/JNK signaling pathway is activated during DZ-514induced methuosis,but inhibition of this pathway does not resuce the methuosis by DZ514.Further,we found that DZ-514 induced methuosis of triple negative breast cancer cells in part by ROS-MKK4-p38 axis.In conclusion,our study demonstrated that compound DZ-514 is a novel small molecule inducer of methuosis,and firstly reported that p38 is involved in the methuosis.DZ-514 has the potential to be a new lead compound to develop new drugs and provide a new therapeutic strategy for apoptotic resistant TNBC cells.