Comparative Study On The The Effects Of Nano-Doxorubicin And Doxorubicin On The Cross-Talk Between Triple-Negative Breast Cancer Cells And Plateletes

Background: Breast cancer is a malignant tumor with the highest morbidity and mortality among women in the world,and it is a serious threat to women’s health.Triple-negative breast cancer(TNBC)is a type of breast cancer whose expression of estrogen receptor,progesterone receptor and human epidermal growth factor receptor2 are all negative,due to the lack of specific receptors on its surface.At present,there is a lack of effective molecular targeted therapy,and chemotherapy is still the main clinical treatment method.Doxorubicin(DOX)is a first-line chemotherapeutic drug,the main anti-tumor mechanism of action is to damage tumor cell DNA.DOX has problems such as high toxicity and tumor resistance,which is a huge obstacle to anti-tumor efficacy.Emerging nanotechnology and nanomaterials provide new strategies and means to overcome these problems of DOX.At present,people have prepared a variety of nanoparticles in the laboratory for the targeted delivery of DOX to tumors,and they have all been reported to have higher efficacy and lower systemic toxicity than free DOX.We also synthesized " Nano-DOX " in our previous research for the targeted delivery of DOX to tumors.But what are the similarities and differences between the effect of DOX in the form of nano-delivery on tumor cells and its mechanism with DOX? At the same time,the occurrence and development of tumors are inseparable from the tumor microenvironment(TME).TME refers to the local biological environment in which tumor cells are located.It is composed of stromal cells,cytokines,and extracellular matrix.They interact closely with tumor cells and continue to affect tumor progression.Platelets are an important and special component of TME,which can interact with tumor cells through soluble factors such as Thromboxane A2(TXA2).So what are the similarities and differences between DOX in the form of nano-delivery on the tumor microenvironment,especially the "tumor cell-platelet interaction" ? The answers to the above two questions are of great significance to the nanotherapy of tumors,especially TNBC,but there are currently few research reports.In this topic,we will compare Nano-DOX and free DOX to discuss the above two issues.Objective: Taking mouse TNBC as the research object,1.To investigate and compare the effects of Nano-DOX and free DOX on the " TXA2-mediated TNBC cell-platelet interaction ";2.To explore the mechanism of tumor cell DNA damage as a starting point,for the different effects of DOX and free DOX.Methods: Using mouse TNBC cells(4T1)and mouse-derived platelets as research models,in vitro cell monoculture and co-culture experiments were carried out.Using q RT-PCR,Western-blotting,ELISA,FACS and immunofluorescence staining,DNA ladder and comet assay and other technical means,analyze the viability of 4T1 cells,DNA damage,metastasis ability,TAX2 synthesis and release,and TXA2 receptor expression,Detect the activation of platelets and the release of TAX2.Establish tumor-bearing mice,carry out in vivo experiments,and verify the observation results of in vitro cell experiments through immunohistochemistry and other methods.Results: 1.DOX significantly stimulates cyclooxygenase-1(COX-1),cyclooxygenase-1(COX-2)and thromboxin synthase(TBXAS)in 4T1 cells,TXA2receptor(thromboxane A2 receptor,TXA2-R)expression and TXA2 release;DOX significantly promotes 4T1 cell activation of co-cultured platelets,the latter highly promotes the migration of the former(4T1 cells)by releasing TXA2 and promotes its down-regulation E-cadherin(E-cad)simultaneously up-regulates the expression of matrix metalloproteinase-2(MMP2)and heat shock protein 90(HSP90).2.DOX significantly damages the DNA of 4T1 cells,up-regulates the stimulator of interferon gene(STING),phosphorylated telangiectasia-mutated(ataxia-telangiectasia-mutated,P-ATM),phosphorylation group in tumor cells The expression of protein H2AX(γH2AX)activates ERK and NF-κB signaling;the activation of ERK causes the up-regulation of COX-1 and COX-2,and the activation of NF-κBE also participates in up-regulating the expression of COX-1.3.Compared with DOX,Nano-DOX causes 4T1 cells to cause DNA damage,NF-κB and ERK signal activation,and the upregulation of COX-1,COX-2,TBXAS and TXA2-R is significantly weaker than DOX,Nano-DOX The activated 4T1 cells on platelets are also far inferior to DOX.Conclusion: There is an interaction mediated by TXA2/TXA2-R between 4T1 cells and platelets,which can promote the migration of 4T1 cells.Free DOX can significantly promote the interaction between 4T1 cells and platelets mediated by TXA2/TXA2-R,while this effect of Nano-DOX is significantly weaker than that of free DOX.The effect of DOX stems from its serious damage to the DNA of 4T1 cells,and the DNA damage of tumor cells caused by Nano-DOX is significantly weaker than that of DOX.