Study On The Mechanism Of Combretastatin(A4) Analogue Activating CGAS/STING Pathway In Breast Cancer Cell Proliferation By Causing DNA Damage

Objective:Breast Cancer is the most commonly diagnosed malignancy in women worldwide.The traditional treatment of breast cancer is easy to produce endocrine resistance due to poor tolerance,poor single drug treatment effect,anthracycline drugs used have certain cardiac toxicity,and seriously damage the immune function of patients,which has been difficult to significantly improve the survival prognosis of breast cancer patients,especially those who have occult micrometastasis and tumor cells with therapeutic resistance.Microtube targeting agents(MTA)blocking mitosis is one of the earliest strategies to interfere with the proliferation of cancer cells.At present,it is found that regulating cell death by inducing DNA damage is also one of the anti-tumor effects of some microtubule inhibitors such as paclitaxel and colchicine.Many tubulin inhibitors have been proved to interfere with the mitotic process of cells by inhibiting or promoting the polymerization of tubulin in tumor cells,induce mitotic arrest,and cause the damaged DNA to transfer into the cytoplasm in the form of micronucleus,and play the role of anti-tumor immunity by being recognized by the cytoplasmic DNA receptor cGAS.Developing new microtubule inhibitors,inducing genomic DNA damage in tumor cells and exploring the molecular mechanism of its occurrence have become the focus of anti-tumor drug research in the future.Therefore,the design of drugs targeted to promote the release of genomic DNA from tumor cells,enhance the immunogenicity of tumor cells,and amplify the anti-tumor effect will help to better guide the development of targeted drugs or re-engineer and utilize drugs with known mechanisms of action,so as to play a greater role in tumor research and clinical treatment.Our research group designed and synthesized a series of new small molecular compounds targeting tubulin.Based on the previous activity tests,we selected the compound IJ-1-79 with the best anti-tumor activity as the research object to explore the molecular mechanism of this compound against breast cancer cells in vivo and in vitro.Methods:The inhibitory activity of compound IJ-1-79 on the proliferation of breast cancer cells was detected by MTT method and clone formation test,and the optimal concentration and time were selected;The effect of IJ-1-79 on cell cycle and apoptosis was investigated by flow cytometry;The effect of IJ-1-79 on cell migration was detected by scratch test and Transwell.The inhibition of compound IJ-1-79 on tubulin polymerization was detected by molecular docking,immunofluorescence assay and Western blot;Comet assay,immunofluorescence staining and Western Blot were used to detect the effect of IJ-1-79 on DNA damage in breast cancer cells.The antitumor effect of IJ-1-79 in vivo was tested by xenograft tumor model.qRT-PCR and Western Blot were used to detect the effect of IJ-1-79 on the expression level of key mRNA of cGAS/STING signal pathway.Results:(1)IJ-1-79 has inhibitory effect on the growth of various breast cancer cells,among which,it is more toxic to breast cancer cells MDA-MB-468 and MDAMB-231,and its effect is proportional to the action time and dose gradient.(2IJ-1-79 can effectively inhibit the proliferation and migration of breast cancer cells,block the cell cycle in G2/M phase,and effectively induce apoptosis.(3)IJ-1-79 has a significant inhibitory effect on tubulin polymerization and can significantly reduce the expression level of tubulin.In addition,IJ-1-79 can cause DNA damage in breast cancer cells and DNA damage markers γ The expression level of H2AX increased.(4)IJ-1-79 has obvious anti-tumor effect in vivo,and its effect is slightly better than the positive drug CA-4.(5)IJ-1-79 can significantly up-regulate the level of expressionc GAS/STING signaling pathway and its downstream molecule NF-κB、TNF α.Conclusion:In the early stage of the design of this project,structural modification was carried out based on CA-4 to synthesize and screen out the compound IJ-1-79 with the best anti-tumor activity.In vitro pharmacodynamics experiments showed that IJ-1-79 could effectively inhibit the proliferation and migration of breast cancer cells,and induce tumor cell cycle arrest and apoptosis.In vivo pharmacodynamics experiments showed that IJ-1-79 had good antitumor activity in vivo.The mechanism study shows that IJ-1-79 can inhibit the polymerization of tubulin in tumor cells,interfere with mitosis,induce cell DNA damage,and active cGAS/STING signal pathway to play an anti-tumor role.