Mechanism Of Targeting EP2 Tanshinone ?A Nanoparticles To Inhibit Invasion And Metastasis Of Colorectal Cancer

Objective : First,the research team established tanshinone ?A nanoparticles(EP2-TS?A-NP)with good water solubility and active targeting of EP2 receptors.Then,the characterization of EP2-TS?A-NP was examined,and its effect on colorectal cancer Lo Vo cells and its therapeutic effect on subcutaneous xenograft models in nude mice were examined.Therefore,the mechanism of EP2-TS?A-NP targeting EP2 receptor to inhibit invasion and metastasis of colorectal cancer was discussed.Method:1.Immunohistochemistry was used to detect the specific expression of EP2 receptor in colorectal cancer tissues,paracancerous tissues,lung metastasis tissues and liver metastasis cells.The colorectal cancer EP2 receptor high expression cell line Lo Vo cells were screened by Western Blot.EP2 receptor agonist(Butaprost),inhibitor(PF-04418948),PGE2 were applied to colorectal cancer Lo Vo cells,and the expression of EP2,?-arrestin1(total),?-arrestin1(nuclear)and MMP9 protein was detected by Werstern blot,to investigate the regulation of ?-arrestin1 by EP2 receptors.2.The cytotoxicity of tanshinone ?A(TS?A)on colorectal cancer Lo Vo cells was observed by CCK-8 method.The effect of TS?A on the migration ability of colorectal cancer Lo Vo cells was determined by scratch test.3,through the emulsion solvent evaporation method,TS?A can be made into EP2-TS?A-NP(tanshinone ?A nanoparticles targeting EP2 receptor).The characterization and analysis of EP2-TS?A-NP were performed by electron microscopy and zeta potential.4.In vitro anti-tumor experiments: CCK-8 was used to compare the cytotoxicity of EP2-TS?A-NP and TS?A on colorectal cancer Lo Vo cells.The targeting effect of EP2-TS?A-NP on colorectal cancer Lo Vo cells was determined by fluorescence analysis.The effects of EP2-TS?A-NP and TS?A on the migration of colorectal cancer Lo Vo cells were compared using a scratch test.5.Colorectal cancer Lo Vo cells were divided into 6 groups: control group,TS?A group,TS?A nanoparticle group(TS?A-NP group),EP2-TS?A-NP group,empty nanoparticle group(NP group),positive control group(EP2 receptor inhibitor PF-04418948 group,abbreviated EP2 below),Western Blot detection of EP2-TS?A-NP for human intestinal cancer Lo Vo cells EP2,?-arrestin1(total),?-arrestin1(nuclear),MMP9 protein Regulate the role of EP2-TS?A-NP in inhibiting the invasion and metastasis of colorectal cancer.6.Anti-tumor experiments in vivo: A nude mouse model of subcutaneous transplantation was established and randomly divided into 6 groups: tail control group: control group;TS?A group;TS?A-NP group;EP2-TS?A-NP group;NP group;EP2 group.The drug was administered every other day,and the tumor growth,tumor necrosis and survival time of each group were compared.The expression of EP2,?-arrestin1(total),?-arrestin1(nuclear)and MMP9 protein in tumor tissues was detected,to explore the effect of EP2-TS?A-NP on targeted therapy of colorectal cancer.Results:The EP2 receptor can specifically express on the cell membrane of colorectal cancer cells and regulate the EP2 receptor.It is found that the EP2 receptor may induce the invasion and metastasis of colorectal cancer cells by promoting the entry of ?-arestin1 into the nucleus.The cytotoxicity experiment showed that tanshinone ?A was cytotoxic,and the scratch test showed that TS?A significantly inhibited the migration ability of intestinal cancer Lo Vo cells,thereby inhibiting the invasion and metastasis of intestinal cancer Lo Vo cells.The solubility of EP2-TS?A-NP was successfully improved,and the particle size distribution of EP2-TS?A-NP was uniform under electron microscope.The particle size was about 180 nm±9.8 nm,and the morphology was spherical.Zeta potential analyzer analyzed EP2-The zeta potential of TS?A-NP is 30 m V,indicating that the stability of EP2-TS?A-NP is good.Using fluorescence analysis technology,EP2-TS?A-NP has a good effect of targeting EP2.In vitro,EP2-TS?A-NP is more cytotoxic than TS?A and can better inhibit the invasion and metastasis of colorectal cancer cells.In vivo experiments,EP2-TS?A-NP was significantly effective by comparing the volume,tumor weight,and tumor survival of subcutaneous xenograft tumors in each group.Western Blot protein assay showed that compared with other groups,EP2-TS?A-NP may regulate ?-arrestin1 and down-regulate MMP9 protein expression level by targeting EP2 receptor.Conclusion:Compared with TS?A,EP2-TS?A-NP has good solubility,targeting and sustained release,has better therapeutic effect on colorectal cancer,and prolongs the survival of tumor-bearing nude mice.The mechanism by which EP2-TS?A-NP inhibits invasion and metastasis of colorectal cancer may inhibit the invasion of metastasis of colorectal cancer by targeting the EP2 receptor,inhibiting the entry of ?-arrestin1 into the nucleus,and reducing the expression of MMP9 protein.