The Effect Of 2-deoxyglucose-enhancing Sonodynamic Therapy On Breast Cancer And Its Mechanism

Background and purposeAs a most common malignancies,severely breast cancer threatened women's health.Its high recurrence and metastasis could not be satisfied with the traditionary therapies,charging by the abnormal glucolytic metabolism which was preferly adopted by tumour in comparison with normal tissues.Thereout,2-deoxyglucose(2DG)was orientatedly proposed to inhibit the rate-limiting enzyme of glycolytic for tumor therapy.While,studies had shown that that alone inhibition cannot attain a significant anti-tumor effect and its side-effect also limit clinical application.Sonodynamic therapy(SDT)is a promising cancer therapy.And,it has been primarily confirmed that mitochondria is the target of SDT.Suggestively,a dual-targeting inhibition of energetic will be a more available approach accompanying drug dosage and side effects reduction,further achieve synergetic efficient in breast cancer remedy.Methods and resultsPart 1:Effect of 2DG combined with DVDMS-SDT in breast cancer cellsMTT,Caclein AM/PI staining joint with fluorescent microscopy,Annexin V-FITC/PI apoptosis analysis and Western blot detection,DCFH-DA and Rho123 staining combined with flow cytometry were applied to determine cell survival,apoptosis,intracellular reactive oxygen species and the change of mitochondrial membrane potential.The results showed that 2DG combined DVDMS-SDT could significantly inhibit the survival of MBA-MB-231,MCF-7 and 4T1 breast cancer cells;however,there is no obvious effect on human vascular endothelial cell HUVEC,which preliminarily proves the safety and effect of this experiment.Further Annexin V-FITC/PI apoptosis detection and Western blot analysis found that combined treatment could significantly increase the expression of p-Caspase 3 and induce apoptosis of 4T1 cells.Besides,DCFH-DA and Rho123 stain presented abundant reactive oxygen production which further caused the damage of mitochondrial membrane potential,suggesting that ROS plays an important role during the process.Part 2:2DG combined DVDMS-SDT affected 4T1 cell proliferation and migrationBasing on cloning,cell adhesion,wound healing assay and transwell inquiried into the combined theraputic effect on cell increment,adhesion,migration and invasion;scanning electron microscope and flow cytometry were used to analyze the cell surface structure and cycle.The results showed that 2DG combined DVDMS-SDT could significatly inhibit cell proliferation.Furthermore,cellular adhesion,migration and invasion were also confined.In addition,the scanning electron microscopy showed that the 4T1 cells were severely deformed after 2DG combined with DVDMS-SDT,and the microvilli structure almost disappeared.The cell cycle progression has undergone significant changes.It is speculated that the microstructure destruction of the cell surface and the blocking of cell cycle are involved in inhibiting the proliferation and adhesion,migration and invasion of 4T1 cells,which may affect the metastasis of tumor cells in vivo.Part 3:The influence of 2DG combined DVDMS-SDT on 4T1 cellular metabolismFirstly,the localization of the acoustic sensitizer DVDMS in 4T1 breast cancer cells was analyzed.Seahorse Bioscience XFp Extracellular Flux Analyzer,ATP detection kit with chemiluminescence detector,immunofluorescence,laser confocal microscope and Western blot were applied for exploring cellular oxidative phosphorylation,ATP generation,HK2 subcellular localization and key enzymes expression of glycolysis.Results showed that DVDMS is mainly located in the mitochondria.Oxidative phosphorylation and ATP generation was partly suppressed in alone 2DG or DVDMS-SDT groups,which hindering a cooperation treatment of 2DG united DVDMS-SDT might express obvious inhibition.Hexokinasse II(HK2)is mainly located in the outer membrane of mitochondria and further to function.Laser confocal microscope results presented that the bondage of HK2 with mitochondria was significantly inhibited followed the combination therapy,thus controlling glycolysis.Otherwise,the expression of key-enzymes in glycolysis were also down-regulated.Part 4:The function of 2DG united with DVDMS-SDT for 4T1 transplanted tumorIn this study,a model of 4T1 tumor-bearing mice was established and its response was investigated suffering the combined treatment by using HE staining and lung nodules observation,TUNEL apoptosis detection,immunohistochemical analysis and safety evaluation.The results showed that 2DG combined DVDMS-SDT can significantly reduce the expression of PCNA in tumor tissues,cause tumor tissue injury and apoptosis,and then inhibit the 4T1 transplanted tumor growth.In addition,immunohistochemical analysis showed that the expression of Glutl and HK2 protein was significantly down-regulated after combined treatment,inhibiting the production capacity of tumor glycolysis.The number of pulmonary nodules in transplanted tumor mice showed that 2DG combined with DVDMS-SDT significantly inhibited tumor metastasis.In addition,it was found that the treatment model had relatively high safety by the analysis of the contents of GOP,GPT and urea nitrogen and the histological morphology of the main organs.ConclusionGenerally,this topic for 2DG joint DVDMS-SDT cytotoxic effect of different breast cancer,4T1 inhibition of cell proliferation and migration,the changes of energy metabolism and antitumor effect of 4T1 transplantation tumors and explore the treatment of several aspects,such as safety,the result shows that 2DG joint DVDMS-SDT is a highly efficient,safe,has potential clinical application mode of anti-tumor,extremely direction for clinical treatment of breast cancer provides a new treatment and experimental basis.