Mechanism Of The Combination Of Hydroxychloroquine And 2-Deoxyglucose Inhibiting The Proliferation Of Breast Cancer Cells

Canine mammary tumor is the most common tumor in canine tumors,and considered as an appropriate model for studying human breast cancer.Triple-negative breast cancer(TNBC),one of the most aggressive subtype of breast cancer,grows fast and may have spread when detected,compared to other breast cancer subtypes.Methods based on regulate cell metabolism are the promising therapeutic strategy to combat TNBC.2-Deoxyglucose(2-DG)can be used in anti-tumor research by inhibiting glycolysis and promoting the endoplasmic reticulum stress(ERS)pathway.However,its clinical use is limited by doseside effects,especially the protective autophagy induced by it promotes cell survival to a certain extent.Studies have shown that 2-DG combined with autophagy blockers may be an effective treatment strategy.Hydroxychloroquine(HCQ),an FDA-approved autophagy inhibiting drug,which has been administered as auxiliary drugs in oncology clinical trials.Therefore,the combination of 2-DG and HCQ may have a better inhibitory effect on cancer cells,while the biological effects of 2-DG combined with HCQ in the treatment of breast cancer still need to be further studied.Based on this,this study used canine breast cancer as a mammary tumor model to explore the effect of the combination of 2-DG and HCQ in breast cancer,and explore its biological mechanism,hoping to provide a new strategy for breast cancer treatment.In this study,4T1(mouse origin)and CMT-7364(canine origin),both of which belong to TNBC cells,were used as mammary tumor cell models for in vitro experiments.CCK-8,Wound-Healing migration assay,flow cytometry,Western blot and immunofluorescence staining tests were used to verify the effects of the drug on the multiplication,migration and apoptosis of TNBC cells.Detect the changes of organelles in TNBC cells after drug treatment by transmission electron microscopy technology.The effects of drugs on the ERS pathway and autophagy pathway were verified by immunofluorescence staining and western blot.In order to verify the therapeutic effect of the drug in vivo,BALB/c mice was used to establish a orthotopic xenograft model with 4T1 cells.On the 12 th day after the successful modeling,the mice with obvious orthotopic tumors in the abdomen were randomly divided into 5 groups(5 mice per group).Mice in 5 groups were treated with normal saline,HCQ(50 mg/Kg),2-DG(100 mg/kg),2-DG(50 mg/Kg)+ HCQ(50 mg/Kg),2-DG(100 mg/kg)+ HCQ(50 mg/Kg),respectively.During the drug test,each group of drugs were injected intraperitoneally every 2 days,and the mice body weight and tumor volume were recorded at the same time.Finally,mice were euthanized after 25 days of continuous administration,tumors were harvested and weighed,and major vital organs from each group were harvested for H&E staining to analyze the inhibitory effect of the drugs on the proliferation and metastasis of TNBC cells in vivo.The results of 4T1 and CMT-7364 cells in vitro showed that HCQ combined with 2-DG can further inhibit the viability and migration and induce apoptosis of breast tumor cells,compared with other individual drugs.The combination of 2-DG and HCQ can significantly reduce transplanted tumor size and tumor cell metastasis of lung and liver in vivo.The test results in vitro and in vivo both showed that compared with the group of 100 mg/Kg 2-DG treated alone,50 mg/Kg 2-DG combined with HCQ could achieve a similar tumor suppressor effect,significantly reducing the effective dose of 2-DG.Further,the mechanism of action of the combination of 2-DG and HCQ was analyzed at the cellular level.HCQ suppressed autolysosome formation and terminated the autophagy process induced by 2-DG-mediated ERS,resulting in the continuous accumulation of misfolded proteins in endoplasmic reticulum which generated sustained ERS through PERK-e IF2α-ATF-4-CHOP axis,and triggered the transformation from survival process to cell death.In summary,the combined use of HCQ and 2-DG can inhibit autophagy,produce continuous ERS,and induce the increase of downstream pro-apoptotic proteins and the decrease of anti-apoptotic proteins,thus playing an anticancer role.The effective concentration of 2-DG can be effectively reduced by the addition of HCQ,thereby reducing the side effects of high-dose 2-DG.Our research reinforced the research interest of metabolic disruptors in triple-negative breast cancer and emphasized the potential of the combination of 2-DG and HCQ as an anticancerous treatment.