Role Of Glucose Metabolism Reprogramming In Silibinin-induced Breast Cancer Cell Autophagic Death

Background and Objectives:With the highest incidence among women,breast cancer has become a serious threat to women’s physical and mental health disorders.Although the comprehensive treatment and individual treatment of breast cancer are becoming more and more perfect,and the prognosis of early breast cancer is better.However,for advanced breast cancer,the prognosis of patients is poor.Postoperative adjuvant therapy has certain limitations and needs to be improved.For example,chemotherapy is prone to drug resistance,radiotherapy is likely to cause cardiopulmonary injury,and endocrine therapy has limited target patients.As a solid tumor,breast cancer cells follow the "Warburg effect".Breast cancer cells still give priority to the use of aerobic glycolysis under aerobic environment.This type of metabolism provides a large amount of energy and intermediate products for its biosynthesis,which in turn promotes tumor proliferation and migration.Studies have shown that fatal autophagy occurs when tumor cells are deficient in energy supply.Silibinin has been reported to induce glucose metabolism reprogramming and fatal autophagy of tumor cells in breast cancer.However,the effect of glucose metabolism reprogramming on autophagy of breast cells is not clear.Therefore,this study intends to construct breast cancer cells and animal models to observe the effect of silibinin on the growth of breast cancer.Then we will explore the effect of silibinin on the energy metabolism of breast cancer cells,and the possible mechanisms that mediate autophagy and cell death.Methods:(1)The effect of silibinin on the activity of breast cancer cells was detected by MTT and LDH methods.Glycolysis and energy production of breast cancer cells were detected by pyruvate,lactic acid and ATP kits,and the expression levels of glycolysis key enzymes HKⅡ,PFKP and PKM2 were detected by Western Blotting(WB)assay.The mortality of breast cancer cells was detected by LDH method supplemented with exogenous pyruvate.(2)The expression of autophagy marker protein LC3B and autophagy substrate P62 in breast cancer cells was detected by WB;the death rate of breast cancer cells was detected by LDH after adding autophagy inhibitor;the expression of autophagy-related proteins in breast cancer cells was detected after exogenous pyruvate supplementation.(3)The expression of BNIP3 protein in breast cancer cells was detected by WB;the death rate of breast cancer cells was detected by LDH after BNIP3 was knocked out by si RNA.The expression of BNIP3 protein in breast cancer cells was detected after adding autophagy inhibitors 3-MA and Baf-A1.(4)Breast cancer transplantation model was established in nude mice.Silibinin was injected intraperitoneally and the changes of tumor volume were observed.The expressions of glycolysis,autophagy-related proteins and BNIP3 in tumor tissues were detected by WB.Results:(1)Silibinin down-regulated the viability of breast cancer cells and increased the mortality of breast cancer cells;silibinin down-regulated the expression of key enzymes of glycolysis and down-regulated the products(pyruvate)and end products(lactic acid)in glycolysis of breast cancer cells;supplementation of exogenous pyruvate inhibited the increase of mortality of breast cancer cells induced by silibinin.(2)Silibinin upregulated LC3B protein and down-regulated the expression of P62 protein in breast cancer cells;autophagy inhibitor inhibited the death rate of breast cancer cells;supplementation of exogenous pyruvate inhibited the increase of LC3B protein and decrease of P62 protein.(3)Silibinin caused the increase of BNIP3 protein in breast cancer cells;BNIP3 knockout cells decreased the death rate caused by silibinin.With the addition of autophagy inhibitor,the accumulation of intracellular BNIP3 was inhibited.(4)In vivo experiment,the expression of related proteins in breast cancer treated with silibinin was significantly smaller than that in the control group,and the expression level of related proteins in tumor tissue was consistent with that of cell experiment.Conclusions:(1)Silibinin can induce breast cancer cell death and glucose metabolism reprogramming.(2)Silibinin mediated autophagic death by inducing glucose metabolism reprogramming in breast cancer cells.(3)Silibinin induced BNIP3 accumulation via autophagy,which led to breast cancer cell death.