Study On The Anti-breast Cancer Effect And Mechanism Of Metformin Hydrochloride

Background&Object:Breast cancer is a serious public problem affecting human health.Metformin hydrochloride has been used as a first-line drug for diabetes treatment,which has been used in clinical practice for decades.With the continuous improvement of scientific research level and the development of technology,more studies have been carried out on the hypoglycemic and other pharmacological activities of metformin hydrochloride,such as cardioprotection,improvement of blood lipids and possible anti-tumor effects.Pre-clinical studies have shown that metformin hydrochloride can affect the morbidity and mortality of cancer.Therefore,this research studied the pharmacological activity of metformin hydrochloride against breast cancer,and studied the effect of glucose on its'anti-tumor activity.This study can promote the clinical application or adjuvant therapy of metformin hydrochloride for breast cancer.Methods:?1?Study on effect and mechanism of metformin on breast cancer in mice:Forty Kunming mice were randomly divided into saline group?CT?and metformin hydrochloride group?MT?.The model of breast cancer in mice was established by inoculating 4T1 breast cancer cells.MT group mice were given metformin hydrochloride solution daily at a dose of 200 mg/kg.CT group mice were given the normal saline every day.The experimental period was 14 days.The mice were observed daily for general conditions,and the weight and tumor volume of mice were recorded daily.Metabolomics was used to study the metabolic reprogramming of plasma in mice induced by metformin hydrochloride.?2?1 MTT assay showed that after metformin hydrochloride was applied to breast cancer cells?MCF-7?for 24 h,the IC₅₀ value was 30.70±1.21 mM;after metformin hydrochloride was applied to breast cancer cells?MCF-7?in a series concentration of glucose?5.0 mM,7.5 mM,10.0 mM,12.5 mM,15.0 mM?media for24 h,the IC₅₀ values were 21.94±0.68 mM,24.88±0.55 mM,30.70±1.21 mM,31.10±1.39 mM and 93.39±5.01 mM,respectively.The results of apoptosis detected by flow cytometry showed that the apoptotic rate of MCF-7 cells was?7.40±0.85?%,which was significantly higher than that of the control group?P<0.05?.Moreover,the glucose concentration in the medium affected the apoptosis of metformin-induced cells,and the apoptotic rate was inversely proportional to the glucose concentration.The results of cell cycle detected by flow cytometry showed that MCF-7 cells were arrested in G1 phase after metformin hydrochloride treatment for 24 h,the results of the effect on metformin-induced cell cycle arrest by glucose concentration showed that the effect of cell cycle arrest was most pronounced at a glucose concentration of10.0 mM.Metabolomics analysis of the inhibitory effect of metformin hydrochloride on the proliferation of MCF-7 cells showed that after treatment with metformin hydrochloride for 24 hours,the content of citric acid,malic acid and glutamine in MCF-7 cells decreased significantly,indicating that the energy metabolism pathways such as TCA cycle and glutamate and glutamine metabolism were regulated in MCF-7 cells.Further study on the effect of glucose concentration on the inhibition proliferation of MCF-7 cells by metformin,it was found that low glucose further enhanced the regulation of metformin hydrochloride on TCA cycle.2 The results of ELISA in the detection of citrate synthase content in MCF-7 cells showed that the citrate synthase content in MCF-7 cells decreased in the normal glucose concentration?10.0 mM?environment after treated with metformin hydrochloride,while the citrate synthase content in MCF-7 cells increased in low glucose medium.?3?Mechanism analysis of the anti-breast cancer effect of metformin:Differentially expressed genes in breast cancer were screened by using bioinformatics tools,and it was analyzed by combined with the metabolomic study of metformin hydrochloride inhibiting breast cancer proliferation in vitro and in vivo,the mechanism of metformin hydrochloride against breast cancer was analyzed.Results:?1?The results of metformin on breast cancer in mice show that the volume of tumors in saline group increased from 310.6±20.8 mm³ to 411.3±40.8 mm³,and that in metformin hydrochloride group decreased from 373.5±22.3 mm³ to 185.8±29.4mm³?P<0.05?.Metabolomics results of plasma in mice bearing breast cancer tumor revealed that when compared with the control group,the main differential compounds were citric acid,glutamine,aldosterone,vitamin B6 and other metabolites.These differential compounds were involved in the tricarboxylic acid cycle,glutamate and glutamine metabolism and steroid hormone biosynthesis.?2?1 MTT assay showed that after metformin hydrochloride was applied to breast cancer cells?MCF-7?for 24 h,the IC₅₀ value was 30.70±1.21 mM;after metformin hydrochloride was applied to breast cancer cells?MCF-7?in a series concentration of glucose?5.0 mM,7.5 mM,10.0 mM,12.5 mM,15.0 mM?media for24 h,the IC₅₀ values were 21.94±0.68 mM,24.88±0.55 mM,30.70±1.21 mM,31.10±1.39 mM and 93.39±5.01 mM,respectively.The results of apoptosis detected by flow cytometry showed that the apoptotic rate of MCF-7 cells was?7.40±0.85?%,which was significantly higher than that of the control group?P<0.05?.Moreover,the glucose concentration in the medium affected the apoptosis of metformin-induced cells,and the apoptotic rate was inversely proportional to the glucose concentration.The results of cell cycle detected by flow cytometry showed that MCF-7 cells were arrested in G1 phase after metformin hydrochloride treatment for 24 h,the results of the effect on metformin-induced cell cycle arrest by glucose concentration showed that the effect of cell cycle arrest was most pronounced at a glucose concentration of10.0 mM.Metabolomics analysis of the effect of glucose concentration in the medium on the effect of metformin hydrochloride on MCF-7 cells showed that after treatment with metformin hydrochloride for 24 hours,the content of citric acid,malic acid and glutamine in MCF-7 cells decreased significantly,indicating that the energy metabolism pathways such as TCA cycle and glutamate and glutamine metabolism were regulated in MCF-7 cells.Further study on the effect of glucose concentration on the inhibition proliferation of MCF-7 cells by metformin,it was found that low glucose further enhanced the regulation of metformin hydrochloride on TCA cycle.2The results of ELISA assay for citrate synthase in MCF-7 cells showed that the content of citrate synthase in MCF-7 cells decreased in the normal glucose concentration?10.0 mM?environment after treated with metformin hydrochloride,while the citrate synthase content of MCF-7 cells increased in low glucose medium.?3?2336 differentially expressed genes in breast cancer were screen by using bioinformatics tools,and it was analyzed combined with the metabolomic results of tumor-bearing mice and breast cancer cells MCF-7 after metformin hydrochloride treated.It was found that metformin hydrochloride can change the TCA cycle,glutamic acid and glutamine metabolism.Conclusion:?1?Metformin hydrochloride has anti-breast cancer activity;?2?Metformin hydrochloride may inhibit the proliferation of breast cancer and induce apoptosis via regulating TCA cycle and glutamate and glutamine metabolism.