Studies On The Induction Of Autophagy And Anti-tumor Synergism In Breast Cancer Cells By Dual Blocking Of Estrogen And Energy

Background:Breast cancer has become the newest morbidity and mortality among female malignancies worldwide.Clinically,endocrine therapy has a good therapeutic effect on hormone receptor-positive breast cancer patients,but some breast cancer patients still develop drug resistance during the treatment process.How to improve the sensitivity of endocrine therapy and reverse the problem of resistance is one of the hotspots of endocrine therapy for breast cancer.Tumor cells generate energy mainly by aerobic glycolysis by consuming a large amount of glucose than normal tissue cells.Studies have shown that estrogen restriction can induce autophagy in breast cancer cells,and this manifestation is similar to the cell death signaling pathway activated by tumor cell energy restriction;a planty of articles also demonstrated that aerobic glycolysis and Gluts overexpression are related to correlation with drug resistance in malignant tumors.Object: 1.To investigate the effect of estrogen inhibition on the energy metabolism of breast cancer cells.2.To explore the effects of estrogen and energy block on proliferation,apoptosis and autophagy in breast cancer cells.3.To explore the antitumor synergistic effect of BAY-876 combined with TAM in vivo.Methods: Tamoxifen was applied to breast cancer cells MCF-7 and T-47 D cells in a concentration gradient to detect the inhibition of proliferation of the two cells at 24 h,48h,and 72 h.Tamoxifen was used as a single agent to treat the two cells.Differences in post glucose consumption rate,ATP level,and lactic acid production.When the expression of Glut1 was knocked down,tamoxifen was applied to two breast cancer cells separately to observe the sensitivity of breast cancer cells to tamoxifen.Based on the IC50 of tamoxifen monotherapy and IC50 of BAY-876 monotherapy for 24 h,the low-toxicity BAY-876 and tamoxifen drug concentration were selected to use,and the breast cancer cells MCF-7 and T-47 D were 10 ?M.Tamoxifen monotherapy,20?M BAY-876 monotherapy,10?M Tamoxifen and 20 ?M BAY-876 combined treatment alone for 24 h,compared with the negative drug-free treatment group,the cell line apoptosis rate and mitochondrial membrane potential Differences in levels;After 48 hours of treatment,the glucose consumption rate,mitochondrial membrane potential level,and cell submicrostructure of the cell line were different from those of the negative drug-free treatment group;after 6 hours of treatment,the cell lines were compared with the negative drug-free treatment group.The differences in ATP levels and ROS levels;and the expression of PI3K/AKT/m TOR signaling pathway,apoptosis,and autophagy-related proteins in the above two cells and 48 hours of treatment time were analyzed.A nude mouse xenograft model was also established to test the antitumor effect of BAY-876 combined with tamoxifen in vivo.Results: 1.Effect of TAM on the energy metabolism of breast cancer cells Inhibition of breast cancer cell MCF-7 and T-47 D proliferation with different concentrations of TAM(0,2.5,5,10,20,40?mol/L)at different action time(24h,48 h,72h)The effect increased with increasing concentration and time;tamoxifen(0,2.5,5,10?mol/L)at a low concentration gradient when knocking down Glut1 expression significantly inhibited cell proliferation for 24 h.Low doses of tamoxifen increase the glucose consumption of breast cancer cells,but 5,10 ?mol/L TAM does not show a significant effect on glucose consumption of cancer cells;and tamoxifen monotherapy Breast cancer cells significantly increased the amount of lactic acid produced by the cells.2.BAY-876 enhances the proliferation inhibition effect of TAM on breast cancer cells Different concentrations of BAY-876(0,5,10,20,40,80 ?mol/L)were used to treat breast cancer cell MCF-7 and T-47 D at different time(24h,48 h,72h).The proliferation inhibitory effect increases with increasing concentration and time.According to the IC50 of BAY-876 single drug and IC50 of TAM single drug,a low concentration of BAY-876 20 ?M and TAM 10 ?M were selected to act on two breast cancer cells,and BAY-876 combined with TAM inhibited cell proliferation.The effect was further enhanced,and the expression of Ki67 and PCNA in tumor tissues was significantly reduced after the combined administration.3.Effect of BAY-876 enhanced TAM on breast cancer cells mortality Electron microscopy results showed that after using BAY-876,TAM,BAY-876& TAM on breast cancer cells MCF-7 and T-47 D,respectively,compared with the BAY-876 and TAM treatment groups,the BAY-876 combined with the TAM group Apoptotic morphological features such as obvious shrinkage of the nucleus,shrinkage of the cell membrane,and fragmentation of the nucleus are also apparent.The experimental results of Annexin V-FITC method showed that BAY-876 combined with TAM significantly induced breast cancer cell MCF-7 and T-47 D mortality.Western blot results also confirmed this result.Compared with the single administration group,increased the expression of pro-apoptotic proteins Bax and cleaved PARP in the BAY-876 & TAM treatment group,and reduced significantly the expression of the anti-apoptotic protein Bcl-2.4.Effect of BAY-876 combined with TAM on energy metabolism in breastv cancer cells MCF-7 and T-47 D Compared with BAY-876 and TAM single drugs,after BAY-876 combined with TAM acts on breast cancer cells MCF-7 and T-47 D,the cell's glucose uptake is significantly reduced,which further decreases the intracellular ATP level and reducing lactic acid production.At the same time gradually increasing cell ROS levels.The results of JC-1 experiment showed that the intracellular mitochondrial membrane potential level showed a downward trend.It also shows that BAY-876 enhances TAM-induced early apoptosis of breast cancer cells MCF-7 and T-47 D.5.BAY-876 enhances TAM-induced autophagy in breast cancer cells MCF-7 and T-47 D BAY-876,TAM,BAY-876&TAM acted on breast cancer cells MCF-7 and T-47 D after 48 h,compared with the blank group,TAM,BAY-876 acted on breast cancer cells MCF-7 and T-47 D The accumulation of autophagy-related protein LC3-II increased the expression of Beclin-1 and decreased the expression of P62.Electron microscopy showed that compared with the control group,BAY-876 and TAM-treated breast cancer cells had more active autophagy.In the BAY-876 & TAM group,mitochondrial ridges were broken and swollen and deformed,and vacuolation was observed.Compared with the control group,they were significantly increased.The above results suggest that combination therapy enhances autophagy induction.6.BAY-876 enhances TAM-induced changes in MCF-7 and T-47 D energy regulatory signaling pathways in breast cancer cells After 48 hours of treatment,BAY-876 and TAM monotherapy and combined administration decreased the AKT phosphorylation activation state(p-AKT)content,and the signal molecule m TOR phosphorylation inactivation state(p-m TOR)also changed with At the same time,the expression of phosphorylated PI3 K and AKT in tumor tissues decreased,suggested that BAY-876 combined with TAM may inhibit proliferation,induce cell autophagy,and promote apoptosis by down-regulating the PI3 K signaling pathway.7.BAY-876 enhances the anti-tumor effect of TAM in vivo After establishing breast cancer models using T-47 D cells,they are divided into 4 groups: PBS group,BAY-876 group,TAM group,BAY-876 & TAM group.In vivo experiments showed that compared with BAY-876 and TAM alone,BAY-876 combined with TAM significantly inhibited the growth of tumor cells and had a good anti-tumor effect.Conclusion: BAY-876 enhances the sensitivity of TAM to breast cancer cells.This sensitization mechanism may be related to inhibiting Glut1 by down-regulating the PI3 K signaling pathway to induce cell autophagy and apoptosis.At the same time,it was also confirmed in nude mice transplantation tumor experiments that BAY-876 enhanced the antitumor effect of TAM in vivo.