Study On The Molecular Mechanism Of Yiqi Fuzheng Compound Combined With Everolimus By Regulating The Akt/mTOR Signaling Pathway To Inhibit The Proliferation Of Triple-negative Breast Cancer Cells

Objective:To explore the effect of Yiqi Fuzheng Formula（YFF）on the inhibition of triple-negative breast cancer cell proliferation may be through the regulation of Akt/mTOR signaling pathway,and its mechanism may be different or partially overlapped with the mTOR inhibitor Everolimus.Methods:Methyl thiazolyl tetrazolium（MTT）assay was used to detect the MDA-MB-468 cell proliferation inhibition of 10,20,40,80,160 mg/m L FFY and1,10,100,1000 nmol/L Everolimus for 12,24 and 48h,Treat MDA-MB-468 breast cancer cells with IC₅₀YFF of 40 mg/m L and Everolimus 100 nmol/L for 48h,then observe the inhibition of cell proliferation and detecte the cell cycle and apoptosis by flow cytometry;Real-time PCR,Western Blot were used to detect the key molecular and protein levels of Akt,mTOR and PTEN in the Akt/mTOR pathway.The Akt target gene was silenced by siRNA interference technique,and the changes of key proteins in the Akt/mTOR signaling pathway were observed by Western Blot method to confirm the possible target of the YFF.Results:1.Both YFF and Everolimus inhibited the proliferation of MDA-MB-468 breast cancer cells in a dose and time dependent manner.The combination of YFF with IC₅₀40mg/m L and Everolimus 100nmol/L intervened MDA-MB-468 breast cancer cells for 48hours,YFF group and the Everolimus group reduced the survival rate of MDA-MB-468breast cancer cells to 54%vs 51%,respectively.The combination of the YFF and Everolimus reduced the survival rate of MDA-MB-468 breast cancer cells to 34%,the combined.group got the Q value of 43.89>1.15,which indicted they had a synergistic effect,and the inhibition of cell proliferation was more obvious（p<0.05）.2.Flow cytometry showed that the apoptosis rate of the YFF group and Everolimus group was20%and 26%,while the apoptosis rate was increased to 41%after the combination.3.Cell cycle experiments showed that compared with the blank control group,the S-phase cells in the YFF group;the Everolimus group and the combined group decreased by63.8%;66%and 72.6%.The Everolimus group and the combined group reduced G2/M phase cells by 25.5%and 48.4%,respectively（p<0.05）.4.Real-time PCR and Western Blot results show that YFF alone and in combination with Everolimus can inhibit the activity of p-mTOR,p-Akt and p-P70s6k,and increase the expression of PTEN and p-4EBP-1,at the same time,it can significantly reduce the expression of anti-apoptotic protein Bcl-2,increase the expression of proapoptotic protein Bax,and activate Caspase-3.5.After Akt was silenced by siRNA technology,the regulation of PTEN,Akt,P70s6k and 4EBP-1 was blocked by the YFF group and the combination group.Conclusion:YFF can inhibit the proliferation of triple negative breast cancer cells,and combined with Everolimus can increase the inhibitory effect of Everolimus.We conclude that the YFF and the mTOR inhibitor Everolimus have some overlapping mechanisms.The YFF can inhibit the Akt/mTOR signaling pathway via multiple targets to exert its anti-tumor effect,which provides a valuable experim ental basis for the treatment of triple-negative breast cancer by Chinese medicine.