Study On The Anti-breast Cancer And Its Mechanism Of Polydatin Combined With Brusatol In Regulating Nrf2 Signaling Pathway

Objective: The primary objective of this study was to develop a combination treatment strategy that combined the natural small molecule drug polydatin(PD)with brusatol(BRU)and reduced the dose and number of administrations.It can provide a more valuable method for the treatment of triple negative breast cancer(TNBC).The inhibitory effect and mechanism of combined administration on the proliferation of TNBC cells MDA-MB-231 were also studied.Methods: 1.First of all,CCK-8 assay was used to detect the inhibitory effects of PD combined with BRU on the proliferation of MDA-MB-231 cells.Using Calcu Syn software,we calculated the CI value of cells treated with PD and BRU.2.Flow cytometry was further used to analyze the effect of PD in combination with BRU on intracellular reactive oxygen species(ROS)levels.3.Analysis of the association between Nrf2 gene expression and breast cancer patients’ survival in the GEPIA database according to TCGA.4.The changes of Nrf2 protein levels in MDA-MB-231 cells after combined administration of PD with BRU were further investigated by immunofluorescence experiment and Western blot method.Meanwhile,the effect of the combination of drugs on the expression levels of Nrf2 protein in the respective components of nucleus and cytosol was observed again through nuclear and cytoplasmic extraction,and quantitative statistical analysis was carried out.5.The inhibitory effect of PD in combination with BRU treatment on Nrf2 downstream target genes HO-1 and NQO1 at m RNA levels was analyzed by real-time PCR(q-PCR)in MDAMB-231 cells.6.To establish a subcutaneous tumor model in nude mice,observe the inhibitory effect of PD combined with BRU on tumor growth and the safety of the drugs under the condition of lower dose and fewer times of administration.Results: 1.Compared with single drug treatment,the combination of PD and BRU has a more significant effect on inhibiting the growth and proliferation of MDA-MB-231 cells in vitro.And the drug CI value after the combination of the two drugs is less than 1,indicating a synergistic inhibitory effect.2.The combination of PD and BRU significantly increased the ROS levels in MDA-MB-231 cells,which increased about 2.5-fold compared with the control group.3.Breast cancer patients with higher Nrf2 expression have worse survival and worse prognosis.4.PD and BRU synergistically could significantly inhibit Nrf2 protein expression in MDA-MB-231 cells.5.The expression of Nrf2 downstream target genes,HO-1 and NQO1,was down regulated by PD with BRU alone or in combination,but the inhibition of Nrf2/HO-1 and NQO1 antioxidant pathways was more significant in combination treatment.6.The combination of low-dose PD and low-dose BRU exhibited a better inhibitory effect on tumor growth in mice than single drug,with no toxic effects and higher safety.Conclusion: In this study,we developed a therapeutic strategy combining the natural small molecule drug PD with BRU to treat TNBC,and it exerted good antitumor efficacy with a low-dose and fewer administrations.Mechanistically,it significantly increased ROS levels and inhibited cell proliferation in vitro,mainly by inhibiting Nrf2/HO-1 and NQO1 signaling pathways in MDA-MB-231 cells,and again confirmed in vivo that the combination treatment inhibited tumor growth in mice more effectively than monotherapy.The findings can provide a new scheme for treating TNBC clinically.