Molecular Mechanism Of The Inhibitory Effect Of Cryptotanshinone On Proliferation Of ER?+ Breast Cancer Cells Based On BCRP

Bcakground and PurposeBreast cancer is a malignant tumor with high incidence and high mortality in women.Multidrug resistance hanppended in the later stage of endocrine therapy is an important cause of high mortality.Chinese medicine theories believe that the cause of breast cancer lies in deficiency of vital qi and stagnation of liver qi.The critical change in the process of cancer is qi stagnation and blood stasis.In view of the etiology and pathogenesis of breast cancer,xwe believe that Salvia miltiorrhiza is a typical Chinese medicine for promoting blood circulation and removing blood stasis.Its main mechanism is dispelling blood stasis,clearing qi and relieving pain,the pathogenesis and pathogenesis are mutually compatible,which can kill tumors from the root cause.Cryptotanshinone(CPT),one of the active components of the traditional Chinese medicine Salvia miltiorrhiza,has been proven to have multiple anti-breast cancer effects.In previous studies,we found that CPT can act on estrogen receptor alpha(ERa)and has a significant inhibitory effect on MCF-7(ERa+)cells,but not on MDA-MB-23 1(ERa-)cells.At the same time,we also found that breast cancer resistance protein(BCRP),which is closely related to the multidrug resistance of breast cancer,is highly expressed in MCF-7 cells,and there is also an interaction between this protein and ERa.Therefore,based on the above experimental results and findings,this study aims to explore the specific molecular mechanism of CPT inhibition of ER?+breast cancer cell proliferation from the perspective of BCRP.Research Contents and ResultsIn this experiment,we first found that there were significant differences in the intracellular and extracellular concentrations of CPT in CPT-sensitive MCF-7 cells and CPT-insensitive MDA-MB-231 cells.The results of this experiment led us to focus on drug efflux proteins.Through literature research and database analysis,,we used BCRP as a key protein in this study,and confirmed by molecular docking technology that CPT can be recognized and combined by BCRP.We further determined by Western blot,Real-time PCR,immunofluorescence,flow efflux and other experiments that CPT had no significant effect on the expression of BCRP total protein and mRNA in MCF-7 cells and MDA-MB-231 cells,but CPT could Significantly inhibited the efflux function of BCRP in MCF-7 cells,while CPT also inhibited the expression of BCRP on MCF-7 cell membrane and reduced the functional enrichment of BCRP on the cell membrane,while the above experimental phenomena were not detected in MDA-MB-231 cells.After confirming the specific inhibitory effect of CPT on BCRP in MCF-7 cells,we continued to use Westernbloting and cell viability assays to find that CPT can inhibit the proliferation of MCF-7 cells by regulating MAPKs signaling pathway in MCF-7 cells.The main manifestations were phosph orylation inhibition of p38 and ERK1/2,but no changes in related proteins were detected in MDA-MB-231 cells.By constructing shRNA to knock down ERa,westernbloting,flow cytometry and other experiments were used to confirm the important mediation of ERa in the regulation of MAPKs and BCRP by CPT.Based on the above experimental results,we further confirmed that the phosphorylation level of p3 8 plays a key role in the regulation of BCRP by CPT in MCF-7 cells by positive and negative validation of p3 8,ERK1/2 inhibitor and agonist.In addition,we further found that in MCF-7 cells,CPT can specifically reduce the formation of BCRP oligomers by inhibiting p38 activity through non-reducing gel electrophoresis experiments and fluorescence resonance energy transfer(FRET).Finally,we used the breast cancer multidrug resistant cell MCF-7/ADR to find that CPT can significantly inhibit its expression of BCRP and cell membrane functional enrichment.Meanwhile,when CPT combined with BCRP efflux drugs,the sensitivity of tumor cells to chemotherapeutic drugs would enhanced and inhibit its proliferation.Conclusion and SignificanceBased on the above results,we found that the significant inhibition of CPT on ERa+breast cancer cells is closely related to the expression of BCRP.CPT can inhibit the phosphorylation of downstream p38 by ERa,reduce the formation of BCRP oligomers,and reduce its enrichment on the cell membrane surface.At the same time,the efflux function is inhibited,resulting in the accumulation of CPT in tumor cells and then play the role of multi-target inhibition of tumor growth.This study clarified the molecular mechanism by which CPT can inhibit the proliferation of ERa+breast cancer cells by regulating the functional activity of BCRP protein,and provides a preliminary research basis for the application of CPT as a natural inhibitor of BCRP and reversing the multidrug resistance of breast cancer.