Z-Ligustilide Sensitizes Tamoxifen-resistant Breast Cancer Cells Through Inhibiting Autophagy And DNA Damage Repair Mechanisms

Objective:Tamoxifen(TAM)has a good therapeutic effect on ER-positive breast cancer patients,but the drug resistance greatly limits the therapeutic effect and application of TAM.Changed in cellular mechanisms such as DNA damage repair,autophagy may have impetus to the emergence of resistance,but the specific mechanism is not fully elucidated.Z-ligustilide(Z-LIG)is one of the main active ingredients of Angelica,Ligusticum Wallichii,and other Umbelliferous plants.In the existing studies,only one literature indicates that Z-LIG can regulate cell autophagy induced by TNF-?,however,its mechanism in autophagy has not been explored.Also,there has been no exploration into Z-LIG's specific role in TAM-resistant breast cancer cels.This research is set out to preliminarily study Z-LIG's mechanism of inhibiting autophagy and sensitization of TAM-resistant ER+ breast cancer cells.Methods:Firstly,impacting by a high concentration of TAM in a short period of time induced TAMresistant breast cancer models.To distinguish drug-resistant strains from drug-sensitive strains,we started by applying SRB to test the effect of TAM on cell viability of drug-resistant/sensitive strains.We then identified the difference between autophagy levels of the two types of strains by applying GFP-LC13.Then,using Western blotting tested mark detection related protein expression with validation.Subsequently,chloroquine(CQ),the autophagy inhibitor,was used to investigate whether the autophagy in the resistant strain had a protective effect on its survival.Finally,we applied Immunoprecipitation to investigate the molecular mechanism of the increase of autophagy level in drug-resistant strains.In order to investigate the role of Z-LIG in regulating autophagy and molecular mechanism,we applied RFP-LC3 and Western blotting to detect the autophagy effect of Z-LIG on different breast cancer cells,the results were verified via CQ method.Finally,we carriedout several experiments including tf-mRFP-GFP-LC3 plasmid transient transfection,the colocalization of LC3 and LAMP1,testing the expression of cathepsin D via Western blotting,and acridine orange(AO)staining to study the mechanism of regulated autophagy by Z-LIG.To explore Z-LIG restore the drug-resistant breast cancer cells' sensitivity to TAM,we firstly tested the impact of Z-LIG alone or combination with TAM on the survival rate of drug-resistant breast cancer cells.Then verified the results by Clone formation experiments and Hoechst33342 stains.Later,Western blotting was used to detect the expression of PARP.We also applied SRB method to examine ZVAD-FMK's impact in recovering survival rate of cells,and thereby exploring if Z-LIG will restore TAM cytotoxicity via Caspase pathway.Additionally,we tested the expression of ?-H2 AX by Western blotting and Immunoflourescence,inspecting the DNA damage of two strains cells and whether Z-LIG would interfere with the DNA repair mechanism.To further study if Z-LIG interfere with DNA damage repair via Nur77,we applied shRNA to inhibit the expression of Nur77,Western blotting and SRB examined the DNA damage and cell viability of TAM-resistant breast cancer cells.Then,ubiquitination degradation inhibitor MG132,CQ,siRNA,inhibit Beclin1,and Z-LIG was added to treat,we examined whether the expression of Nur77 would be restored by Western blotting.Finally,Immunoprecipitation and DNA Affinity Precipitation Assay(DAPA)were applied to study how does Z-LIG interfere with the DNA repair system via Nur77.Results:SRB showed that compared with parental MCF7 and T-47 D,MCF-7TR5 and T-47DTR5 had shown good tolerance to TAM(~ 5?M).Subsequently,we noticed that the green fluorescence of MCF-7 cells had shown a diffuse distribution while MCF-7TR5 cells had shown more numbers of GFP-LC3 puncta.We have seen autophagy markers LC3-II/I and p62 expression were noticeably decreased,the level of BRCA1 and Nur77 also were decreased by Western blotting.In addition,combination of TAM and CQ,the cytotoxicity of TAM could be significantly enhanced.Finally,immunoprecipitation result indicated a rising of the Beclin1-PI3KC3-ATG14 complex which caused autophagy level to rise.After MCF-7TR5 cells had been transfected with mRFP-LC3 plasmids,Z-LIG's treatment significantly increased the puncta of red fluorescence and the number of intracellular autophagosomes.Moreover,when using Z-LIG at a different concentration or using 50?M of Z-LIG for a different length of time,the results showed that Z-LIG increased the expression of LC3-II and p62 in a time-and concentration-dependent manner.The combination of Z-LIG and CQ could also increase the expression level of LC3-II,and p62.Later,by transfecting MCF-7TR5 cells with tfmRFP-GPF-LC3,we found that the treatment of Z-LIG and TAM had increased yellow fluorescence,i.e.,the co-localization of green and red fluorescence.Furthermore,GFP-LC3 and RFP-LAMP1 co-transfected MCF-7TR5 cells,Z-LIG reduced yellow fluorescence.Finally,Western blotting and AO stained showed that Z-LIG reduced the expression of cathepsin D,neutralized pH of lysosome.The experiments have shown that when treating the drug-resistant breast cancer cells by the combination of Z-LIG and TAM,the appotosis of such cells was increased,and the survival rate of drug-resistant cells was reduced as well as their colony formation.However,no change has been found in the expression of activated PARP.Similarly,Z-VAD-FMK did not recover the cell viability.By Western blotting and immunofluorescence,we observed higher level of DNA damage was observed in MCF-7TR5 cells with the decreased BRCA1 and RAD51 level and the increased Ku80 level.Compared with the treatment of TAM alone,the combination would up-regulate the expression of ?-H2 AX.Later,via sh-RNA to inhibit Nur77,both Western blotting and SRB have shown that the combination had reversed function in accumulating expression of ?-H2 AX and downregulating survival rate of MCF-7TR5 cells.Subsequently,we treated MCF-7TR5 cells with MG132,si-RNA to inhibit Beclin1,CQ,and Z-LIG,finding that only the inhibition of autophagy could restore the expression of Nur77.Additionally,Immunoprecipitation has validated that the interaction between Nur77 and Ku80 was attenuated in MCF-7TR5 cells,but Z-LIG was able to enhance this interaction.Finally,DAPA confirmed that Z-LIG was able to inhibit the DNA-terminus with Ku80 and DNA-PKcs respectively.Conclusion:This study shows that with the treatment of TMA,the interaction between Beclin1 and Bcl2 was attenuated,releasing Beclin1,resulting in an increase in binding of Beclin1-PI3KC3-ATG14 complex,inducing autophagy,thereby promoting the survival of breast cancer cells.Moreover,higher level of DNA damage was observed in MCF-7TR5 cells with the decreased BRCA1 and RAD51 level and the increased Ku80 level.Interestingly,Nur77 was selectively degraded by autophagy,which causes the release of Ku80 from the Nur77-Ku80 complex,resulting in the increase of the DNA binding of Ku80 and DNA-PKcs.Meanwhile,Z-ligustilide was shown to inhibit the autophagic flux by blocking the autophagosome-lysosome fusion.Importantly,Z-LIGmediated autophagy inhibition restored Nur77 expression in MCF-7TR5 cells.Furthermore,Z-LIG promoted the interaction of Nur77 with Ku80 and thereby abolished the association of DNA-PKcs with DNA ends.Moreover,Z-LIG sensitized MCF-7TR5 cells in a caspase-independent cell death and enhanced the DNA damage caused by tamoxifen.Together,these findings not only provide important insights into the formation of tamoxifen resistance in breast cancer cells,but also suggest Z-LIG may function as a novel autophagy inhibitor to overcome chemoresistance.