Stellera Chamaejasme Extract Overcomes Breast Cancer Multidrug Resistance By Regulating Autophagy

Objective:Breast cancer has the highest incidence rate and poor prognosis among women nowadays.Chemotherapy is widely used to treat breast cancer clinically,but the multidrug resistance of chemotherapy has restricted the clinical efficacy of chemotherapy for a long time.Therefore,overcoming the multidrug resistance of breast cancer has become one of the most important topics of breast cancer research.Autophagy is the main focus of this article,and its activation is an important mechanism leading to multidrug resistance in breast cancer.In addition,breast cancer microenvironment is rich in adipocytes,which can activate autophagy and enhance drug resistance of breast cancer cells.Therefore,this study will concentrate on the regulation of chamaejasme extract(LD)on autophagy related processes and multidrug resistance of breast cancer cells based on the single cell culture of breast cancer and the co-culture model of breast cancer cells and adipocytes,and explore the related mechanism in the meanwhile.Methods:For the single cell model of breast cancer,MTT was used to detect the survival rate of the 4 kind of breast cancer cell lines after Stellera chamaejasme extract administration,IC50 and doxorubicin resistant factors were calculated.DAPI staining was used to detect the effect of LD on apoptosis characteristics of MCF-7/ADR cells.The effect of LD on the apoptosis of MCF-7/ADR and MDA/ADR cells was detected by flow cytometry.The effect of Stellera chamaejasme extract on the expression of apoptosis-related proteins caspase-8,cleaved caspase-8,caspase-3,cleaved caspase-3 and autophagy-related proteins mTOR,p-mTOR,p62 and LC3B in MCF-7/ADR and MDA/ADR cells was detected by western blot.The effects of LD on autopagosomes of MCF-7/ADR cells were detected by MDC staining and LC3 immunofluorescence assay was used to detect the effect of LD on the expression of LC3 in MDA/ADR cells.GFP-LC3B-mcherry was used to detect the effect of LD on autophagy flux of MCF-7/ADR.MTT assay and flow cytometry was used again to detect the effects of LD and autophagy inhibitor chloroquine on the survival and apoptosis rate of MCF-7/ADR and MDA/ADR cells.The effects of LD on the contents of lactic acid,glucose and ATP in the supernatant of MCF-7/ADR and MDA/ADR cells were detected by the assay kit.For the co-culture model of adipocytes and breast cancer cells,MTT was used to detect the effects of adriamycin and LD on the survival rate of MDA-MB-231 cells and MDA/ADR cells.Flow cytometry was used to detect the effects of LD on the apoptosis of MDA/ADR cells.Western blot was used to detect the effect of LD on the expression of autophagy-related proteins,p62,LC3B in MDA-MB-231 cells and MDA/ADR cells.The effect of LD on lipid droplets of MDA/ADR cells was detected by oil red O staining.The effect of LD on lipid droplet autophagy of MDA/ADR cells was detected by co-localization of bodipy fluorescence and LC3 immunofluorescence.The effects of LD on the content of lactic acid,glucose and FFA were detected by the assay kits.RTPCR was used to detect the effects of LD on adipocyte differentiation markers FABP4 and inflammatory factors IL-6,IL-1β and TNF-α in the co-culture system.In vivo experiment,the model of breast cancer transplantation tumor was established.Breast cancer cells were inoculated subcutaneously on the back of nude mice as the control group,and the inguinal fat pad as the model group to simulate the breast cancer fat microenvironment.On this basis,the inhibitory effect of LD on breast cancer transplantation tumor was studied.Besides,western blot was used to detect the expression level of LC3 and p62.Results:In the study on the single cell model of breast cancer,MTT showed that the two drug resistant breast cancer cell lines had higher IC50 values for doxorubicin compared with their corresponding sensitive cell lines.LD had significant inhibitory effects on the proliferation of MCF-7/ADR,MDA/ADR and MCF-7,MDA-MB-231.The IC50 value of LD on drug resistant cell lines were lower than that of sensitive cell lines.DAPI staining results showed that LD could induce apoptosis characteristics such as nuclear lysis in MCF-7/ADR.The flow cytometry results showed that LD in each concentration group could significantly increase the apoptosis rate of MDA/ADR and MCF-7/ADR breast cancer drug resistant cell lines.Western blot results showed that LD could significantly increase the ratio of cleaved-caspase-3/caspase-3 and cleavedcaspase-8/caspase-8 ratio of two breast cancer resistant cell lines;The ratio of pmTOR/mTOR in the two cells was significantly decreased,the ratio of LC3B-II/LC3BI was up-regulated,and the expression level of p62 was increased.MDC staining results showed that the positive fluorescence region of MCF-7/ADR cells increased after LD treatment.LC3 immunofluorescence results showed an increase in the positive fluorescence region of MDA/ADR cells was induced by LD.The results of administration of GFP-LC3B-mcherry after transfection showed that the number of yellow spot fluorescence increased significantly after the administration of LD on MCF-7/ADR cells.MTT results showed that both LD and CQ could significantly reduce the survival rate of breast cancer cells seperately,and they have a synergistic effect on the effect on the survival rate of drug resistant breast cancer cell lines.The detection results of energy metabolites in the supernatant showed that LD could significantly reduce the content of lactic acid and ATP in the supernatant of two breast cancer-resistant cell lines,and significantly increase the content of glucose in the supernatant.In the study of breast cancer cells in the co-culture model,MTT results showed that the survival rate of breast cancer cells in the adipocyte-conditioned medium and co-culture systems was significantly increased,and the survival rate of the co-culture group after the administration of adriamycin was significantly higher than that of the control group.LD could significantly reduce the survival rate of breast cancer cells in the two cell models.At the same time,the combined effect of LD and chloroquine on the proliferation inhibition was significantly stronger than they were used alone.The results of flow cytometry showed that compared with the control group treated with doxorubicin,the survival rate of the co-culture group treated with doxorubicin was significantly higher,while LD significantly increased the apoptosis rate of breast cancer cells in the model group,and there was also a synergistic effect with chloroquine.The results of oil red O staining showed that co-culture could significantly increase the content of lipid droplets in breast cancer cells,while LD could significantly reduce the content of lipid droplets in breast cancer cells when simultaneous administration of co-culture but increase lipid droplets when drug administration is after co-cultivation.The results of fluorescence co localization of Bodipy and LC3 showed that co-culture could significantly increase the level of lipid droplet autophagy in breast cancer cells,and LD could block the binding process of autophagosome and lysosome or lysosome acidification process,so that lipid droplets and autophagosomes in drug resistant breast cancer cells could not be decomposed in time and accumulated continuously.RT-PCR results showed that The expression level of FABP4 in adipocyte was significantly decreased when co-cultured with MDA-MB-231 and MDA/ADR.After treatment with LD,the transcription level of FABP4 was significantly increased.After co-culture with two breast cancer cells,The transcription level of IL-6,IL-1β and TNF-α was significantly increased.After the effect of LD,the transcription level of inflammatory factors was significantly lower than that of the model group.Western blot results showed that for MDA-MB-231 and MDA/ADR cells,co-culture could significantly reduce the expression level of p62 and increased the ratio of LC3B-II/LC3B-I.After treatment with LD,the level of p62 increased,and the ratio of LC3B-II/LC3B-I increased.The energy metabolism detection results showed that compared with the control group,the glucose content in the supernatant of the two breast cancer cell model groups decreased,the lactic acid content significantly increased,and the glucose content in the LD administration group significantly increased.In vivo experiment showed that compared with the control group,the quality of transplanted tumor in the model group was significantly increased in the control+ADR、control+LD low dose group and the control+LD high dose group.Compared with the model group,the transplanted tumor quality in the model+ADR,model+LD low dose,and M+LD high dose groups significantly decreased.Compared with the control+adriamycin group,the tumor inhibition rate in the model+adriamycin group was lower.Conclusion:Based on the all the experimental results above,we consider that LD can play an anti-drug resistance role by inhibiting the autophagy process of drug-resistant breast cancer cells,therefore making the autopagosomes accumulate,inhibiting energy metabolism,promoting apoptosis,and thus overcome the multidrug resistabce of breast cancer.Moreover,LD could not only inhibit autophagy and energy metabolism of breast cancer cells in co-culture system,but also lead to apoptosis,It can also prevent fat cells from changing into CAA phenotype,reduce the secretion of inflammatory factors and adipose factors,thereby reducing the risk of breast cancer malignancy.Finally,in nude mice transplanted tumor models,LD can also effectively inhibit tumor growth.Through the overall regulation of the co-culture system,it can regulate the microenvironment of breast cancer and inhibit the occurrence and development of breast cancer.The above effects of LD reflect the characteristics of multiple targets,multiple pathways and overall regulation of traditional Chinese medicine,indicating that it has the potential to be further developed in the future.