Effect And Mechanism Of Exosomes Mediated Breast Cancer Cells On Doxorubicin Resistance

Background and ObjectiveBreast cancer is one of the most common malignant tumors among women worldwide,and it is also the second leading cause of female-related cancer death.With the improvement of early diagnosis and advanced treatment strategies,the mortality rate of breast cancer has decreased significantly.Current treatment strategies for breast cancer usually combine surgery with a variety of adjuvant therapies,such as chemotherapy,hormone therapy,radiotherapy,targeted therapy.Most patients respond to initial treatment within a certain period of time,but some breast cancers develop into more aggressive forms of cancer,which are usually resistant to chemotherapy and radiotherapy.Therefore,overcoming metastasis and recurrence of breast cancer,which leads to poor prognosis,is the main challenge for breast cancer treatment.In recent years,with the introduction of new drugs such as endocrine therapy drugs and molecular targeted drugs,great progress has been made in the treatment of breast cancer,but chemotherapy is still the main treatment strategy for triple negative breast cancer.Doxorubicin plays an important role in first-line chemotherapy of breast cancer,but drug resistance remains the main obstacle to successful treatment.With the in-depth study of the mechanism of chemoresistance,the transfer of drug resistance information from exosomes derived from cancer cells as gene cargo carriers has gradually attracted the attention of researchers.Exosomes are small vesicles secreted by most cells with a diameter ranging from 40 to 100 nm.They contain various active molecules,including proteins,lipids,DNA and RNA.Exosomes,as mediators of cell-to-cell communication,shuttle in the tumor microenvironment and are absorbed by surrounding cancer cells or stromal cells,and can transmit information by releasing content,thus causing the proliferation,invasion,metastasis and drug resistance of cancer cells.Studies have shown that tumor-derived or tumor-associated exosomes are important mechanisms regu Lating tumor resistance,and they can confer resistance to sensitive cells by transmitting molecules such as RNA and proteins.This article mainly explores the role and preliminary molecular mechanism of exosomes in the transmission of doxorubicin in breast cancer cells,and aims to provide new ideas for the molecu Lar mechanism and therapeutic strategies of doxorubicin resistance in breast cancer.Methods1.Breast cancer parental cell line(MCF-7),doxorubicin-resistant breast cancer cell line(MCF-7/Dox),sensitive cell line co-cultured with doxorubicin-resistant supernatant(MCF-7/EXO)were emplored as the cell models in the present study.2.The effects of doxorubicin on the proliferative activity of MCF-7,MCF-7/EXO and MCF-7/ADR cells were detected by CCK8 assay 3.The effects of doxorubicin on cell apoptosis of MCF-7,MCF-7/EXO and MCF-7/ADR cells were observed by fluorescence microscope.4.Exosomes in the cell supernatants were extracted by ultracentrifugation,and the quantity of exosomes was determined by transmission electron microscopy,BCA and Di I labeling assay.Expression levels of exosome-specific molecules CD63 and Flotillin-1 were detected by Western blot.5.The uptake of MCF-7/Dox cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy.6.Western blot was used to detect the expression levels of multidrug resistance protein(ATP-binding cassette subfamily B member 1,ABCB1)in MCF-7,MCF-7/EXO and MCF-7/ADR cells.7.Statistical analysis was performed using SPSS 21.0 software.The measurement data were analyzed by ANOVA.The LSD-t method was used to compare the two groups.The data were expressed as x ± s.The difference was statistically significant at P < 0.05.Results 1.The cell proliferation assay showed that the IC50 of MCF-7/EXO cells to doxorubicin was 0.83±0.09 μmol·L-1,which was significantly higher than MCF-7 cells which was 0.15±0.05 μmol·L-1(P<0.01),and the drug resistance increased by 5.5 times.2.Apoptosis assay showed that apoptosis of MCF-7 cells were induced significantly after doxorubicin treatment(P<0.001),but MCF-7/EXO cells were not significantly different(P> 0.05).3.Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells(P<0.05).4.PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells.5.Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells.Conclusions1.Doxorubicin-resistant breast cancer cells can secrete a large number of exosomes.2.After co-cultured with sensitive cells,the exosomes in the supernatant of doxorubicin-resistant breast cancer cells can be efficiently taken up by sensitive cells.3.Exosomes can transmit doxorubicin resistance to sensitive breastcancer cells,and the mechanism may be related to exosome-mediated ABCB1 protein transport.