Effect Of Exosomes On Psoralen Reversing Breast Cancer Multidrug Resistance

Objective:1.7 million women worldwide are diagnosed with breast cancer each year.In developing countries,breast cancer is the most common cancer in women and is the leading cancer that causes women to die.The incidence of breast cancer in Asia is also on the rise.Breast cancer as the first killer of modern women,surgery is a key means of local treatment,and systemic chemotherapy is still an indispensable treatment,but breast cancer cells to obtain resistance to chemotherapy drugs,often make breast cancer cells through a variety of ways to effectively escape chemotherapy,and ultimately lead to treatment failure.The acquired resistance mechanism has been a hot topic.Recent studies have found that exosomes plays an important role in the transmission of drug resistance between drug-resistant tumor cells and drug-sensitive cells.Psoralen is a traditional Chinese medicine preparation with a precise reversal of multidrug resistance and has fewer side effects.This research will study the function of the exosomes(EXOs)in transfering drug resistance information between breast cancer cells and whether psoralen reversing breast cancer multidrug resistance through exosomes,to provide an important theoretical basis on reversing the tumor resistance.Methods:1.Breast cancer cell line MCF-7 and MCF-7/ADR were selected as experimental cell.Exosomes derived from MCF-7/ADR were isolated from the supernatants by ultracentrifugation.2.Transmission electron microscopy(TEM)was used to observe the morphology of isolated exosomes.3.Using the inverted fluorescence microscope observed interaction process between ADR/EXO and MCF-7 cell;4.Cells uptake of adriamycin are observed by the Laser confocal microscope.5.CCK-8 assay was used to detect the changes of adriamycin IC50 value of MCF-7 after co-cultured with ADR/EXO.6.The effect of ADR/EXO on MCF-7 MDR1 mRNA was determined by RT-PCR.7.Western blot analysis was used to examine P-gp expression.8.The different formation and secretion of exosomes were detected by im-munofluorescence and transmission electron microscopy.9.A statistical analysis was using the SPSS 17.0 statistical soft-ware.Results:1.Under the transmission electron microscope,the isolated EXOs were round or oval in shape with a diameter between 40-100 nm.2.ADR/EXO labeled with PKH67 can be uptake by MCF-7 cells.3.After incubation with ADR/EXO,adriamycin IC50 of sensitive cell line MCF-7 cells increased 2.62 times(P<0.05);4.MCF-7 cells co-cultured with MCF-7/ADR cells in the Transwell model for 60h,adriamycin fluorescence intensity was weaker than MCF-7.5.The expression of MDR1 mRNA in MCF-7+ ADR/EXO group was significantly higher than MCF-7 group,showing statistically significant differences(P<0.05).6.Compared with the MCF-7 groups,co-cultured with ADR/EXO for 12h,P-gp relative expression increased 4.7 times(P<0.05);Co-cultured with ADR/EXO for 72h,P-gp relative expression increased 3.4 times(P<0.05).7.We found a marked abundance of exosomes on the surface of MCF-7/ADR cells by transmission electron microscopy compared to MCF-7 cells,and exosomes generation significantly decreased after psoralen treatment.Immunofluorescent staining for the exosomes marker CD63 in MCF-7/ADR cells further confirmed that showed that psoralen treatment decreased the exosomes.Conclusion:1.Our results suggest that the exosomes from breast cancer cells have the function of transmitting the drug-resistant message,sensitive cells can acquire drug resistance from the exosomes of drug resistant cells.2.P-gp can be transferred between breast cancer cells,not only can be in the form of protein,but also in the form of mRNA.3.In this study,we have proved that psoralen could influence the formation and secretion of exosomes and induce the reduction of resistance transmission via exosomes.