Effects Of Breast Cancer Cell-derived Exosomes Mediated EGFR On MAPK/ERK And PI3K/Akt Pathways And Expression Of Autocrine VEGF In Huvecs

Objective：To study the effects of Breast cancer MDA-MB-231cells-derivedexosomes mediated EGFR on MAPK/ERK and PI3K/Akt pathways andexpression of autocrine VEGF in HUVECs. To explore the mechanism oftumor cell-derived exosomes in tumor angiogenesis, and provide atheoretical basis for elucidating the important role of tumor cell-derivedexosomes in the tumor microenvironment.Methods：1. Breast cancer MDA-MB-231cells and HUVECs were used as theresearch model. MDA-MB-231cells-derived exosomes were isolated andpurified from the culture supernatants by ultracentrifugation and sugardensity ultracentrifugation. Flow cytometry (FCM) was used to examinethe distribution of cells cycles of the HUVECs cells.2. To observe the effects of exosomes derived from MDA-MB-231cells on expression of EGFR and its downstream MAPK/ERK and PI3K/Akt signaling pathway in HUVECs. The expression and location ofEGFR and phosphorylated-EGFR in HUVECs with exosomes derived fromMDA-MB-231cells co-cultured were detected by Immunofluorescence.The activity of ERK1/2and Akt in HUVECs with exosomes derived fromMDA-MB-231cells co-cultured were detected by Western blot.3. To investigate the effects of exosomes derived from MDA-MB-231cells on expression of autocrine VEGF in HUVECs. The expression ofautocrine VEGF in culture supernatants of HUVECs with exosomesderived from MDA-MB-231cells co-cultured were detected by ELISA.The effects of exosomes derived from MDA-MB-231cells on mRNA andprotein expression of VEGF were detected by RT-PCR and Western blot,respectively. The protein expression of VEGFR2and p-VEGFR2weredetected by Western blot.Results：1. HUVECs were cultured in low concentration of newborn bovineserum for cell cycle synchronization, then fresh complete medium weregiven so that cells reentering the cell cycle. Flow cytometry showed that200μg/mL exosomes can increase S-phase of the cell in HUVECs, anddecrease G1-phase cell in HUVECs in cycle, and reduced the rate of G1/Sphase.2. Immunofluorescence showed that the positive EGFR and p-EGFRwere mainly found in cytoplasm and membrane of HUVECs with 200μg/mL exosomes derived from MDA-MB-231cells co-cultured. At thesame time, Western blot result showed that the expression of p-ERK1/2andp-Akt protein were increased in HUVECs in a classical time-dependentmanner. In contrast, the expression of p-ERK1/2and p-Akt protein weredecreased in HUVECs pre-treated with50μg/mL EGFR inhibitor ZD1839.3. ELISA result showed that the concentration of VEGF in culturedHUVECs with exosomes co-cultured were significantly different from theuntreated HUVECs. RT-PCR and Western blot exhibited that the VEGFmRNA and protein expression had significantly increased in HUVECs withexosomes co-cultured. Meanwhile, the expression of p-VEGFR2proteinwas increased in HUVECs. But VEGF mRNA and protein expression weredecreased and the phosphorylation of p-VEGFR2inhibited in HUVECspre-treated with50μg/mL EGFR inhibitor ZD1839.Conclusion：1. Breast cancer MDA-MB-231cells-derived exosomes promoted theexpression of EGFR in HUVECs, and increased S-phase cell in HUVECs.With the transfer of MDA-MB-231cells-derived exosomes mediatedEGFR, the activation of MAPK/ERK and PI3K/Akt signaling pathwayswere associated with HUVECs proliferation and tumor angiogenesis.2. Breast cancer MDA-MB-231cells-derived exosomes mediatedEGFR, promoted the expression of autocrine VEGF in HUVECs, increasedthe phosphorylation of VEGFR2, and activating the VEGF/VEGFR2 signaling pathway. They may be associated with the regulation in tumorangiogenesis.