HMGB1 Involved In Breast Cancer Development By Regulating The Differentiation Of Mdscs

Objective:1. To analyze the relation between HMGB1 and MDSCs in breast cancer by detecting the expression of HMGB1 and the percentage of MDSCs.2. To investigate the mechanism of HMGB1 regulating the differentiation of myeloid-derived suppressor cells by analyzing the relationship between HMGB1 and MDSCs.Methods:1. The percentage of MDSCs in spleen and peripheral blood of normal and tumor-bearing mice were measured by flow cytometry; the expression of HMGB1, i NOS and ARG1 in normal and tumor tissue of tumor-bearing mice were detected by q RT-PCR and western blotting.2. The cells were harvested from the bone marrow and spleen of healthy mice and cultured with HMGB1, GM-CSF+IL-6, GM-CSF+IL-6+HMGB1, GM-CSF+IL-6+HMGB1+EP respectively in vitro; the percentage of MDSCs, DCs and macrophages were measured by flow cycometry after 48 hours.3. The phosphorylation level of ERK1/2, STAT3, NF-κB and P38 were accessed by western blotting in 0, 15, 30, 60, 90 and 120 minutes; bone marrow cells pre-cultured with PDTC, U0126, Niclosamide and SB203580 for 1 hour, the percentage of MDSCs were measured by flow cytometry after HMGB1 stimulation for 48 hours.4. Two groups of mice were treated intraperitoneally with EP or PBS given every three days after successfully established the models; the percentage of MDSCs in tumor and spleen were accessed by flow cytometry after treated 25 d and 40 d.5. Sorted M-MDSCs and G-MDSCs from the femurs of healthy BALB/c mice were stimulated with HMGB1 and the percentage of MDSCs, DCs and macrophages were measured by flow cytometry.Results:1. The percentage of MDSCs in the peripheral blood and spleen of tumor-bearing mice were higher than in the normal mice; comparing with normal tissues, the expression of HMGB1,i NOS and ARG1 were higher in tumor tissues.2. Flow cytometry results showed that the proportion of MDSCs in HMGB1 group, GM-CSF+IL-6 group, GM-CSF+IL-6+HMGB1 group were increased, while DCs and macrophages decreased by comparing with NC group after culturing for 48 hours; interestingly, comparing with GM-CSF+IL-6+HMGB1 group, the percentage of MDSCs was reduced but the proportion of DCs and macrophages were increased in EP-treated group.3. The expression of phosphorylated ERK1/2, P38 and NF-κB were increased gradually with 250ng/m L HMGB1 stimulation in 0 to 120 minute except for p-STAT3; compared with control group, the percentage of MDSCs were significantly reduced in U0126, SB203580 and PDTC groups, but not in Niclosamide group.4. Comparing with PBS group, the tumor volume, weight and the proportion of MDSCs in tumor and spleen were significantly decreased in EP group.5. Sorted M-MDSCs and G-MDSCs from the healthy mice were stimulated with HMGB1 for 48 hours in vitro; the results showed that the percentage of MDSCs were increased and the proportion of DCs and macrophages were reduced in HMGB1-stimulated group, but there were no significant differences between groups.Conclusions:1. HMGB1 was over-expressed and the percentage of MDSCs was increased in breast cancer, otherwise, there was a positive correlation between HMGB1 and MDSCs.2. HMGB1 regulated the differentiation of MDSCs through NF-κB, ERK1/2 and P38 MAPK signaling pathways, and HMGB1 blockade can reduce the accumulation of MDSCs and delay the development of tumors.