Eugenol triggers CD11b+Gr1+ myeloid-derived suppressor cell apoptosis via endogenous apoptosis pathway

Abstract

To study the effect and underlying molecular mechanism of eugenol on CD11b⁺Gr1⁺ myeloid-derived suppressor cells (MDSCs). The effect of eugenol on the inhibition of immortalized MDSC cell line MSC-2 and murine peritoneal macrophages was detected by MTT. Flow cytometry was used to detect the pro-apoptosis effect of eugenol on MDSCs. The expression levels of apoptosis-related proteins were detected by western blot. Eugenol has a selective inhibitory effect on MDSCs in a dose-dependent manner, which activates an endogenous apoptosis pathway, leading to apoptosis. Eugenol promotes the apoptosis of MDSCs via the intrinsic pathway.

Fig. 1. MDSCs in splenocytes of wild-type mice. (A) Splenocytes from CT-26 tumour-bearing mice (5 × 10⁴ per well in a 96-well culture plate) were treated with various concentrations of eugenol for 24 hours and then stained with fluorescent antibodies (anti-CD11b, anti-Gr1) and subjected to a fluorescence-activated cell sorting analysis on the flow cytometer (BD Calibur). Each group is set with four repeat wells. Data were analysed using FlowJo 7.6. (B) A bar chart was generated using Graph Pad Prism 5.0. *p < 0.05, **p < 0.01.

Fig. 2. The effect of eugenol on MSC-2 cells and macrophages. (A & B) MSC-2 cells (0.6 × 10⁴ per well in 96-well culture plates). Each group is set with four repeat well; (C) macrophages (1 × 10⁵ per well in 96-well culture plates) were treated with various concentrations of eugenol for 24 hours. Cell viability was analysed by MTT assay. DMEM was used as the control. *p < 0.05, ***p < 0.001, and ns represents non-statistically significant differences.

Fig. 3. Flow cytometry analysis of cells stained with Annexin V-FITC and PI. (A) MSC-2 cells were treated with eugenol for 24 hours and then harvested and processed by Annexin V-FITC and PI staining followed by flow cytometry analysis. Each group is set with four repeat wells. The fluorescence pattern of Annexin V-FITC and PI-stained MSC-2 cells after 24 hours of treatment. (B) Percentages of Annexin V positive cells following different treatments. (C) Levels of bax, cytochrome C, caspase-9, cleaved caspase-3, BCL-2 and caspase-8 (no bands were detected) in total cell lysates were determined by western blotting. The expression of β-actin served as a control.

Fig. 4. MDSCs in splenocytes of TLR4 knockdown mice. (A) Splenocytes from TLR4 knockout tumour-bearing mice (5 × 10⁴ per well in 96-well culture plates) were treated with eugenol at different concentrations for 24 hours and analysed by flow cytometry. Each group is set with four repeat wells. (B) The data analysis is shown in a bar chart. In addition, ns represents non-significant differences.

Fig. 5. The apoptotic signalling pathway of MDSCs by eugenol.