Panoply™ Human CXCR2 Knockdown Stable Cell Line

The CXCL8-CXCR1/CXCR2 signaling pathway may form a complex crosstalk between different cell types within the ovarian tumor microenvironment, thereby regulating distinct cell behaviors. Here, researchers aimed to investigate the expression pattern of CXCL8 in the ovarian tumor microenvironment and its impact on endothelial-mesenchymal transition (EndMT) and ferroptosis in endothelial cells. In vitro experiments were conducted using the human monocytic cell line THP-1 and the human umbilical vein endothelial cell line PUMC-HUVEC-T1. Ferroptosis was induced using erastin. Results showed that tumor-associated macrophages are the primary source of CXCL8 in the tumor microenvironment. CXCL8 treatment promoted the nuclear translocation of NF-κB p65 in endothelial cells and promoted p65 phosphorylation via CXCR2, suggesting activation of NF-κB signaling. CXCL8 enhanced TGF-β1-induced EndMT in PUMC-HUVEC-T1 cells through the NF-κB signaling pathway and upregulated the expression of SLC7A11 and GPX4. These trends were significantly attenuated by CXCR2 knockdown or SB225002 treatment. TPCA-1 reversed CXCL8-induced upregulation of SLC7A11 and GPX4. CXCL8 protected endothelial cells from erastin-induced ferroptosis. However, knockdown of CXCR2 largely abolished this protective effect. In summary, CXCL8 activates the NF-κB signaling pathway in endothelial cells in a CXCR2-dependent manner. The CXCL8-CXCR2/NF-κB axis enhances EndMT and activates the expression of SLC7A11 and GPX4, thereby protecting endothelial cells from ferroptosis.

Because CXCL8 stimulates the expression of SLC7A11 and GPX4, the researchers explored its potential protective effect against erastin-induced ferroptosis. Erastin treatment significantly inhibited NF-κB p65 phosphorylation, which was partially rescued by CXCL8 treatment (Figure 1a). However, this CXCL8-induced rescue effect was abolished in CXCR2 knockdown cells (Figure 1b). CXCL8 treatment increased the basal GSG/GSSG ratio (Figure 1c), reduced ROS (Figure 1d) and lipid ROS levels, and enhanced cell viability (Figure 1f). Furthermore, CXCL8 attenuated the erastin-induced decrease in GSG/GSSG, reduced erastin-induced ROS (Figure 1d) and lipid ROS (Figures 1e, g, i), and preserved cell viability after erastin treatment (Figure 1f). These protective effects of CXCL8 were abrogated in CXCR2 knockdown cells (Figures 1c–i).

Figure 1. CXCL8 protects endothelial cells from erastin-induced ferroptosis. (Ji H, et al., 2023)