The MPRO Clone 2.1 cells were with or without treatment of CXCR2 antagonist, SCH 527123, for 4 hours. Higher Levels of CXCR2 Ligands To assess the relationship between the IL-17/CXCR2 axis and chemotherapy resistance, the murine breast cancer cell collection Cl66, cells resistant to doxorubicin (Cl66-Dox), or cells resistant to paclitaxel (Cl66-Pac) were used. The resistant malignancy cells express more CXCR2 ligands than their parent cell.9 Significantly higher levels of CXCL1 (Determine?1A) and CXCL5 (Physique?1B) were observed in Cl66-Dox and Cl66-Pac cells compared with parental controls. Quantitative RT-PCR was used to quantify the expression of (Physique?1C) and (Physique?1D) in these cells. Both chemotherapy-resistant cell lines and parent cells showed expression of and at the mRNA level. To confirm these findings at the protein level, enzyme-linked immunosorbent assay was performed to detect IL-17 secreted by Cl66, Cl66-Dox, and Cl66-Pac cells (Physique?1C) and immunoblotting was performed for IL-17R levels in parent and resistant cells by Western blot analysis (Physique?1D). Cl66, Cl66-Dox, and Cl66-Pac cells showed positive protein expression of IL-17 and IL-17R. These results suggest that malignancy cells express IL-17R and might respond to IL-17 activation. Open in a separate window Physique?1 Expression levels of and in the parent, Cl66-Dox, and Cl66-Pac cell lines. A and B: Levels of CXCL1 (A) and CXCL5 (B) in the supernatant of Cl66, Cl66-Dox, Brazilin and Cl66-Pac, as determined by enzyme-linked immunosorbent assay (ELISA). C: Quantitative RT-PCR for the expression of and level of IL-17 in the supernatant of Cl66, Cl66-Dox, and Cl66-Pac, as determined by ELISA. D: Quantitative RT-PCR for the expression of and level of IL-17R in the Cl66, Cl66-Dox, and Cl66-Pac, and its confirmation by Western blot analysis. The values are fold switch (unpaired ligands, were evaluated in the tumors created by parent Brazilin Cl66 and drug-resistant (Cl66-Dox Brazilin or Cl66-Pac) cells. Cl66-Pac tumor lysates exhibited significantly higher mRNA levels of ELF3 (Physique?2A), (Physique?2B), (Physique?2C), and (Physique?2D) in comparison with tumors formed by the parent Cl66 cell collection (Physique?2). Insignificant higher levels of (Supplemental Physique?S1A) and CXCR2 (Supplemental Physique?S1B) were also observed in Cl66-Pac tumors in comparison with the parent Cl66 cells. Also, all of the tumors (Cl66, Cl66-Dox, and Cl66-Pac) expressed the and (Supplemental Physique?S1, C and D). Cl66-Pac tumors expressed the highest mRNA levels of and compared with Cl66 and Cl66-Dox tumors (differences are not significant). Together, higher expression levels of ligands, ligands. Quantitative RT-PCR for the expression of ligands (A), (B), (C), and (D) in main tumors generated from Cl66, Cl66-Dox, and Cl66-Pac. The values are fold switch (Cq; unpaired < 0.05 and ??< 0.01 versus Cl66-Dox; ?< 0.01 and ???0.001 versus Cl66-Pac. Cl66, Cl66-Dox, and Cl66-Pac cells were further treated with 10 ng/mL recombinant IL-17 for 24 hours, then the supernatant was collected and another chemotactic assay was performed with differentiated MPRO Clone 2.1 cells (neutrophils). The MPRO Clone 2.1 cells were with or without treatment of CXCR2 antagonist, SCH 527123, for 4 hours. The resistant cells recruited higher numbers of neutrophils in comparison with the parent cells (Physique?8, BCD). Overall, the IL-17Ctreated tumor cells recruited higher numbers of neutrophils; and targeting CXCR2 in these cells significantly inhibited the chemotaxis (Physique?8, BCD). These results suggest that IL-17 promotes chemotaxis of neutrophils through secretion of CXCR2 ligands, and blocking of CXCR2 signaling in the neutrophils can inhibit this IL-17Cdependent neutrophil recruitment. The interactions between neutrophils and malignancy cells were also examined. The differentiated HL60 cells were cocultured with Cl66, Cl66-Dox, and or Cl66-Pac cells. When cocultured with malignancy cells, HL60 cells expressed Th17 priming factor, (Supplemental Physique?S4, A and B),40,41 and (Supplemental Determine?S4C). G-CSF is the crucial regulator for neutrophil mobilization from bone marrow to the blood.13 However, the protein levels of these factors were below the detection levels of the enzyme-linked immunosorbent assay packages used (catalog figures DY206-05, DY1290-05, and DY214-05; R&D Systems; data not shown). Discussion Breast cancer is one of the most common malignancy types.
