Icariin inhibits tumor-associated neutrophil recruitment via the CXCL8-CXCR2 axis and restrains pro-metastatic programs in colorectal cancer

Figure 1 SMAD4-deficient colorectal cancer cells SW480 recruit more C-X-C motif chemokine receptor 2+ tumor-associated neutrophils. A: The relative expression of protein SMAD4, C-X-C motif chemokine ligand 1 (CXCL1), and CXCL8; B: The mRNA relative expression of CXCL1 and CXCL8; C: The proportion of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils in the lower chamber. Results are illustrated as mean ± SD. ^aP < 0.05 vs the normal group, ^bP < 0.05 vs the Caco-2 group. CXCL: C-X-C motif chemokine ligand; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; CXCR: C-X-C motif chemokine receptor.

Figure 2 Icariin decreases SMAD4 protein expression and recruitment of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils. A: The mRNA relative expression of SMAD4, C-X-C motif chemokine ligand 1 (CXCL1), and CXCL8; B: The relative expression of protein SMAD4, CXCL1, and CXCL8; C: The proportion of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils in the lower chamber. Results are illustrated as mean ± SD. ^aP < 0.05 vs the control group. ICA: Icariin; CXCL: C-X-C motif chemokine ligand; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; CXCR: C-X-C motif chemokine receptor.

Figure 3 Icariin affects the biological behavior of colorectal cancer cells relying on the concentration. A: Cell counting kit-8: Cell proliferation; B: Flow cytometry: Apoptosis; C: Transwell: Cell invasion; D: Scratch assay: Cell migration. Results are illustrated as mean ± SD. ^aP < 0.05 vs the control group. ICA: Icariin.

Figure 4 Icariin represses epithelial-mesenchymal transition in colorectal cancer cells. A: The relative expression of protein E-cadherin, α-catenin, and vimentin; B: The mRNA relative expression of interleukin-1β and tumor necrosis factor-α. Results are illustrated as mean ± SD. ^aP < 0.05 vs the control group. ICA: Icariin; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β.

Figure 5 Icariin affects the biological behavior of colorectal cancer cells via up-regulating SMAD4 expression. A: The mRNA relative expression of SMAD4, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 8; B: Cell counting kit-8: Cell proliferation; C: Flow cytometry: Apoptosis; D: Transwell: Cell invasion; E: Scratch assay: Cell migration. Results are illustrated as mean ± SD. ^aP < 0.05 vs the small interfering SMAD4-negative control group, ^bP < 0.05 vs the small interfering SMAD4 group, ^cP < 0.05 vs the icariin + small interfering-negative control group. ICA: Icariin; CXCL: C-X-C motif chemokine ligand; siSMAD4: Small interfering SMAD4; NC: Negative control.

Figure 6 Icariin represses epithelial-mesenchymal transition in colorectal cancer cells as well as recruitment of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils, and promotes immune activation by up-regulating SMAD4 expression. A: The relative expression of protein E-cadherin, α-catenin, and vimentin; B: The proportion of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils in the lower chamber. Results are illustrated as mean ± SD. ^aP < 0.05 vs the small interfering SMAD4-negative control group, ^bP < 0.05 vs the small interfering SMAD4 group, ^cP < 0.05 vs the icariin + small interfering-negative control group. ICA: Icariin; siSMAD4: Small interfering SMAD4; NC: Negative control; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; CXCR2: C-X-C motif chemokine receptor 2.

Figure 7 Icariin inhibits recruitment of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils predominantly via the C-X-C motif chemokine ligand 1-C-X-C motif chemokine receptor 2 axis. A and B: The proportion of C-X-C motif chemokine receptor 2+ tumor-associated neutrophils in the lower chamber. Results are illustrated as mean ± SD. ^aP < 0.05 vs the control group, ^bP < 0.05 vs the icariin group. ICA: Icariin; CXCL: C-X-C motif chemokine ligand; CXCR2: C-X-C motif chemokine receptor 2.

Figure 8 Icariin may suppress migration and invasion of colorectal cancer cells through downregulating C-X-C motif chemokine ligand 8 expression. A: Transwell: Cell invasion; B: Scratch assay: Cell migration. Results are illustrated as mean ± SD. ^aP < 0.05 vs the control group. ICA: Icariin; CXCL: C-X-C motif chemokine ligand.

Figure 9 Icariin was able to inhibit tumor growth, accelerate apoptosis of cancerous tissues, and improve pathological morphology in colorectal cancer mice. A: Tumor growth; B: Histological observation; C: Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Results are illustrated as mean ± SD. ^aP < 0.05 vs the model group. ICA: Icariin; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Figure 10  Icariin impacts N1/N2 polarization of neutrophils in the cancer tissues of colorectal cancer mice. A: The positive rate of SMAD4, CD66b+, C-X-C motif chemokine ligand 1 (CXCL1), CXCL8, and C-X-C motif chemokine receptor 2 detected by immunohistochemistry; B: Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NI-type (CXCL10 and intercellular adhesion molecule 2) and N2-type (arginase 1 and matrix metalloproteinase 9) markers in tumor tissue samples. Results are illustrated as mean ± SD. ^aP < 0.05 vs the model group. ICA: Icariin; CXCL: C-X-C motif chemokine ligand; CXCR2: C-X-C motif chemokine receptor 2; Icam2: Intercellular adhesion molecule 2; Arg1: Arginase 1; MMP9: Matrix metalloproteinase 9.

Figure 11  Icariin suppresses epithelial-mesenchymal transition in colorectal cancer mice. A: The positive rate of protein E-cadherin and vimentin detected by immunohistochemistry; B: The relative expression of protein matrix metalloproteinase 2 and matrix metalloproteinase 9; C: Inflammatory factor levels. Results are illustrated as mean ± SD. ^aP < 0.05 vs the model group. ICA: Icariin; MMP: Matrix metalloproteinase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β.