Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: Primer sequences for CX3CL1/CX3CR1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: CX3CL1/CX3CR1 is overexpressed in human prostate cancer tissues and cell lines. CX3CL1 was overexpressed in the paracancerous tissues of spinal metastasis (SM) and healthy spines, as demonstrated by (A) western blotting, (B) densitometric analysis of western blotting, and RT-qPCR of the CX3CL1 mRNA expression level; GAPDH was as a loading control. * P<0.05, ** P<0.01 vs. respective control. n=12. (C) The concentration of CX3CL1 in the blood of patients with prostate cancer with SM, primary tumors and healthy cases was measured by ELISA. ** P<0.01 vs. primary; ## P<0.01 vs. healthy. CX3CR1 was overexpressed in prostate cancer with SM compared with primary cancer tissues as demonstrated by (D) western blotting, (E) densitometric analysis of western blotting, and RT-qPCR of the CX3CR1 mRNA expression level; GAPDH was used as a loading control. * P<0.05 vs. respective control. n=12. CX3CR1 expression in VCaP cells (derived from SM) was increased compared with 22RV1 cells (derived from primary tumors) and RWEP-1 cells (healthy prostate epithelial cells), as demonstrated by (F) western blotting and (G) densitometric analysis. ** P<0.01 vs. 22RV1; ## P<0.01 vs. RWEP1. (H) The mRNA expression of CX3CR1 in VCaP cells was increased compared with 22RV1 and PC-3 cells. * P<0.05 vs. 22RV1; ## P<0.01 vs. VCaP. (I) Clinical samples were collected and examined by immunohistochemical staining with CX3CL1 and CX3CR1 antibodies. Representative staining images are presented (×100 magnification). (J) Immunohistochemical staining of CX3CL1 and CX3CR1 is presented in healthy human spinal tissue, limb osseous tissues, primary prostate cancer and healthy prostate tissues (×100 magnification). SM, spinal metastasis; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif chemokine receptor 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: Expression of CX3CL1/CX3CR1 in human tissues. Clinical samples of (A) paracancerous tissues, (B) healthy bone tissues and (C) tumor tissues were collected and examined by western blotting using CX3CL1 and CX3CR1 antibodies. GAPDH was used as a loading control. CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif chemokine receptor 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: CX3CR1 promotes cell proliferation and inhibits cellular apoptosis. Stably-overexpressing CX3CR1 and control cell lines (VCaP and PC-3) were constructed by lentivirus infection. Specific siRNA was used to inhibit the expression of CX3CR1 (KD) in VCaP and PC-3 cells. The expression of CX3CR1 was determined by (A) western blotting and reverse transcription-quantitative polymerase chain reaction analysis (B, left: VCaP; right: PC-3). Cell counting kit-8 assays was used to demonstrate VCaP and PC-3 cell proliferation following lentivirus infection (C, left: VCaP; right: PC-3) or specific siRNA transfection (D, left: VCaP; right: PC-3). * P<0.05, ** P<0.01 vs. respective NC group. The experiment was repeated three times. Annexin V-FITC/PI assays were used to detect the cellular apoptosis of (E) VCaP and (F) PC-3 cells following lentivirus infection or siRNA transfection. The experiment was repeated three times. * P<0.05, ** P<0.01. OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif chemokine receptor 1; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Stable Transfection, Construct, Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Transfection, Over Expression, Negative Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: CX3CL1 induces cell migration and invasion. Cell migration was measured by scratch wound assay in (A) CX3CL1-treated, (B) CX3CR1 overex-pression and (C) CX3CR1-knockdown group at time point of 96 h. Representative images are presented (left panel; ×100 magnification). The results were summarized from three independent experiments (right panel). * P<0.05, ** P<0.01 vs. respective control. Cell invasion was determined by Transwell assay in (D) CX3CL1-treated, (E) CX3CR1 overexpression and (F) CX3CR1-knockdown group. Representative images are presented (upper panel; ×200 magnification). The results were summarized from three independent experiments (lower panel). * P<0.05 vs. respective control. (G) Cells were fixed and stained with F-actin in CX3CL1 treated, CX3CR1 overexpression and CX3CR1 knockdown group, the representative images are shown (magnification, ×400). OE, overex-pression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Migration, Scratch Wound Assay Assay, Transwell Assay, Over Expression, Staining, Negative Control

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: Kinases associated with the CX3CL1/CX3CR1 axis. The association between kinases relevant to CX3CL1 in the Src/focal adhesion kinase signaling pathway were predicted using the Ingenuity Pathway Analysis database. Src, proto-oncogene tyrosine-protein kinase Src; PI3K, phosphatidylinositol 3-kinase; ITGB3, integrin β-3; EGFR, epidermal growth factor receptor; PTK2B, protein-tyrosine kinase 2-β.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques:

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Expressing, Western Blot, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: EGFR, Src and FAK inhibitors reverse cell migration induced by CX3CL1 in PC-3 cells. (A) Cell migration was measured by scratch wound assay. Representative images are presented (×200 magnification). (B) The results were summarized from three independent experiments. * P<0.05 vs. without CX3CL1; # P<0.05 vs. non-inhibitor-treated group. CX3CL1, C-X3-C motif chemokine ligand 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: Migration, Scratch Wound Assay Assay

Journal: International Journal of Oncology

Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

doi: 10.3892/ijo.2018.4487

Figure Lengend Snippet: CX3CL1/CX3CR1 facilitates the spinal metastasis of prostate cancer in vivo . (A) A total of 1×10 6 PC-3-control or PC3-CX3CR1-overexpressing cells were resuspended in 200 µ l serum-free medium and injected into the left ventricles of mice. After 6–8 weeks, the mice received a positron emission tomography scan. If a suspected spinal metastasis was found, the lesion underwent a further micro-CT scan. The micro-CT scans illustrated local destruction (arrow) in the affected vertebrae of the tumorigenic mice. H&E staining and immunohistochemical staining of CX3CL1, CX3CR1 and AR is presented in (B) paracancerous tissues, tumor tissues and (C) healthy spine tissues (×100 magnification). CT, computed tomography; H&E, hematoxylin and eosin; CX3CL1, C-X3-C motif chemokine ligand 1; CX3CR1, C-X3-C motif chemokine receptor 1.

Article Snippet: The sections were incubated in H 2 O 2 (3%) for 10 min, and blocked in 5% goat serum (cat. no. AR1010; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 60 min at room temperature, followed by anti-CX3CL1 or anti-CX3CR1 or androgen receptor (AR; 1:500; cat. no. 5153; CST) antibodies at 37°C for 60 min.

Techniques: In Vivo, Injection, Positron Emission Tomography, Micro-CT, Staining, Immunohistochemical staining, Computed Tomography