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1.
Figure 6

Figure 6. Model for the mechanism of osteopontin-dependent MMP-2 up-regulation in hepatocellular carcinoma.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

The results of the present study show that osteopontin up-regulates SDF-1α, CXCR4, and MMP-2 via integrin αvβ3 and CD44v6, as well as PI-3K/Akt and JNK. These data are consistent with an osteopontin-induced autocrine loop of SDF-1α/CXCR4 activation that leads to tumor invasion, in part via MMP-2 secretion.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.
2.
Figure 4

Figure 4. The rhOPN-induced expression of CXCR4 and MMP-2 depends on the PI3K/Akt and JNK pathways.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

rhOPN induced the phosphorylation of Akt (A), p38 (B) and JNK (C), but not ERK1/2 (D) in SMMC7721 and HepG2 cells. The cells (1×106 cells/ml) were left untreated or stimulated with rhOPN (50 nM) for 30 min and total cell lysates were subjected to Western blotting analysis. After pretreatment of SMMC7721 cells (E) or HepG2 cells (F) with PD98059 (ERK inhibitor, 100 µM), SB203680 (p38 inhibitor, 100 µM), SP600125 (JNK inhibitor, 100 µM), LY294002 (PI-3K inhibitor, 100 µM) or DMSO for 45 min, the cells were treated with rhOPN (50 nM) for 48 hours and total cell lysates were subjected to Western blotting analysis for MMP-2 or CXCR4.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.
3.
Figure 1

Figure 1. SDF-1α, CXCR4 and MMP-2 expression are induced by rhOPN in SMMC7721 and HepG2 cells.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

SMMC7721 cells (A) or HepG2 cells (B) were stimulated with various concentrations of rhOPN for 48 hours, the cells were collected, and SDF-1α, CXCR4 and MMP-2 were detected by Western blotting assay. (C) HepG2 cells were stimulated with 50 nM rhOPN for increasing time frames, the cells were collected, and SDF-1α, CXCR4 and MMP-2 were detected by Western blotting assay. (D) SDF-1 ELISA of culture supernatants (SMMC7721 and HepG2) after 0–72 hours of rhOPN (50 nM). (E) MMP-2 activity was analyzed by gelatin zymography after stimulation with 50 nM rhOPN for 60 hours in the SMMC7721 and HepG2 cell lines. *denotes P<0.05 versus control. The results presented are representative of at least three independent experiments.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.
4.
Figure 2

Figure 2. Effects of the SDF-1α/CXCR4 axis on rhOPN-induced MMP-2 expression and activity.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

(A) Verification by Western blotting of the miRNA knockdown of CXCR4 showed a significant reduction of the CXCR4 protein in all clones (1-4, 2-4, 3-1, 4-4). After blocking the SDF-1α/CXCR4 axis with miRNA-CXCR4 and inhibitors (SDF-1 neutralizing antibody at 100 ng/ml, CXCR4 inhibitor 12G5 at 50 µg/ml, or CXCR4 inhibitor AMD3100 at 500 ng/ml), the cells were stimulated by rhOPN in serum-free medium for 60 hours, the cells were collected and analyzed by Western blotting in SMMC7721 cells (C) and in HepG2 cells (F). The supernatants of SMMC7721 cells (B) and HepG2 cells (E) were analyzed by gelatin zymography. (D) and (G) show the densitometric ratio of MMP-2 protein/α-tubulin. (H) Western blotting was used to assay the MMP-2 expression induced by rhOPN (50 nM) or/and SDF-1 (30 nM) for 48 hours. * denotes P<0.05 versus control. The results presented are representatives of at least three independent experiments.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.
5.
Figure 5

Figure 5. The rhOPN-induced SMMC7721 cells invasion is mediated by CXCR4.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

The invasion assay was set up in transwell chambers. Cell culture inserts with 8.0-µm pore diameter were used to separate the top and bottom chambers. 60 µl of ECM gel solution was added to the upper compartment of each cell culture insert and dried overnight under laminar air flow. SMMC7721 parent cells, vector controls, and CXCR4 miRNA clones (1-4, 2-4) were plated onto the membrane of the top chamber, and rhOPN was administered to the bottom chamber. After 48 hours, the cells that had invaded to the lower surface of the membrane were enumerated. (A) Bright-field image of cells migrated to the bottom of chambers on the inserts (200× original magnification). (B) Quantification of cell invasion. The open bars represent no osteopontin, the filled bars represent rhOPN treatment. In each chamber, six fields were counted at 200× magnification for each condition by two investigators. * indicates P<0.05 versus control. The data are representative of three experiments.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.
6.
Figure 3

Figure 3. Integrin αvβ3 and CD44 mediated OPN-induced CXCR4 expression in SMMC7721 and HepG2 cells.. From: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines.

FACS analysis using monoclonal antibodies to αvβ3 integrin (left) and CD44 (right) was done for SMMC7721 cells (A) and HepG2 cells (C), stimulated by rhOPN for 24 hours. The grey area represents isotype control, while the dark line represents the control and the grey line represents the experimental group. SMMC7721 (E) and HepG2 (F) cells were treated with rhOPN (50 nM), in the presence of neutralizing antibodies to integrin αvβ3 or CD44v6, or control IgG. After 60 hours, the cells were collected and Western blotting was performed to detect CXCR4. (B), (D), (G) and (H) are quantitative evaluations. The results are shown as mean ± standard deviation (n = 3). * denotes P<0.05 compared to rhOPN treatment in the absence of antibody.

Rihua Zhang, et al. PLoS One. 2011;6(8):e23831.

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