Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: Activation of Dickkopf-1 (DKK-1) in hepatic stem cell-like hepatocellular carcinoma (HpSC-HCC). ( A ) Heatmap images of 793 genes differentially expressed between HpSC-HCC and mature hepatocyte-HCC (MH-HCC). HpSC-HCC and MH-HCC cases are indicated as yellow or blue boxes, respectively. Each cell in the matrix represents the expression level of a gene in an individual sample. Orange and blue cells depict high and low expression levels, respectively, as indicated by the scale bar. Genes encoding epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP), and DKK-1 were activated in HpSC-HCC. ( B ) qRT-PCR analysis of genes encoding EpCAM and DKK-1 in sorted EpCAM +/− Huh7 cells. ( C ) DKK-1 levels were higher in EpCAM + cells than in EpCAM − cells as assessed by Western blot analysis ( D ) qRT-PCR analysis of DKK-1 expression in HpSC-HCC (red circle) and MH-HCC (blue circle). ( E ) Serum DKK-1 levels in HpSC-HCC (red circle) and MH-HCC (blue circle). ( F ) Kaplan–Meier curves of overall survival in HpSC-HCC (red line) and MH-HCC (blue line). ( G ) Kaplan–Meier curves of recurrence-free survival in HpSC-HCC (red line) and MH-HCC (blue line).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: Serum Dickkopf-1 (DKK-1) levels as a diagnostic marker for hepatocellular carcinoma (HCC). ( A ) Serum DKK-1 levels were significantly higher in patients with HCC than in those without HCC. ( B ) A receiver operating characteristic (ROC) curve of serum DKK-1, alpha-fetoprotein (AFP), and protein induced by vitamin K absence-II (PIVKA-II) levels. The optimal cutoff value of serum DKK-1 levels for the diagnosis of HCC was 262.2 pg/mL. The area under the ROC curve of serum DKK-1 levels was 0.708. The sensitivity and specificity of DKK-1 were 80.5% and 53.2%, respectively. ( C , D ) The correlation (r) between serum DKK-1 and serum AFP levels was 0.44 ( p < 0.001; C ), whereas that between serum DKK-1 and serum PIVKA-II levels was 0.27 ( p < 0.001; D ).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques: Diagnostic Assay, Marker, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: Relationship between serum Dickkopf-1 (DKK-1) levels and clinicopathological characteristics of hepatocellular carcinoma (HCC) and liver function. ( A ) Serum DKK-1 levels were elevated according to HCC size ( p < 0.001). ( B ) No difference was observed between solitary and multinodular HCC cases. ( C ) Serum DKK-1 levels were higher in the massive/diffuse type than in the nodular type, according to Eggel’s classification. ( D , E ) Serum DKK-1 levels were elevated in portal vein invasion (Vp)-positive patients compared with that in Vp-negative patients ( p < 0.001; D). Serum DKK-1 levels were elevated in Vp3/4 patients compared with those in Vp- or Vp1/2 patients (*** p < 0.001 and * p < 0.05; E ). ( F , G ) Serum DKK-1 levels were significantly higher in advanced-stage patients classified according to the BCLC ( F ) and UICC stages ( G ). ( H , I ) Serum DKK-1 levels were not related to hepatic reserve assessed by using the Child–Pugh score ( H ) and albumin-bilirubin (ALBI) grade ( I ).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: Overall (3-year) survival. ( A ) The receiver operating characteristic (ROC) curve analysis for survival showing the cutoff value of serum Dickkopf-1 (DKK-1) levels to be 365.9 pg/mL; area under the ROC curve (AUC) is 0.601, sensitivity is 71.9%, and specificity is 52.9%. ( B ) Kaplan–Meier survival analysis, indicating statistically significant correlation of elevated serum DKK-1 levels (≥365.9 pg/mL) with worse prognosis ( p < 0.001). ( C , D ) In both albumin-bilirubin (ALBI) grade 1/2a (good liver function) and 2b/3 (poor liver function) cohorts, the high-serum DKK-1 group showed a statistically significant worse prognosis ( p < 0.001). ( E , F ) The prognosis was significantly worse in patients with elevated DKK-1 levels in early stages treated by surgical resection ( p = 0.045) as well as in those in an advanced stage treated by systemic therapy ( p = 0.024).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: Dickkopf-1 (DKK-1) expression in resected hepatocellular carcinoma (HCC) tissues. ( A , B ) The staining area was evaluated and scored on four levels (none = 0, focal = 1, multifocal = 2, and diffuse = 3; A ). The staining intensity was evaluated and scored on three levels (none = 0, mild = 1, and strong = 2; B ), and the sum of both levels was used as the immunostaining score (0–5). ( C ) The Kaplan–Meier survival analysis indicates that DKK-1-high patients showed worse recurrence-free survival than DKK-1-low patients with statistical significance ( p = 0.026). ( D ) The overall survival rate did not differ between the high- and low-DKK-1 groups.

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: mRNA and protein expression of DKK-1 in HCC cell lines and in Huh7 and Hep3B cells knocked down for DKK-1 using siRNA. ( A ) DKK-1 mRNA expression was higher in Huh7 and Hep3B cells than in HLE and HLF cells ( p < 0.001). ( B ) DKK-1 protein levels in the culture supernatant were significantly higher in Huh7 and Hep3B cells than in HLE and HLF cells ( p < 0.05), as evaluated using ELISA. ( C ) DKK1 mRNA levels were significantly decreased in Huh7 and Hep3B cells following siRNA transfection (* p = 0.013 and *** p < 0.001, respectively; n = 3 for each group). ( D ) DKK-1 protein levels in the culture supernatant were also significantly decreased in Huh7 and Hep3B cells following siRNA transfection ( p < 0.001; n = 4 for each group). Proliferation, migration, and invasion in Huh7 and Hep3B cells. ( E ) The proliferation of Huh7 and Hep3B cells was significantly suppressed by DKK-1 knockdown at 24, 48, and 72 h ( p < 0.001 at 24, 48, and 72 h; n = 6 for each group. ( F ) Migration capacity was attenuated by DKK-1 knockdown in Huh7 and Hep3B cells with statistical or borderline significance ( p = 0.017 and p < 0.001, respectively; n = 3 for each group). ( G ) Invasion capacity was attenuated by DKK-1 knockdown in Huh7 and Hep3B cells with borderline statistical significance (** p = 0.069 and p < 0.001, respectively; n = 3 for each group).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Migration, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma

doi: 10.3390/ijms23052801

Figure Lengend Snippet: The effect of Dickkopf-1 (DKK-1) on angiogenesis and tumorigenesis. ( A ) Time-lapse image analysis of tube formation using human umbilical vein endothelial cells. DKK-1 treatment slightly accelerated tube formation compared with the control 2 h after treatment. This effect was abolished by the administration of anti-DKK-1 neutralizing antibodies. ( B , C ) Co-administration of anti-DKK-1 neutralizing antibody with Huh7 cells suppressed tumor growth in NOD/SCID mice compared with control IgG ( n = 3 for each group).

Article Snippet: Quantitative PCR probes for DKK-1 (Hs00183740_m1) were procured from Applied Biosystems (Foster City, CA, USA).

Techniques: Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 1. In vitro evaluation of the proper effect of the BHQ880 antibody over Dkk1 protein neutralization. (A) Representative images of B1a/7TGC cell reporter line activation when stimulated by the GSK-3 inhibitor CHIR99021 (positive control) or by increasing doses of recombinant mouse Wnt3a (mWnt3a), and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. Non-treated (NT); # vs. 0 ng/mL mWnt3a. (B) Representative images of B1a/7TGC cell reporter line inactivation when previously stimulated cells with 150 ng/mL of mWnt3a were turned off through the addition of increasing doses of recombinant rDkk1, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; # vs. 0 ng/mL rDkk1. (C) Representative images of the BHQ880 neutralizing effect over rDkk1 protein in the B1a/7TGC reporter cells stimulated with 150 ng/mL of mWnt3a, together with 250 ng/mL of rDkk1 and increasing doses of BHQ880 antibody, and data obtained from the evaluation of the percentage of GFP+ cells after 48 hours. *vs. NT; & vs. Wnt3a; # vs 0 ng/mL BHQ880. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between groups. In all cases, scale bars = 50 μm and the data are presented as the mean + SEM. **p <0.01; ***p <0.001; #p <0.05; ##p <0.01; ###p <0.001; &&p <0.01.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: In Vitro, Neutralization, Activation Assay, Positive Control, Recombinant

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 2. Protein expression of Dkk1 in the healthy and injured spinal cord. The figure shows representative images from the immunohistochemical evaluation of Dickkopf-1 (Dkk1) protein expression in the non-lesioned (NL) (n = 5) spinal cord (A) and after SCI at 24 hours post-injury (hpi) (n = 4), and 3 days post-injury (dpi), 7 dpi (n = 4), 14 dpi (n = 5), 28 dpi (n = 3), and 42 dpi (n = 5) at the lesion epicenter (B-G). Dashed line squares in A-G indicate the areas shown in the corresponding higher magnification images. Scale bars = 500 μm. Inserts scale bars = 50 μm. (H) Densitometric analysis of Dkk1 immunolabeling in the NL spinal cord and at the injury epicenter after SCI from 24 hpi to 42 dpi. A one-way ANOVA followed by a Bonferroni post-hoc test was used to assess the existence of significant differences between NL and the different times post-injury. In all cases, the data are presented as the mean + SEM. **p <0.01; ***p <0.001.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Expressing, Immunohistochemical staining, Immunolabeling

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

doi: 10.1016/j.biopha.2024.116792

Figure Lengend Snippet: Fig. 3. Analysis of the circulating levels of Dkk1 in Serum samples from non- lesioned (NL) and lesioned rats at 24 hours after damage (SCI). The figure shows data obtained from the quantitative analysis of serum circulating Dkk1 protein levels measured by ELISA in NL animals (n = 9) and injured animals 24 hours after SCI (n = 9). No significant differences were observed between groups. A two-tailed t test was used to determine the existence of significant differences between groups. The data represent the mean ± SEM.

Article Snippet: Finally, to validate the specificity of the Dkk1 primary antibody used in these experiments, we pre-adsorbed the antibody with its corresponding blocking peptide (Proteintech, AG15110) at a 20-fold weight/ weight excess following the protocol previously described [51], and we observed that pre-adsorption abolished Dkk1 immunostaining, thus confirming antibody specificity (Fig. S1).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 3. Upregulation of Dkk1 expression in pagetic osteoblasts and BMSCs. (A) The relative expression of Dkk1 was determined by real-time PCR, and values are expressed relative to the mean level of expression in the nonpagetic groups, which have been normal- ized to 1. The number of samples was the same as in Fig. 2. (B) The concentration of secreted Dkk1 protein in conditioned media samples as determined by ELISA. The con- ditioned media samples were collected dur- ing the last 72 h of incubation from the cell cultures described above. Data in A and B are presented as mean ± SE. ap < 0.05 and bp < 0.01 vs. control. (C) Correlation be- tween levels of Dkk1 mRNA in cells and se- creted Dkk1 protein in conditioned media. Levels of Dkk1 mRNA are expressed rela- tive to the expression of ribosomal RNA. The correlation was calculated using Deming (model II) linear regression (r 0.88, p < 0.0001).

Article Snippet: A total of 100 l of standard or sample was loaded per well and incubated overnight at 4°C, washed, and incubated with biotinylated goat antihuman Dkk1 IgG (R&D Systems) diluted to a concentration of 0.2 g/ml in dilution buffer.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 7. Schema showing possible effects and interactions of the changes in osteoblast gene expression shown in this study. Solid lines denote production of a factor by that cell, and broken lines indicate a regulatory influence of a factor. The overproduction of IL-1 and IL-6 by the pagetic osteoblast will result in osteoclast proliferation, leading to further increases in IL-6 levels in the bone marrow microenvironment. Increased pro- duction of Dkk1 by the pagetic osteoblast will further increase IL-6 levels and reduce osteoblast proliferation. This combination of effects could account for the development of lytic lesions in early phase Paget’s disease. Over time, both the excess of Dkk1 and of IL-6 will result in increased differentiation of osteoblasts, thus promoting mineralization. This could contribute to the development of sclerosis in longer-standing pagetic lesions.

Article Snippet: A total of 100 l of standard or sample was loaded per well and incubated overnight at 4°C, washed, and incubated with biotinylated goat antihuman Dkk1 IgG (R&D Systems) diluted to a concentration of 0.2 g/ml in dilution buffer.

Techniques: Gene Expression

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 3. Upregulation of Dkk1 expression in pagetic osteoblasts and BMSCs. (A) The relative expression of Dkk1 was determined by real-time PCR, and values are expressed relative to the mean level of expression in the nonpagetic groups, which have been normal- ized to 1. The number of samples was the same as in Fig. 2. (B) The concentration of secreted Dkk1 protein in conditioned media samples as determined by ELISA. The con- ditioned media samples were collected dur- ing the last 72 h of incubation from the cell cultures described above. Data in A and B are presented as mean ± SE. ap < 0.05 and bp < 0.01 vs. control. (C) Correlation be- tween levels of Dkk1 mRNA in cells and se- creted Dkk1 protein in conditioned media. Levels of Dkk1 mRNA are expressed rela- tive to the expression of ribosomal RNA. The correlation was calculated using Deming (model II) linear regression (r 0.88, p < 0.0001).

Article Snippet: Microtiter plates (Greiner) were coated with 100 l of anti-Dkk1 polyclonal antibody (R&D Systems) at a concentration of 1 g/ml in PBS, pH 7.2.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Differential gene expression in cultured osteoblasts and bone marrow stromal cells from patients with Paget's disease of bone.

doi: 10.1359/jbmr.061108

Figure Lengend Snippet: FIG. 7. Schema showing possible effects and interactions of the changes in osteoblast gene expression shown in this study. Solid lines denote production of a factor by that cell, and broken lines indicate a regulatory influence of a factor. The overproduction of IL-1 and IL-6 by the pagetic osteoblast will result in osteoclast proliferation, leading to further increases in IL-6 levels in the bone marrow microenvironment. Increased pro- duction of Dkk1 by the pagetic osteoblast will further increase IL-6 levels and reduce osteoblast proliferation. This combination of effects could account for the development of lytic lesions in early phase Paget’s disease. Over time, both the excess of Dkk1 and of IL-6 will result in increased differentiation of osteoblasts, thus promoting mineralization. This could contribute to the development of sclerosis in longer-standing pagetic lesions.

Article Snippet: Microtiter plates (Greiner) were coated with 100 l of anti-Dkk1 polyclonal antibody (R&D Systems) at a concentration of 1 g/ml in PBS, pH 7.2.

Techniques: Gene Expression