δ-catenin regulates Rho GTPase activity in LECs.

<p>HMVEC-LLy cells were transfected with an empty vector or δ-catenin expression vectors for 48 hours. Cell lysate was collected and analyzed for activation of Rac1 (Panel A), Cdc42 (Panel B), and RhoA (Panel C). Activation of Rac1 and Cdc42 was determined by using a PAK1- pull-down assay while active RhoA was pulled down with Rhotekin-agarose beads. For knockdown experiments, HMVEC-LLy cells were transfected with shRNA for δ-catenin or control shRNA constructs for 48 hours. Cell lysate was collected and analyzed for activation of Rac1 (Panel D), Cdc42 (Panel E), and RhoA (Panel F) by Western blotting. HMVEC-LLy cells transfected with a vector control or δ-catenin expression vector plus a vector control or Rac1N17 were incubated on Matrigel for 24 hours. The number of vascular branch points was counted in 10 randomly selected fields under microscopy (Panel G). All experiments were repeated three times. Mean and SE were plotted. * p<0.05. </p

Loss of δ-catenin impairs lymphangiogenesis and tumor growth in an experimental metastasis mouse model.

<p>3LL cells (1x10⁵) were injected into tail vein of wild type littermates and δ-catenin null mice. Two weeks after the injection, lungs were excised from mice and images were taken (Panel A). Arrows point to tumor metastases in lungs. Lung weight (Panel B) and lung surface metastases (Panel C) were quantified and graphed. *p<0.05. Tumor metastases were sectioned and stained with an antibody against Lyve-1 (Panel D). Lyve-1 positive vascular structures were counted in 10 randomly selected high power fields under microscopy and graphed (Panel E). *p<0.05. Representative images were shown. This experiment was repeated twice and each time 5 mice per group were used. </p

Additional file 1: of Dynamic Hyaluronan drives liver endothelial cells towards angiogenesis

Table S1. Sequences of primers used for quantitative real time PCR on mice. (DOCX 25 kb

Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

<div><p>The role of nuclear receptor PXR in detoxification and clearance of xenobiotics and endobiotics is well-established. However, its projected role in hepatic cancer is rather illusive where its expression is reported altered in different cancers depending on the tissue-type and microenvironment. The expression of PXR, its target genes and their biological or clinical significance have not been examined in hepatic cancer. In the present study, by generating DEN-induced hepatic cancer in mice, we report that the expression of PXR and its target genes CYP3A11 and GSTa2 are down-regulated implying impairment of hepatic detoxification capacity. A higher state of inflammation was observed in liver cancer tissues as evident from upregulation of inflammatory cytokines IL-6 and TNF-α along with NF-κB and STAT3. Our data in mouse model suggested a negative correlation between down-regulation of PXR and its target genes with that of higher expression of inflammatory proteins (like IL-6, TNF-α, NF-κB). In conjunction, our findings with relevant cell culture based assays showed that higher expression of PXR is involved in reduction of tumorigenic potential in hepatic cancer. Overall, the findings suggest that inflammation influences the expression of hepatic proteins important in drug metabolism while higher PXR level reduces tumorigenic potential in hepatic cancer.</p></div

PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A.

<p>Real-time PCR analysis was performed for <i>Bcl-xl</i>, <i>Bcl-2</i>, <i>Cdk2</i> and <i>Cdk4</i> with total RNA extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Levels of inflammatory proteins are enhanced in DEN-induced hepatic cancer.

<p>A. Real-time PCR analysis was performed for <i>Tnf-α</i>, <i>p65</i>, and <i>Stat3</i> with total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell extracts of control and DEN-induced hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse TNF-α, anti-mouse P65, and anti-mouse IL-6 antibody. Bands of human and mouse P65 were detected at 65 KDa and 60 KDa respectively. β-Actin antibody served as control; C. The relative endogenous TNF-α, P65 and IL-6 protein expressions in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with seven samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Correlations between the reduced levels of PXR and PXR-regulated CYP3A11 DME with enhanced levels of inflammatory proteins in hepatic cancer.

<p>Relative protein levels of TNF-α, P65, and IL-6 were correlated with PXR and CYP3A11 protein levels using Pearson’s correlation coefficient (r) in scattered plot. Respective p-value (p) represents the significance between the correlations.</p

Assessment of expression levels of nuclear receptor PXR, CAR and RXR-α in DEN-induced hepatic cancer.

<p>A. Real-time PCR analysis was performed for PXR, CAR and RXR-α genes with the total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell lysate of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse PXR antibody, anti-mouse CAR antibody and anti-mouse RXR-α antibody. β-Actin antibody served as control; C. The relative endogenous protein expression levels of PXR, CAR and RXR-α in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice. The values are represented the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Altered expression of PXR-regulated genes in DEN-induced hepatic cancer.

<p>A. Total RNA was extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. Real-time PCR analysis was performed for <i>Cyp3A11</i>, <i>Gsta2</i> and <i>Mrp3</i>. For <i>Mdr1</i> semi-quantitative PCR was performed. GAPDH served as an internal control. B. Cell lysates of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse Cyp3a11 antibody (upper panel). β-Actin antibody served as control (lower panel); C. The relative endogenous Cyp3a11 protein expression in control and DEN-induced hepatic cancer was quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. The P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Overexpression of FLAG-tagged mPXR gene at protein level and its effect on tissue histology.

<p>A. Liver tissue extracts of Control and transgenic mice were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-FLAG antibody and anti-mouse PXR antibody (upper panels). β-Actin antibody served as control (lower panel). B. Control and transgenic mice were sacrificed and their livers were removed. Paraffin sections were prepared from control and transgenic mice livers harvested in the experiment and immunodetection were performed with anti-FLAG antibody (indicated by arrows). The immunodetected tissues were viewed and recorded at 20X magnification using a light microscope. The experiments were performed with three samples of each of the controls and the transgenic mice.</p