Neuropilin-2 Mediated β-Catenin Signaling and Survival in Human Gastro-Intestinal Cancer Cell Lines

NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI) cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p<0.01). Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of β-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p<0.05). Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01). Collectively, our results demonstrate that tumor cell–derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells

Effect of several factors on the mechanical properties of pressure-sensitive adhesives used in transdermal therapeutic systems

The effects of coating thickness, type of adhesive and type and concentration of enhancer on the mechanical properties of two acrylic pressuresensitive adhesives (PSAs) were investigated using a 2 factorial design and an optimization technique. Sixteen formulations containing 0% or 10% of either caprylic acid or methyl laurate in two different PSAs. namely Duro-Tak® 87-2196 and Duro-Tak® 87–2097, were prepared. The adhesive properties of these laminates were evaluated by applying the 90° Dynamic Adhesive Strength Peel Test (90° DASPT) and 180° Release Liner Peel Test (180° RLPT). Coating thickness, concentration of enhancer, and type of adhesive did affect the 90° DASPT. For the 180° RLPT, the most significant factors were coating thickness and concentration of enhancer, with a strong interaction observed between the two. Coating thickness and concentration of enhancer were also used to create mathematical models that correlated these factors with the mechanical properties of the PSAs. For this purpose the optimization technique 32 was applied. It was found that the correlation of the above factors can be adequately described with polynomial equations, which can be used for predicting the mechanical properties of the laminates containing the above PSAs and methyl laurate (0%–10%)

Effect of NRP-2 on β-catenin expression and function.

<p>(A) Assessment of TCF reporter activity using the β-catenin-responsive TOPflash or mutant FOPflash reporters. Luciferase activities were measured after transient transfection of the reporter plasmids into shcntr and shNRP-2 CNDT 2.5 cells (B) Increased proteasome-mediated degradation of β-catenin in shNRP-2 cells. shcntr and shNRP-2 CNDT 2.5 cells were treated with 30 µM MG132 or equal volume of DMSO (a solvent for MG132) for 2 hours. Cell lysates were analyzed by immunoblotting using anti-β-catenin antibody (‘φ’, no treatment; ‘Ub’, ubiquitinated β-catenin). (C) Immunoblot analysis showing restoration of β-catenin level in the cytoplasmic and nuclear fractions after treatment with 30 µM MG132 for 2 hours. (D) Decreased levels of phosphorylated GSK3β in NRP-2 knockdown cells. Immunoblot analysis of phosphorylated GSK3β (at Ser-9) and GSK3β. (E) Restoration of β-catenin level after blocking GSK3β activity in NRP-2 knockdown cells. shcntr or shNRP-2 CNDT 2.5 knockdown cells were treated with increasing concentrations of LiCl for 24 hours and harvested for immunoblot analysis to detect β-catenin.</p

Assessment of NRP-2 expression in human gastric cancer tissues and cell lines.

<p>(A) Immunohistochemical staining for NRP-2 expression in representative tissue sections (20X) of normal human gastric mucosa and gastric cancer specimens (B) Immunoblot analysis of NRP-2 expression in six human GI cancer cell lines. Vinculin served as an internal loading control. (C) Generation of stable CNDT 2.5 cell lines with NRP-2 knockdown. Immunoblot analysis of NRP-1 and -2 expression in CNDT 2.5 cells transfected with shcntr or shNRP-2 plasmids (shNRP-2 clones; C6 and C10). Vinculin served as a loading control. (D) MTT assay results. Growth rates were no different between the control cells and NRP-2 knockdown clones. Bars indicate SEM. (E) Top: Mean number of cells that migrated in a Boyden chamber assay. Bottom: Representative images (10X) of migration assays. (F) Top: Mean number of cells that invaded in BioCoat Matrigel invasion chamber assay. Bottom: Representative images (10X) of invasion assays.</p

Effect of NRP-2 knockdown on expression of known metastatic genes.

<p>(<i>A</i>) Autoradiographic image of the array membrane showing differential expression of metastatic genes in shcntr (<i>left</i>) and shNRP-2 (<i>right</i>) cells. Circled spots indicate the position of the S100A4 gene. (B) Validation of S100A4 protein expression level in cells by immunoblot analysis. Actin served as a loading control. (C) Reduction of the steady-state level of β-catenin by NRP-2 knockdown. β-catenin expression in shcntr and shNRP-2 cells was determined by immunoblotting. Vinculin served as a loading control. (D) Verification of reduced β-catenin expression in shNRP-2 cells by immunofluorescence staining. shcntr and shNRP-2 CNDT 2.5 cells growing in chamber slides were fixed and immunostained with anti-β-catenin antibody (red). Nuclei were counterstained with DAPI (blue). (E) Validation of β-catenin reduction in a second NRP-2 knockdown gastric cancer cell line. NCI-N87 gastric cancer cells were infected with lentivirus containing shcntr or shNRP-2 constructs. Immunoblot analysis showed reduction in β-catenin expression in NCI-N87 NRP-2 knockdown cells. Actin served as a loading control. (F) β-catenin level in the cytoplasmic (Cyto) and nuclear (Nuc) fractions of shcntr and shNRP-2 cells. HSP90 (cytoplasmic) and LaminB (nuclear) served as fractionation controls.</p

Hypothetical schema for NRP-2-mediated activation of β-catenin signaling in gastrointestinal cancer cells.

<p>Left: In a GI cancer cell, increased inactive GSK3β inhibits destruction complex function, leading to stabilization of β-catenin that is free to translocate to the nucleus and activate transcription of target genes. Right: In a GI cancer cell with NRP-2 knockdown, the active destruction complex phosphorylates β-catenin and targets it for proteasome-mediated degradation, resulting in decreased activation of downstream target genes.</p

Effect of NRP-2 on in vivo growth of GI cancer cells.

<p>(A) Decreased tumor incidence and mean tumor volume after NRP-2 knockdown. Tumor incidence (10 mice) after liver injection with CNDT 2.5 shcntr cells or one of two shNRP-2 clones. (B) Top: Final liver tumor volumes in mice injected with shcntr and shNRP-2 clones. Bottom: Representative images of tumors. (C) Immunoblot analysis of NRP-2 in tumors from mice injected with CNDT 2.5 shcntr or shNRP-2 C6 cells [‘T1’; Tumor #1; ‘T2’; Tumor #2]. β-Actin served as a loading control.</p