Pancreatic cancer cell-derived vascular endothelial growth factor is biologically activein vitro and enhances tumorigenicityin vivo

Abstract

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator that acts by binding to high-affinity transmembrane receptors. Although both VEGF and its receptors are overexpressed in human pancreatic ductal adenocarcinoma (PDAC), this malignancy is not generally considered to be highly vascular. It is not known, therefore, whether the abundance of VEGF in PDAC is biologically relevant. To address this issue, we measured the angiogenic effects of pancreatic cancer cell-derived VEGF in an in vitro endothelial cell proliferation assay and characterized the consequences of suppressing VEGF expression on pancreatic tumor growth in an athymic nude mouse model. We found that human pancreatic cancer cell lines secrete large quantities of biologically active VEGF into conditioned medium (CM). Stable transfection of an anti-sense VEGF 189 (AS-VEGF 189 ) expression construct into PANC-1 pancreatic cancer cells resulted in decreased VEGF expression and secretion, a decreased capacity of the resultant CM to enhance endothelial cell proliferation and a significant attenuation of tumor cell proliferation in vitro. Furthermore, when injected into athymic nude mice, AS-VEGF 189 -expressing cells exhibited an 80% decrease in tumor growth compared with control cells. These results support the hypothesis that VEGF promotes pancreatic cancer growth in vivo and suggest that anti-VEGF therapy may be useful in the treatment of this disease.

Perlecan and Tumor Angiogenesis

“…More than 1300 articles have been published in this field during the past 5 years. It is reported that many tumor cell lines can synthesize VEGF in vitro, such as mouse RT101 tumor cells (our unpublished data), human and rat prostate carcinoma cell lines (Chen et al 2000), human glioma (Im et al 1999), chondrosarcoma (Furumatsu et al 2002), renal cell carcinoma (Shi and Siemann 2002), and pancreatic cancer cell lines (Luo et al 2001). VEGF mRNA level is upregulated in a wide variety of human tumors, including breast, lung, kidney, gastrointestinal tract, bladder, and ovarian carcinomas (reviewed in Ferrara 1999), and the serum level of VEGF is increased in many cancer patients (Talks and Harris 2000).…”

“…Overexpression of VEGF stimulates angiogenesis in human ovarian cancer xenografts (Duyndam et al 2002) and enhances tumorigenicity of U251 MG glioma cells in vivo (Ke et al 2002). Inhibition of tumor growth and angiogenesis in animal models has been demonstrated by neutralizing anti-VEGF antibody (Borgstrom et al 1998), reduction of VEGF expression by antisense cDNA (Im et al 1999;Luo et al 2001) or antisense oligonucleotides (Shi and Siemann 2002), soluble Flt-1 (Goldman et al 1998;Hoshida et al 2002), dominant negative Flk-1 (Millauer et al 1994), anti-KDR antibody (Sweeney et al 2002), Flk-1/KDR kinase inhibitor SU5416 (Fong TA et al 1999;Takamoto et al 2001), anti-VEGF 189 or anti-Flk-1 or Flt-1 ribozymes (Oshika et al 2000;Pavco et al 2000), and cytotoxic T-lymphocytes engineered to target Flk-1 (Niederman et al 2002). A synthetic peptide that blocks VEGF-KDR interaction or derives from the second Ig-like domain of Flt-1 can abolish angiogenesis in rabbit corneal and chick chorioallantoic membrane angiogenesis models, respectively (Binetruy-Tournaire et al 2000;Tan et al 2001).…”

Myoferlin plays a key role in VEGFA secretion and impacts tumor‐associated angiogenesis in human pancreas cancer

“…BxPC-3 cells were selected as a model, as they are known to produce more VEGFA than PANC-1 cells. 25 Thus, we measured the VEGFA concentration in conditioned medium and VEGFA gene expression in BxPC-3 cells after myoferlin-silencing. VEGFA concentration in the conditioned medium was determined by ELISA 48 hrs after siRNA transfection and normalized to the cell number.…”

Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

“…One tumor fragment was snap frozen in liquid nitrogen and stored at -80°C. Another tumor fragment was embedded in OCT, frozen in liquid nitrogen, and stored at -80°C for subsequent staining with anti-CD31 antibody (49). The third tumor fragment was fixed in formalin and embedded in paraffin for subsequent immunohistochemical staining.…”

“…First, for tumor tissues frozen in OCT, sections (5 μm thick) were mounted on poly-l-lysine-coated glass slides and airdried overnight at 23°C (49,50). Frozen tissue sections were fixed in acetone for 5 min, and endogenous peroxidase activity was blocked by incubation for 10 min with 3% hydrogen peroxide.…”