Hepatic expression ofANG2 RNA in metastatic colorectal cancer

Abstract

We examined the RNA content of the gene encoding angiopoietin (Ang)-2, a modifier of angiogenesis, in hepatic metastases of colorectal cancer (CRC) to explore the role of this protein in neovascularization of metastatic foci. Metastatic CRC exhibited notable blood flow and tumor vessel formation at tumor frontiers. Reverse-transcription polymerase chain reaction assays indicated that the ANG2 RNA content was greater in metastatic CRC than in primary CRC. Investigation of metastatic foci using laser capture microdissection revealed that the RNA content of ANG2, but not ANG1, increased from the bordering liver region to the periphery of the metastatic disease, and also from the periphery to the intermediate portion of the metastatic lesion; immunohistochemical analysis confirmed that there was a corresponding gradual increase in Ang-2 protein expression. Tie-2, a receptor for angiopoietins, was preferentially expressed in the bordering liver region rather than in metastatic CRC. Vascular endothelial growth factor (VEGF) also exhibited an expression pattern similar to that of Ang-2, and there was a significant correlation between the RNA content of ANG2 and that of VEGF in dissected samples (P ‫؍‬ .002). Western blot analysis suggested that expression of Ang-1, Ang-2, Tie-2, and VEGF may be regulated at a transcriptional level. C olorectal cancer (CRC) is a common malignancy worldwide. Although 84%-92% of patients with CRC are treated with surgical resection, more than half of these patients subsequently develop disease recurrence, 1 the most common type of recurrence being CRC metastatic to the liver, which often is associated with mortality. 2,3 Metastasis of CRC to the liver is a complex multistep process, which includes adherence of metastatic cells to endothelial cells (ECs), invasion across the endothelial basement membrane, cell proliferation, and neovascularization. 4 It was reported that tumor vessels appeared in human liver metastases when metastatic foci grew to 200 m in diameter and that the density of tumor vessels increased as tumor size increased. 5 Angiogenesis is essential for tumor growth and expansion, because the resulting blood vessels supply malignant cells with sufficient oxygen and nutrition. 6,7 Therefore, interruption of this process is considered to be a strategy for preventing CRC metastases to the liver.Recent studies have focused on novel endothelial growth factors such as angiopoietins (Ang), which are ligands for the endothelium-specific tyrosine kinase receptor Tie-2. 8 -10 Ang molecules play crucial roles in normal vascular development and in embryonic angio-

Hypoxia-induced up-regulation of angiopoietin-2 in colorectal cancer

“…of necrosis, in accordance with our previous study (10). Considering the similar expression patterns of Glut-1 and the role of Ang-2 in tumor angiogenesis, it is reasonable to attribute this phenomenon to tissue hypoxia, an environmental factor relating to several tumor biological characteristics including angiogenesis.…”

“…LCM was performed in frozen tissue samples using the LM200 LCM system (Arcturus Engineering, Santa Clara, CA) as described previously (10).…”

“…Complementary DNA (cDNA) was generated using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Semiquantitative analyses for the expression of Ang-2 and Glut-1 mRNA were performed by the duplex RT-PCR techniques, as described previously (10). We used ß-actin as the internal standard.…”

Antisense to Cyclin D1 Inhibits Vascular Endothelial Growth Factor–Stimulated Growth of Vascular Endothelial Cells: Implication of Tumor Vascularization

“…RNA extraction was carried out with TRIzol reagent in a single-step method and cDNA was generated with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Semiquantitative analyses of the expression of VEGF RNA were done using the duplex reverse transcription-PCR technique as described previously (30). h-Actin was used as the internal standard.…”

Ep-CAM is a significant prognostic factor in pancreatic cancer patients by suppressing cell activity

“…Complementary DNA was generated from 1 mg RNA with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) as previously described (Tsujie et al, 2003). The qRT-PCR assays were carried out using the Light Cycler (Roche Diagnostics, Mannheim, Germany) as previously described (Ogawa et al, 2004) and the amount of target gene expression was calculated. The target gene expression was normalized relative to the expression of glyceraldehydes-3-phosphate dehydrogenase, which was used as an internal control.…”