Transfer of the sFLT-1 Gene in Morris Hepatoma Results in Decreased Growth and Perfusion and Induction of Genes Associated with Stress Response

Abstract

Purpose: Inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Therefore, monitoring of antiangiogenic approaches with functional imaging and histomorphometrical analyses are desirable to evaluate the biological effects caused by this treatment modality.Experimental Design: Using a bicistronic retroviral vector for transfer of the soluble receptor for the vascular endothelial growth factor (sFLT) hepatoma (MH3924A) cell lines with sFLT expression were generated. In human umbilical vein endothelial cells cultured with conditioned medium of sFLT-expressing hepatoma cells, the inhibitory action of secreted sFLT was determined using a Coulter counter and a thymidine incorporation assay. Furthermore, in vivo experiments were done to measure the effects on tumor growth and perfusion. Finally, the tumors were examined by immunohistochemistry (including computerassisted morphometry) and DNA chip analysis.Results: Stable sFLT-expressing hepatoma cells inhibited endothelial cell proliferation in vitro. In vivo, growth and perfusion, as measured by H 2 15 O positron emission tomography, were reduced in genetically modified tumors. However, the immunohistochemically quantified microvascularization and macrovascularization, as indicated by CD31-and a-actin-positive area, revealed no significant changes, whereas the number of apoptotic cells was increased in sFLT-expressing tumors, although not significantly. DNA chip analysis of tumors with gene transfer showed an increase of genes related to apoptosis, signal transduction, and oxidative stress.Conclusion: Our results suggest that sFLT expression inhibits tumor growth and perfusion and enhances expression of apoptosis-related genes in this model. Enhanced expression of genes for signal transduction, stress, and metabolism indicates tumor defense reactions.

The Soluble VEGF Receptor sFlt1 Contributes to Endothelial Dysfunction in CKD

“…2 However, there is only indirect evidence for the role of sFlt-1 on endothelial cell apoptosis because the authors used sFlt-1 gene transfer in hepatoma and renal cell carcinoma models. 24,25 Herein, we proved this effect by using recombinant protein and showed that the proapoptotic effect was reversed by neutralization of sFLt-1. The proapoptotic activity found in patient serum and shown by recombinant sFlt-1 probably contributes to the antiangiogenic activity described above.…”

TNF-α and the IFN-γ-inducible protein 10 (IP-10/CXCL-10) delivered by parvoviral vectors act in synergy to induce antitumor effects in mouse glioblastoma

“…45 Briefly, the tumors were either paraffin-embedded or cryofixed in liquid nitrogen-cooled isopentane and stored at À80 1C before further processing. Then 6-mm sections were cut at À20 1C with a Microm Microtome, placed on Superfrost microscope slides (Fisher Scientific, Schwerte, Germany) and exposed to 4% paraformaldehyde.…”

“…A previously described custom-developed computerassisted image analysis device 45 was used to quantify necrotic area of the paraffin-embedded, hematoxylineosin-stained tumor sections having the largest crosssectional diameter. The ratio of the necrotic area to the total tumor area was calculated and expressed as a percentage.…”

Identification and Evaluation of a New Tumor Cell-Binding Peptide, FROP-1

“…The human HPV16 immortalized keratinocyte cell line HPV16-GM (Dr. Pascal Tomakidi, University Hospital Heidelberg, Germany) was cultured in keratinocyte growth medium 2 with supplements (PromoCell). Human umbilical vein endothelial cells (HUVECs) were isolated as described in the literature (15). HUVEC cultivation was performed on gelatin (1%)-coated cell culture flasks using medium 199 (Invitrogen) containing 20% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, 100 IU/mL streptomycin, and 2 ng bFGF/mL (Roche Diagnostics).…”