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. 2004 Apr;164(4):1225-32.
doi: 10.1016/s0002-9440(10)63210-5.

Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo

Affiliations

Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo

Riichiro Abe et al. Am J Pathol. 2004 Apr.

Abstract

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. In this study, we have investigated both the in vitro and in vivo growth characteristics of human malignant melanoma G361 cell lines, stably transfected to overexpress human PEDF. Expression levels of PEDF proteins in melanoma cell lines G361 and A375 were comparable with that of human cultured melanocytes, whereas vascular endothelial growth factor levels in two tumor cell lines were much stronger than that in normal melanocytes. Overexpression of PEDF was found to significantly inhibit tumor growth and vessel formation in G361 nude mice xenografts. Furthermore, in vitro proliferation rates of G361 cells were decreased in PEDF-transfected cells. PEDF proteins showed dose-dependent induced growth retardation and apoptotic cell death in nontransfected G361 cells, which were completely prevented by treatment with antibodies against the Fas ligand. Our present study highlights two beneficial effects of PEDF treatment on melanoma growth and expansion; one is the suppression of tumor angiogenesis, and the other is induction of Fas ligand-dependent apoptosis in tumor cells. PEDF therefore might be a promising novel therapeutic agent for treatment of patients with melanoma.

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Figures

Figure 1
Figure 1
Immunofluorescence staining (A) and Western blot analysis (B) of G361, A375, and normal melanocytes. A: Immunofluorescence staining for PEDF was performed as described in Materials and Methods. The inset indicates negative staining without primary antibody. B: Expression levels of PEDF, VEGF, and α-tubulin in cell lysates were analyzed as described in Materials and Methods. Scale bar, 10 μm.
Figure 2
Figure 2
Western blot analysis of conditioned medium from G361 cells, stably transfected with the human PEDF expression vector (lane 2) or with the expression vector alone (lane 3). Purified PEDF proteins (0.2 μg) were used as a positive control (lane 1).
Figure 3
Figure 3
Growth of G361 xenografts in nude mice. G361 cells, stably transfected with the human PEDF expression vector or with the expression vector alone, were injected intradermally into the flanks of five 6-week-old female athymic nude mice. The tumor volumes were calculated as described in Materials and Methods. *, P < 0.0001 compared with control cells.
Figure 4
Figure 4
Tumor vessels in G361 xenografts. Typical photomicrographs of immunofluorescent staining for CD31 (green) in G361 xenografts. Nuclei were stained with propidium iodide (red). G361 cells, stably transfected with the human PEDF expression vector (A) or with the expression vector alone (B). Bottom: The quantitative analysis of the fluorescent-positive area (per 1000 μm2) in tumors. *, P < 0.0001 compared to control cells. Scale bar, 50 μm.
Figure 5
Figure 5
Growth and apoptosis of G361 cells in vitro. A: Growth rates of G361 cells, stably transfected with the human PEDF expression vector or with the expression vector alone in vitro. Growth (B) and apoptosis (C) of nontransfected G361 cells. Cells were incubated with or without the indicated concentrations of PEDF proteins in the presence or absence of 10 μg/ml of monoclonal antibody against Fas ligand. *, P < 0.005 compared to the value without treatments.
Figure 6
Figure 6
Apoptotic cells in G361 xenografts. Typical photomicrographs of apoptotic cells using TUNEL assay (green) in G361 xenografts. Nuclei were stained with propidium iodide (red). G361 cells, stably transfected with the human PEDF expression vector (A) or with the expression vector alone (B). Bottom: The quantitative analysis of the apoptotic cell percentage in tumors. *, P < 0.005 compared to control cells. Scale bar, 50 μm.

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