PURPOSE: Our previous experiments have shown that low dose angiostatin results in decreased hepatic micrometastasis in a mouse model of uveal melanoma. The purpose of these experiments is to evaluate the effect of angiostatin on in vitro migration of melanoma cells and to explore the in vivo mechanism of angiostatin in our model. METHODS: For in vitro studies, quantitative RT-PCR was used to detect VEGF and PEDF mRNA in mouse B16LS9 melanoma cells and Mel290 human uveal melanoma cells with or without supplemental 0.1 mug/ml murine or human recombinant angiostatin. A wound healing assay was used to measure cellular migration in these two groups of cells. For the in vivo mechanism, aliquots of tissue culture B16LS9 cells treated with or without 0.1 mug/ml murine angiostatin were heterotopically inoculated into the posterior compartments of the right eyes of C57BL/6 mice. Frozen hepatic tissue was prepared and stained with hematoxylin using an RNase-free technique. Hepatic micrometastatic uveal melanoma cells were obtained by laser capture microdissection (LCM). Levels of VEGF and PEDF mRNA were detected by real time RT-PCR in the hepatic micrometastases. RESULTS: After in vitro treatment of the cell lines with angiostatin, the ratio of VEGF/PEDF mRNA significantly decreased in the B16LS9 (0.88+/-0.11 [mean+/-standard deviation] versus 2.70+/-0.15 in the control group; p=0.00006) and Mel290 (0.12+/-0.02 versus 0.68+/-0.04 in the control group; p=0.00346). However, the absolute VEGF mRNA and PEDF mRNA did not significantly change (p>0.08 for both cell lines). The migration assay showed significantly decreased migration at 24 h and 48 h after angiostatin treatment for both B16LS9 (p<0.01) and Mel290 (p<0.01) cell lines. For the in vivo experiments, pretreatment with angiostatin resulted in a decreased VEGF/PEDF mRNA ratio in B16LS9 cells compared to controls (0.0274+/-0.0070 versus 0.1726+/-0.0313; p=0.0014). Additionally, there was significantly increased PEDF mRNA (2.14+/-0.12 versus 0.30+/-0.05 in the control group; p=0.00002) in the liver metastases after pretreatment with angiostatin. CONCLUSIONS: Angiostatin inhibits the migration of melanoma cells in vitro. Angiostatin significantly decreases the ratio of VEGF/PEDF mRNA level in vitro and in hepatic micrometastatic melanoma cells. Angiostatin increases PEDF mRNA in melanoma metastases.
PURPOSE: Our previous experiments have shown that low dose angiostatin results in decreased hepatic micrometastasis in a mouse model of uveal melanoma. The purpose of these experiments is to evaluate the effect of angiostatin on in vitro migration of melanoma cells and to explore the in vivo mechanism of angiostatin in our model. METHODS: For in vitro studies, quantitative RT-PCR was used to detect VEGF and PEDF mRNA in mouse B16LS9 melanoma cells and Mel290 humanuveal melanoma cells with or without supplemental 0.1 mug/ml murine or human recombinant angiostatin. A wound healing assay was used to measure cellular migration in these two groups of cells. For the in vivo mechanism, aliquots of tissue culture B16LS9 cells treated with or without 0.1 mug/ml murineangiostatin were heterotopically inoculated into the posterior compartments of the right eyes of C57BL/6 mice. Frozen hepatic tissue was prepared and stained with hematoxylin using an RNase-free technique. Hepatic micrometastatic uveal melanoma cells were obtained by laser capture microdissection (LCM). Levels of VEGF and PEDF mRNA were detected by real time RT-PCR in the hepatic micrometastases. RESULTS: After in vitro treatment of the cell lines with angiostatin, the ratio of VEGF/PEDF mRNA significantly decreased in the B16LS9 (0.88+/-0.11 [mean+/-standard deviation] versus 2.70+/-0.15 in the control group; p=0.00006) and Mel290 (0.12+/-0.02 versus 0.68+/-0.04 in the control group; p=0.00346). However, the absolute VEGF mRNA and PEDF mRNA did not significantly change (p>0.08 for both cell lines). The migration assay showed significantly decreased migration at 24 h and 48 h after angiostatin treatment for both B16LS9 (p<0.01) and Mel290 (p<0.01) cell lines. For the in vivo experiments, pretreatment with angiostatin resulted in a decreased VEGF/PEDF mRNA ratio in B16LS9 cells compared to controls (0.0274+/-0.0070 versus 0.1726+/-0.0313; p=0.0014). Additionally, there was significantly increased PEDF mRNA (2.14+/-0.12 versus 0.30+/-0.05 in the control group; p=0.00002) in the liver metastases after pretreatment with angiostatin. CONCLUSIONS:Angiostatin inhibits the migration of melanoma cells in vitro. Angiostatin significantly decreases the ratio of VEGF/PEDF mRNA level in vitro and in hepatic micrometastatic melanoma cells. Angiostatin increases PEDF mRNA in melanoma metastases.