Angiostatin decreases cell migration and vascular endotheliumgrowth factor (VEGF) to pigment epithelium derived factor (PEDF) RNAratio in vitro and in a murine ocular melanoma model

[摘要] Purpose: Our previous experiments have shown that low doseangiostatin results in decreased hepatic micrometastasis in a mousemodel of uveal melanoma. The purpose of these experiments is to evaluatethe effect of angiostatin on in vitro migration of melanoma cells and toexplore the in vivo mechanism of angiostatin in our model.Methods: For in vitro studies, quantitative RT-PCR was used todetect VEGF and PEDF mRNA in mouse B16LS9 melanoma cells and Mel290human uveal melanoma cells with or without supplemental 0.1 μg/mlmurine or human recombinant angiostatin. A wound healing assay was usedto measure cellular migration in these two groups of cells. For the invivo mechanism, aliquots of tissue culture B16LS9 cells treated with orwithout 0.1 μg/ml murine angiostatin were heterotopically inoculatedinto the posterior compartments of the right eyes of C57BL/6 mice.Frozen hepatic tissue was prepared and stained with hematoxylin using anRNase-free technique. Hepatic micrometastatic uveal melanoma cells wereobtained by laser capture microdissection (LCM). Levels of VEGF and PEDFmRNA were detected by real time RT-PCR in the hepatic micrometastases.Results: After in vitro treatment of the cell lines withangiostatin, the ratio of VEGF/PEDF mRNA significantly decreased in theB16LS9 (0.88±0.11 [mean±standard deviation] versus 2.70±0.15in the control group; p=0.00006) and Mel290 (0.12±0.02 versus0.68±0.04 in the control group; p=0.00346). However, the absoluteVEGF mRNA and PEDF mRNA did not significantly change (p>0.08 for bothcell lines). The migration assay showed significantly decreasedmigration 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 vivoexperiments, pretreatment with angiostatin resulted in a decreasedVEGF/PEDF mRNA ratio in B16LS9 cells compared to controls (0.0274±0.0070 versus 0.1726±0.0313; p=0.0014). Additionally, there wassignificantly increased PEDF mRNA (2.14±0.12 versus 0.30±0.05 inthe control group; p=0.00002) in the liver metastases after pretreatmentwith angiostatin.Conclusions: Angiostatin inhibits the migration of melanoma cells invitro. Angiostatin significantly decreases the ratio of VEGF/PEDF mRNAlevel in vitro and in hepatic micrometastatic melanoma cells.Angiostatin increases PEDF mRNA in melanoma metastases.

[学科分类] 生物化学/生物物理