Low dose adjuvant angiostatin decreases hepatic micrometastasis in murine ocular melanoma model

Abstract

Purpose: To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma.

Methods: Angiostatin and plasminogen expression was detected in three murine melanoma cell lines (Queens, B16F10, and B16LS9). The three cell lines were heterotopically inoculated into the posterior compartment (PC) of the right eyes of C57BL/6 mice. After enucleation, the mice were given injections of 100 microl PBS and low dose (0.1 microg/microl) or high dose (0.3 microg/microl) murine recombinant angiostatin every day for 14 days after enucleation. The mice were sacrificed at 21 days post-enucleation and hepatic micrometastases were counted. In vitro migration/invasion assays were performed with low (0.1 microg/microl) and high (50 microg/microl) concentration angiostatin supplementation. Quantitative RT-PCR detected mRNA and Western analysis determined protein expression of VEGF for all cell lines. Evaluation of TdT mediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of the micrometastases determined apoptosis and proliferation ratios.

Results: There was a decrease in micrometastasis in the low dose group for Queens (p<0.05), B16F10 (p<0.05), and B16LS9 melanoma (p<0.01) cell lines. Two of the cell lines (B16F10 and B16LS9) elaborated plasminogen and were able to cleave plasminogen into K1-K4 (angiostatin). There was a decrease in the in vitro migration and invasion after supplementation with low concentration compared to high concentration angiostatin (p<0.01). VEGF mRNA and protein expression decreased in all cells lines in low concentration angiostatin, with the greatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios were increased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low dose angiostatin.

Conclusions: There were significantly fewer micrometastases in treated compared to controls with low dose compared to high dose angiostatin. This treatment results in apoptosis in the micrometastases. The mechanism appears to be related an anti-migratory effect and altered VEGF expression by melanoma cells.