Low dose adjuvant angiostatin decreases hepatic micrometastasisin murine ocular melanoma model

[摘要] Purpose: To investigate the effect of different doses of adjuvantangiostatin affecting hepatic micrometastasis in a murine model ofmetastatic ocular melanoma.Methods: Angiostatin and plasminogen expression was detected inthree murine melanoma cell lines (Queens, B16F10, and B16LS9). The threecell lines were heterotopically inoculated into the posteriorcompartment (PC) of the right eyes of C57BL/6 mice. After enucleation,the mice were given injections of 100 μl PBS and low dose (0.1 μg/μl) or high dose (0.3 μg/μl) murine recombinant angiostatinevery day for 14 days after enucleation. The mice were sacrificed at 21days post-enucleation and hepatic micrometastases were counted. In vitromigration/invasion assays were performed with low (0.1 μg/μl) andhigh (50 μg/μl) concentration angiostatin supplementation.Quantitative RT-PCR detected mRNA and Western analysis determinedprotein expression of VEGF for all cell lines. Evaluation of TdTmediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of themicrometastases determined apoptosis and proliferation ratios.Results: There was a decrease in micrometastasis in the low dosegroup 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 andinvasion after supplementation with low concentration compared to highconcentration angiostatin (p<0.01). VEGF mRNA and protein expressiondecreased in all cells lines in low concentration angiostatin, with thegreatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios wereincreased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low doseangiostatin.Conclusions: There were significantly fewer micrometastases intreated compared to controls with low dose compared to high doseangiostatin. This treatment results in apoptosis in the micrometastases.The mechanism appears to be related an anti-migratory effect and alteredVEGF expression by melanoma cells.

[学科分类] 生物化学/生物物理