Gelatinase expression in retinoblastoma: modulation of LH(BETA)T(AG) retinal tumor development by anecortave acetate

Abstract

Purpose: Gelatinases, matrix metalloproteinase (MMP)-2, and MMP-9 are known for their importance in angiogenesis and tumor biology. The purpose of this study was to test the hypothesis that anecortave acetate (AA) decreases transgenic retinoblastoma (RB) tumor burden by modulating gelatinase activity.

Methods: To assess the possible gelatinase modulation after AA treatment, a single subconjunctival injection of AA (300 microg) was delivered to the right eyes of 10-week-old LH(BETA)T(AG) mice. Eyes were evaluated for gelatinase expression and activity by gel and in situ zymography at 24 hours, 48 hours, and 1 week after treatment.

Results: Gel zymography of whole eye extracts and in situ zymography of retinal tumors showed strong gelatinase expression and activity within transgenic RB tumors. AA treatment in RB transgenic mice resulted in a significant decrease of gelatinase activity 1 week after AA treatment. Surprisingly, there was an initial transient upregulation of MMP-9 activity in whole eye extracts at 24 and 48 hours after AA treatment in both LH(BETA)T(AG) transgenic and wild-type mice. This increase was not observed in the tumors.

Conclusions: As suggested by our data, inhibition of gelatinase activity appears to be a mechanism of action of AA. AA treatment results in a decrease in gelatinase activity that correlates with the significant decrease in tumor burden shown by the authors' previous studies. However, the significance of the initial, transient upregulation of gelatinase by AA injection is unknown, and further studies are warranted. Combining antiangiogenic agents with multiple mechanisms of action has the potential to enhance RB tumor control.

Figure 1.

AA modulates MMP-2 and MMP-9 activity in LH_BETAT_AG transgenic RB. A single subconjunctival injection of 300 mg AA was administered in the right eye (T) of the LH_BETAT_AG transgenic RB mice, while the right fellow eye (F) remained untreated. Whole eye extracts were analyzed by gel zymography to measure MMP-2 () and MMP-9 (■) activities at 24 hours, 48 hours, and 1 week after injection. Top: gelatin zymogram from a representative experiment. Mouse recombinant MMP-9 and MMP-2 were used as positive controls (M). Bottom: average results of three independent gel zymograms. Error bars represent standard deviations from the mean. WT, wild-type; RB, LH_BETAT_AG.

Figure 2.

MMP-9 expression and activity in LH_BETAT_AG mice 24 hours after AA treatment. Top: gelatin zymogram from a representative experiment. Mouse recombinant MMP-2 and MMP-9 were used as positive controls. Bottom: average results of three independent gel zymograms. Saline was used as a vehicle control in both wild-type (WT) and LH_BETAT_AG (RB) mice. Bars represent gelatinase activity relative to untreated controls. Error bars represent standard deviations from the mean.

Figure 3.

Gelatinase activity is decreased by AA treatment in RB tumors in LH_BETAT_AG transgenic mice. Retinal tumors in saline-treated eyes contained high levels of gelatinase activity. The activity was reduced after treatment with the gelatinase inhibitor PHEN. Tumor-specific gelatinase activity decreased at both 24 hours and 1 week after injection.

Figure 4.

Retinal tumors in LH_BETAT_AG mice have increased MMP-9 expression and gelatinolytic activity. Green: gelatinolytic activity; red: MMP-9 expression.