Abstract
Purpose: :
To determine the effects of pigment epithelium derived factor (PEDF) on Stat3 activation. Stat3 is a pro-metastatic transcription factor that requires phosphorylation at Tyr-705 in order to dimerize, translocate to the nucleus, and bind DNA. In cases of metastatic uveal melanoma, the most common form of eye cancer in adults, the liver is the predominant site of metastasis. PEDF is a secreted protein in the liver that inversely correlates with metastatic tumor growth as shown in PEDF-/- C57BL/6 mice. We hypothesize that PEDF has an inhibitory effect on Stat3 phosphorylation thus preventing metastatic melanoma growth.
Methods: :
Using bovine retinal endothelial cells (BREC) in serum-free media, an established cell line for studying Stat3, phosphorylation of Stat3 at Tyr-705 was induced by one hour in hypoxic conditions at 1% O2. Control cells were grown at 20% O2. Cells were treated with 50ng/mL PEDF, either for 30minutes prior to hypoxia or at the midpoint of 1 hour in hypoxia. Separately, Stat3 was activated by 20ng/mL VEGF for 30 minutes versus non-treated control cells. To test the effect of PEDF on VEGF-mediated Stat3 phosphorylation, cells were pre-treated with 50ng/mL PEDF for 5 minutes before VEGF treatment. Additionally, cells were treated with 50ng/mL PEDF alone for either 5, 15, 30, or 60 minutes. Protein was collected, and western blots were performed.
Results: :
Hypoxia and VEGF treatment both upregulated Stat3 phosphorylation versus control. PEDF treatment, both before and during hypoxia, and before VEGF treatment, returned phospho-Stat3 to control levels. PEDF alone, however, upregulated Stat3 phosphorylation versus control.
Conclusions: :
PEDF has an inhibitory effect on Stat3 activation in both hypoxic and VEGF-rich culture conditions. Conversely, PEDF alone promotes Stat3 activation. Thus, PEDF treatment may not have an anti-metastatic effect unless the system is hypoxic or if VEGF is already present.
Keywords: phosphorylation • melanoma • growth factors/growth factor receptors