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Konrad R Koch, Nasrin Refaian, Deniz Hos, Martina Becker, Claus Cursiefen, Ludwig M Heindl; Tumor-derived vascular endothelial growth factor (VEGF) D stimulates ocular melanoma cell proliferation in an autocrine manner. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):220.
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© ARVO (1962-2015); The Authors (2016-present)
Self-sufficiency in growth signaling is considered one of the hallmarks of cancer. Recently, tumor derived VEGF-A was shown to sustain uveal melanoma cell proliferation apart from primary proangiogenic properties. Prolymphangiogenic growth factors like VEGF-D might also act on malignant cells provided that VEGF receptors 2 or 3 (VEGF-R2, -R3) are simultaneously expressed. It was the goal of this in vitro study to provide evidence whether autocrine tumor-driving VEGF-D signaling is present in ocular melanoma.
Primary melanoma cells of uveal (MEL-202, MEL-270, OM-431) and conjunctival (CRMM-1, CRMM-2, CM2005.1) origin were analyzed for VEGF-D, VEGF-R2, and VEGF-R3 expression by RT-PCR, ELISA (VEGF-A protein) and immunocytochemistry (VEGF receptors 2, 3, each tested with two different primary antibodies). Proliferation of melanoma cells incubated with a neutralizing anti-VEGF-D antibody (anti-D, ≤ 10 µg/mL; R&D Systems, Wiesbaden, Germany), or with a selective VEGF-R3 inhibitor SAR131675 (≤ 50 nM; Selleckchem, Munich, Germany) was assessed by MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide) assays. Lymphatic endothelial cells co-incubated with recombinant VEGF-D (300 ng/mL ) and anti-VEGF-D or SAR131675 served as positive control. Statistical analyses were performed using Jonckheere's trend test.
All OM cells expressed VEGF-D, VEGF-R2, VEGF-R3 mRNA and protein. In all cell lines, incubation with anti-D led to a dose-dependent decrease in proliferation, which was statistically significant for 4 melanoma cell lines (MEL-202, OM-431, CRMM-1, CM2005.1) and LECs (p<0.018, respectively). Treatment with increasing doses of SAR131675 significantly inhibited the proliferation of VEGF-D co-treated LECs (p=0.016), while not statistically affecting the proliferation of any melanoma cell line (p>0.05, respectively).
Autocrine VEGF-D signaling seems to be present in uveal and conjunctival melanoma. The lack of proliferative inhibition by VEGF-R3 inhibitor SAR131675 suggests the assumption that proliferative tumor cell stimulation is more likely driven by VEGF-D / VEGF-R2 signaling. Further studies are needed to clarify whether the ascertained VEGF-R3 expression provides a functional impact for ocular melanoma cells.
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