Phosphomimetic mutants of pigment epithelium-derived factor with enhanced anti-choroidal melanoma cell activity in vitro and in vivo.

PURPOSE: Currently, choroidal melanoma is chemoresistant and there is no routine adjuvant chemotherapy for it. We investigated whether pigment epithelium-derived factor (PEDF) and its triple phosphomimetic mutants could more efficiently suppress melanoma tumor growth and metastasis, as well as how the triple phosphomimetic mutants act as antitumor agents. METHODS: Phosphomimetic mutants of PEDF were constructed by site mutagenesis. Lentiviruses carrying wild type (WT) PEDF, S24E114E227A (EEA)-PEDF, and S24E114E227E (EEE)-PEDF were produced in 293 fast-growing, highly transfectable (FT) cells and used to infect human choroidal melanoma cell line (OCM-1). The growth, migration, invasion and metastasis abilities of OCM-1 cells expressing WT-PEDF, EEA-PEDF or EEE-PEDF were investigated in vitro and in vivo, while the underlying mechanism of PEDF phosphomimetic mutants were investigated via Western blotting. RESULTS: OCM-1 cells infected with lentiviruses carrying WT-PEDF, EEA-PEDF, and EEE-PEDF displayed reduced proliferation, migration and invasion abilities, and were more prone to apoptosis. Cell media containing WT-PEDF, EEA-PEDF, or EEE-PEDF protein inhibited the tube forming capacity of human umbilical vein endothelial cells (HUVEC) in vitro. OCM-1 cells expressing WT-PEDF, EEA-PEDF, or EEE-PEDF displayed significantly reduced tumor growth and metastasis in the melanoma xenograft of nude mice models, with the PEDF mutants displaying much stronger effects than the wild type. The antitumor effects of PEDF are associated with the inhibition of VEGF and nuclear factor kappa-B (NF-κB) expression, as well as further inhibition of Akt phosphorylation. CONCLUSIONS: The phosphomimetic mutants of PEDF showed enhanced anti-melanoma activity by directly affecting tumor cells and indirectly affecting angiogenesis. These findings encourage the development of PEDF mutants as innovative anticancer agents.

Adsorption of apo- and holo-tear lipocalin to a bovine Meibomian lipid film.

Adsorption of apo- and holo-tear lipocalin (Tlc) to bovine Meibomian lipid film was studied. A Langmuir trough was used for these studies and the adsorption of protein was observed by recording changes in the pressure with time (pi-T profile). The films were photographed at different stages of adsorption by doping Meibomian lipids with a fluorescently tagged lipid. The results indicated that apo-Tlc adsorbed much more quickly than holo-Tlc to the Meibomian lipid film. Contrary to the expectation that holo-Tlc would release lipids to the surface and surface pressure would be higher, it was found that the surface pressure was higher with the adsorption of apo-Tlc to the surface. Photography of the films showed that apo- and holo-Tlc interacted differently with the Meibomian lipid layer. Adsorption of holo-Tlc resulted in big bright patches and adsorption of apo-Tlc resulted in many small patches along with the big patches. Both forms of Tlc produced a more stable film as indicated by decreased movement of the protein adsorbed films, and a higher maximum surface pressure upon compression of these films compared with Meibomian lipid films alone. Isocyles of apo-Tlc adsorbed films gave a higher surface pressure than that of holo-Tlc. From these results, it is concluded that both apo- and holo-Tlc adsorbed to the Meibomian lipid layer and the delivery of the lipids from Tlc to the outer lipid layer could not be detected by our techniques. Its scavenging role to remove lipids from the corneal surface and bind with them might be beneficial for increasing tear viscosity but whether those lipids are delivered to the outermost lipid layer still remains unclear.