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. 2016 Nov 1;57(14):5864-5871.
doi: 10.1167/iovs.16-19952.

Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition

Jamie S Rush  1 , David P Bingaman  2 , Paul G Chaney  2 , Martin B Wax  3 , Brian P Ceresa  4
Affiliations

Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition

Jamie S Rush et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 μM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition.

Methods: In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology.

Results: Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation.

Conclusions: An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.

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Figures

Figure 1
Figure 1
Receptor tyrosine kinase inhibitor inhibits EGFR-mediated in vitro wound healing. Human telomerase-immortalized corneal epithelial cells were plated on tissue culture dishes with 2-mm-diameter silicone plugs that, when removed, created an acellular area to monitor wound healing. Cells were pretreated for 30 minutes with the indicated concentrations of RTKi or AG1478, followed by 16 hours with the addition of the indicated concentration of EGF or VEGF. (A) Representative micrographs are shown and were used to quantify the in vitro wound healing response. Photographs were used to trace, measure, and quantify the area of the initial wound (outer circle) and the remaining wound (inner circle). Scale bar: 500 μm. (B) Graphical representation of the quantification of multiple dose response experiments. (C) Graphical representation of a time course of in vitro wound healing with media alone, 1.6 nM EGF, 3.2 μM AG1478, 3.0 μM RTKi, 1.6 nM EGF and 3.2 μM AG1478, or 1.6 nM EGF and 3.0 μM RTKi. Representative micrographs are shown in Supplementary Data Figure S1. Data are plotted as the average ± SEM. from three experiments. Data were analyzed using an unpaired Student's t-test. *P < 0.05; **P < 0.01.
Figure 2
Figure 2
Receptor tyrosine kinase inhibitor slows corneal epithelial wound healing in mice. Epithelial wounds (1.5 mm in diameter) were made on the corneas of 8-week-old C57Bl6 mice. Following wounding, PBS, an EGFR-inhibitor (AG1478–1 μM), or the indicated concentrations of RTKi were topically administered to the wounded area. The epithelial wounds were visualized after fluorescein staining using a fluorescent dissecting microscope. (A) Representative images at the time of the initial wound (0 hour), 16 hours, 20 hours, or 40 hours after wounding. (B, C) Graphical representation of the percentage of wound healing at 16 and 24 hours after wounding, respectively. (D) The percentage of wound healed as a function of time. For (B–D), data are plotted as the average ± SEM from 3 to 20 experiments. Data were analyzed using an unpaired Student's t-test. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Menadione treatment slows the kinetics of EGF-mediated EGFR degradation. Human telomerase-immortalized corneal epithelial cells were pretreated with menadione (0, 0.3, or 3.0 μM) for 4 hours. Cells were then incubated with EGF (50 ng/mL) for the indicated periods of time (0–3 hours). Cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted for phosphorylated EGFR (pY1068) (top), total EGFR (middle), or α-tubulin (bottom). Shown is a representative image from an experiment repeated three times.
Figure 4
Figure 4
Vitamin K3 increases in vitro wound healing. Human telomerase-immortalized corneal epithelial cells were plated for an in vitro wound healing assay as described in Figure 1. Before removing the silicone plug, cells were treated with the indicated concentrations of menadione for 4 hours and supplemented with the varying concentrations of RTKi for 30 minutes. Once the silicone plug was removed, the cells were incubated with the indicated concentrations of menadione, RTKi, and EGF. (A) Representative micrographs are shown and were used to quantify the in vitro wound healing response. Photographs were used to trace, measure, and quantify the area of the initial wound (outer circle) and the remaining wound (inner circle). Scale bar: 500 μm. (B) Graphical representation of multiple experiments. Data are plotted as the average ± SEM from three to four experiments. Data were analyzed using an unpaired Student's t-test. *P < 0.05; **P < 0.01.
Figure 5
Figure 5
Vitamin K3 increases in vivo corneal epithelial wound healing. Eight-week-old C57Bl6 mice were subjected to 1.5-mm-diameter corneal epithelial wounds. Forty-eight hours before wounding, mice were topically administered with vehicle, 0.3 μM menadione, 10 μM RTKi, or 10 μM RTKi with 0.3 μM menadione. The epithelial wounds were visualized after fluorescein staining using a fluorescent dissecting microscope. (A) Representative images of the epithelial wounds at various times after staining. (B, C) Quantification of wound healing at 16 hours and 24 hours after wounding, respectively. (D) Kinetics of wound healing over the course of 40 hours. Data were analyzed using an unpaired Student's t-test. *P < 0.05; **P < 0.01.
Figure 6
Figure 6
Vitamin K3 does not affect RTKi inhibition of VEGF. (A) Human retinal endothelial cells were treated with 0, 0.3, or 3 μM menadione for 4 hours, followed by 30 minutes of treatment with varying concentrations of RTKi (1 nM, 10 nM, 100 nM, or 1μM) in menadione. Finally, cells were stimulated with 10 ng/mL VEGF. Cell lysates were prepared resolved by 7.5% SDS-PAGE, transferred to nitrocellulose an immunoblotted for phosphorylated VEGFR2 (top), total VEGFR2 (middle), or α-tubulin (bottom) as a loading control. (B) Human retinal endothelial cells were pretreated with menadione for 4 hours then the indicated concentration of RTKi for 30 minutes, and supplemented with 10 ng/mL VEGF overnight. Viable cells were quantified by Alamar Blue Assay (Thermo Fisher). Data are plated as the fold growth relative to cells treated with no VEGF or RTKi and are shown as the average ± SEM.
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