The fungicide ciclopirox inhibits lymphatic endothelial cell tube formation by suppressing VEGFR-3-mediated ERK signaling pathway

Abstract

Ciclopirox olamine (CPX), an off-patent antifungal agent used to treat mycoses of skin and nails, has recently been demonstrated to be a potential anticancer agent. However, the underlying mechanism is not well understood. Here, for the first time, we show that CPX inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was, in part, associated with inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) expression, as overexpression of VEGFR-3 conferred partial resistance to CPX inhibitory effect on tube formation in lymphatic endothelial cells (LECs), whereas downregulation of VEGFR-3 mimicked the effect of CPX, blocking the tube formation. Further study revealed that CPX did not alter mRNA level, but inhibited protein synthesis and promoted protein degradation of VEGFR-3. In addition, we found that CPX inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2), a downstream effector of VEGFR-3. Overexpression of VEGFR-3 attenuated CPX inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited ERK1/2 phosphorylation in LECs. Ectopic expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) resulted in activation of ERK1/2 and partially prevented CPX inhibition of LEC tube formation. The results suggest that CPX inhibits LEC tube formation at least, in part, through inhibiting VEGFR-3-mediated ERK signaling pathway.

Figure 1. CPX inhibits LEC tube formation in a concentration and time-dependent manner

LECs were treated with CPX (0-5 μM) for 24 h, or CPX (5 μM) for 0-24 h, followed by tube formation assay and morphological analysis, as described in “Materials and methods”. Representative images are shown in (A) and (B), respectively. Bar = 100 μm. The length of tube-like formation was evaluated by NIH Image J software. Quantitative data are presented as mean ± SD (n = 3) in (C) and (D), respectively. *P < 0.05, **P < 0.01, difference vs. control group.

Figure 2. CPX inhibition of LEC tube formation is associated with suppressing VEGFR-3 protein expression

A and B, CPX inhibited protein expression of VEGR-3 in a concentration and-time-dependent manner. LECs, treated with CPX (0-5 μM) for 24 h (A) or CPX (5 μM) for 0-24 h (B), were harvested and subjected to Western blot analysis with antibodies to VEGFR-3. β-tubulin was used as a loading control. C and D, Overexpression of VEGFR-3 partially prevented CPX inhibition LEC tube formation. LEC/V (control) and LEC/VEGFR-3 cells were treated with CPX (5 μM) for 24 h, followed by Western blot analysis with indicated antibodies (C), or tube formation assay as described in “Materials and methods” (D). Quantitative results of tube formation are shown as mean ± SD (n = 3) in (D). *P < 0.05, difference vs. control group; #P < 0.05, difference vs. LEC/V group.

Figure 3. Downregulation of VEGFR-3 mimicks the effect of CPX, blocking LEC tube formation

Lentiviral shRNA to VEGFR-3, but not GFP, downregulated VEGFR-3 protein expression by ~90% in LECs, as detected by Western blotting (A). LECs, infected with lentiviral shRNAs to VEGFR-3 and GFP (control), respectively, were treated with CPX (5 μM) for 24 h, followed by tube formation assay as described in “Materials and methods”. Quantitative results of tube formation are shown as mean ± SD (n = 3) in (B). *P < 0.05, difference vs. GFP shRNA control group. C and D, LECs, infected with half amount of lentiviral shRNA to VEGFR-3 and GFP (control), respectively, were treated with/without CPX (0, 2.5 and 5 μM) for 24 h, followed by Western blotting (C) and tube formation assay (D). Quantitative results of tube formation are shown as mean ± SD (n = 3). *P < 0.05, difference vs. GFP shRNA.

Figure 4. CPX does not alter mRNA expression, but inhibits protein synthesis and promotes protein degradation of VEGFR-3

A, CPX did not affect VEGFR-3 mRNA level. Total RNA was extracted from LECs treated with CPX (0-5 μM) for 24 h (Left panel) or with CPX (5 μM) for 0-24 h (Right panel), followed by semi-quantitative RT-PCR. β-actin was used as a loading control. B, CPX inhibited protein synthesis of VEGFR-3 in LECs. LECs were pretreated with CPX (0-5 μM) for 24 h (Left panel) or with CPX (5 μM) for 0-24 h (Right panel), and then pulsed with ³⁵S-Met/Cys for 4 h, followed by immunoprecipitation with antibodies to VEGFR-3. The immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes, followed by autoradiography. GAPDH served as an internal control. C, CPX promoted protein degradation of VEGFR-3 in LECs. LECs, grown in 10% FBS-DMEM medium, were exposed to cycloheximide (CHX, 50 μg/ml), in the presence or absence of CPX (5 μM), for 0-12 h, followed by Western blot analysis with indicated antibodies.

Figure 5. CPX inhibits VEGFR-3-mediated ERK1/2 pathway

A and B, CPX inhibited phosphorylation of ERK1/2, but not Akt, JNK and p38 MAPK, in LECs in a concentration and time-dependent manner. LECs, treated with CPX (0-5 μM) for 24 h (A) or CPX (5 μM) for 0-24 h (B), were harvested and subjected to Western blot analysis with indicated antibodies. β-tubulin was used as a loading control. C, Overexpression of VEGFR-3 conferred resistance to CPX inhibition of ERK1/2 phosphorylation in LECs. LEC/V (control) and LEC/VEGFR-3 were treated with or without CPX (5 μM) for 24 h, followed by Western blotting with indicated antibodies. D. Downregulation of VEGFR-3 mimicked the effect of CPX, inhibiting phosphorylation of ERK1/2 in LECs. LECs, infected with lentiviral shRNAs to VEGFR-3 and GFP (control), respectively, were treated with CPX (5 μM) for 24 h, followed by Western blotting with indicated antibodies.

Figure 6. CPX inhibition of LEC tube formation is through targeting VEGFR-3-mediated ERK1/2 pathway

A and B, Expression of constitutively active MKK1 attenuated CPX inhibition of ERK1/2 phosphorylation and the tube formation in LECs. LECs, infected with Ad-MKK1-R4F and Ad-GFP (control), respectively, were treated with or without CPX (5 μM) for 24 h, followed by Western blotting with indicated antibodies (A), or by tube formation assay as described in “Materials and methods” (B). Quantitative results are shown as mean ± SD (n = 3) in (B). *P < 0.05, difference vs. control group; ^#P < 0.05, difference vs. Ad-GFP group. C and D, In combination with U0126, CPX did not enhance U0126 inhibition of LEC tube formation. LECs were treated with U0126 (5 μM) or CPX (5 μM) alone, or both for 24h, followed by Western blotting using the indicated antibodies (C), or by tube formation assay as described in “Materials and methods” (D). Quantitative results of tube formation are shown as mean±SD (n = 3). *P < 0.05, difference vs. control group.