Norcantharidin Inhibits Lymphangiogenesis by Downregulating the Expression of VEGF-C and VEGF-D in Human Dermal Lymphatic Endothelial Cells in vitro

Abstract

Aims: To investigate the effects of norcantharidin on the growth and migration of human dermal lymphatic endothelial cells (HDLECs) and further characterize its effect on lymphangiogenesis. Methods: A 3-dimensional fibrin gel lymphangiogenesis model was built. Flow cytometry was used to analyze the rate of apoptosis and necrosis. RT-PCR, immunohistochemistry and immunoblotting assays were used to examine the effect of norcantharidin on vascular endothelial growth factor C (VEGF-C), VEGF-D and VEGF receptor 3 during in vitro lymphangiogenesis. Results: Norcantharidin caused a marked dose and time-dependent inhibition of the growth of HDLECs with an IC₅₀ of 40 nmol/l. The apoptotic rate of HDLECs was 13.21 ± 1.60% 24 h after treatment with 7.5 nmol/l norcantharidin and 42.34 ± 3.80% with 90 nmol/l norcantharidin (p < 0.01 vs. controls in both). Fibrin gel assays showed that norcantharidin (15 nmol/l) reduced the number of tubular structures from 68.4 ± 5.2 in untreated controls to 10.9 ± 2.3 (p = 0.000). RT-PCR, immunohistochemistry and immunoblotting assays showed norcantharidin markedly reduced the expression of VEGF-C and VEGF-D. Conclusion: Norcantharidin inhibits lymphangiogenesis by downregulating the expression of VEGF-C and VEGF-D, suggesting that norcantharidin could be an effective agent for targeting neolymphangiogenesis.