Cryptotanshinone Inhibits Lymphatic Endothelial Cell Tube Formation by Suppressing VEGFR-3/ERK and Small GTPase Pathways

Cryptotanshinone (CPT), isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism remains to be defined. Here, we show that CPT inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was partly attributed to inhibiting expression of VEGF receptor 3 (VEGFR-3) in murine lymphatic endothelial cells (LEC), as overexpression of VEGFR-3 conferred resistance to CPT inhibition of the tube formation, whereas downregulation of VEGFR-3 mimicked the effect of CPT, blocking the tube formation. Furthermore, CPT inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2). Overexpression of VEGFR-3 attenuated CPT inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited ERK1/2 phosphorylation in LECs. Expression of constitutively active MKK1 resulted in activation of ERK1/2 and partially prevented CPT inhibition of LEC tube formation. In addition, CPT also inhibited protein expression and activities of Rac1 and Cdc42 but not RhoA. Expression of constitutively active Rac1 and Cdc42 concurrently, but not Rac1 or Cdc42 alone, conferred resistance to CPT inhibition of LEC tube formation. Taken together, the results suggest that CPT inhibits LEC tube formation, in part, by inhibiting VEGFR-3-mediated ERK1/2 phosphorylation and, in part, by inhibiting expression of the small GTPases.