Cell experiment:

Cell proliferation and DNA synthesis are measured. For cell counting, VSMCs are seeded in 12-well culture plates at 5-6×104 cells/mL and cultured in DMEM with 10% FBS at 37℃ for 24 h. Under these conditions, the cells reach 70% confluence. The medium is then replaced by serum-free medium with Corynoxeine (5-50 μM). The cells are stimulated with 50 ng/mL PDGF-BB, then trypsinized with trypsin-EDTA and counted using a hemocytometer under a microscope. For [3H]-thymidine incorporation experiments, VSMCs are seeded in 24-well culture plates 5000 cells/well and then allowed to grow for 3-4 d in DMEM, and 2 μCi/mL of [3H]-thymidine are added to the medium. The reactions are terminated after 4 h by aspirating the medium and subjecting the cultures to sequential washes on ice with PBS containing 10% trichloroacetic acid and ethanol/ether (1 : 1, v/v). Acid-insoluble [3H]-thymidine is extracted into 250 μL of 0.5 M NaOH/well; this solution is then mixed with 3ml of scintillation cocktail and quantified using a liquid scintillation counter[1].

Corynoxeine, isolated from the hook of Uncaria rhynchophylla, is a potent ERK1/ERK2 inhibitor of key PDGF-BB-induced vascular smooth muscle cells (VSMCs) proliferation.

Corynoxeine is able to inhibit the PDGF-BB-stimulated proliferation of VSMCs through downregulation of PDGF-BB-induced ERK1/2 activation. Pre-incubation of VSMCs with Corynoxeine significantly inhibits PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas Corynoxeine has no effects on mitogen-activated protein kinase (MAPK/ERK)-activating kinase 1 and 2 (MEK1/2), Akt, or phospholipase C (PLC) γ 1 activation or on PDGF receptor beta (PDGF-Rβ) phosphorylation. Corynoxeine inhibits PDGF-BB-induced ERK1/2 activation, in the same concentration range that inhibits VSMC proliferation and DNA synthesis. Corynoxeine inhibits VSMC numbers in response to PDGF-BB with 50% inhibitory concentrations (IC50) of 13.7 μM. Corynoxeine inhibits DNA synthesis in response to PDGF-BB (24 h) with IC50 of 9.2 μM. Pre-treatment of VSMCs with Corynoxeine (5-50 μM) for 24 h results in significant decreases in cell number without any cytotoxicity; the inhibition percentages are 25.0±12.5, 63.0±27.5 and 88.0±12.5% at 5, 20 and 50 μM, respectively. Corynoxeine also significantly inhibits the 50 ng/mL PDGF-BB-induced DNA synthesis of VSMCs in a concentration-dependent manner without any cytotoxicity; the inhibitions are 32.8±11.0, 51.8±8.0 and 76.9±7.4% at concentrations of 5, 20 and 50 μM, respectively[1].

References:
[1]. Kim TJ, et al. Corynoxeine isolated from the hook of Uncaria rhynchophylla inhibits rat aortic vascular smooth muscle cell proliferation through the blocking of extracellular signal regulated kinase 1/2 phosphorylation. Biol Pharm Bull. 2008 Nov;31(11):2073-8.