Antiangiogenic agents from Tripterygium wilfordii for cancer treatment

Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study.T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide.In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM.This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.)...