Triptolide functions as a potent angiogenesis inhibitor Ming-Fang He 1 , Yi-Hsien Huang 2 , Li-Wha Wu 2,3 , Wei Ge 1 , Pang-Chui Shaw 4,5 , Paul Pui-Hay But 1,5 1 Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of China 2 Institutes of Molecular Medicine and Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China 3 Cardiovascular Research Center, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China 4 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of China 5 Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of China Triptolide is a key anti-inflammatory compound of the Chinese herbal medicine Tripterygium wilfordii Hook. f. (Celastraceae). It also possesses potent antitumor activity. In this study, we show that triptolide is an angiogenesis inhibitor based on various angiogenesis assays. The IC 50 in in vitro assays was 45 nM, which was much lower than the plasma concentrations of triptolide in the rat or human administered with T. wilfordii extracts for treating inflammation. When dosed in vivo, triptolide potently inhibited angiogenesis at 100 nM in Matrigel plug assay. Triptolide at 0.75 mg/kg/day significantly blocked tumor angiogenesis and tumor progression in murine tumorigenesis assay. The underlying mechanism of triptolide correlated with downregulation of proangiogenic Tie2 and VEGFR-2 expression in human umbilical vein endothelial cell by semiquantitative RT-PCR and western blot analysis. Although Tie2 inhibition appeared to be a later event as compared with VEGFR-2, Tie2 overexpression significantly attenuated the inhibitory effect of triptolide on endothelial proliferation and network formation. By contrast, Tie2 knockdown mimicked the inhibitory effect of triptolide on endothelial network formation. Our findings suggest that antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor- mediated signaling pathways, and triptolide can be a promising antiangiogenic agent. Tripterygium wilfordii Hook. f. (Celastraceae), also known as Thunder God Vine, is a popular herb in China for the treat- ment of immune-inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus, nephritis and asthma. 1 Extracts from T. wilfordii has entered clinical trials for the treatment of rheumatoid arthritis. 2–5 Characterization of the active components present in this plant identified trip- tolide, a diterpenoid triepoxide, as a key component responsi- ble for most of the immunosuppressive, anti-inflammatory and antiproliferative effects. 6 The content of triptolide was thus used as the quality control marker for most of the extracts and preparations made from T. wilfordii. 7–9 Tripto- lide itself has also been tested in clinical trials for the treat- ment of psoriasis vulgaris, 10 diabetic nephropathy 11 and ne- phritic syndrome 12 in China. Triptolide was first found to have antileukemic activity in 1972. 13 In addition to its potent immunosuppressive and anti- inflammatory activities, its antitumor activity has attracted much interest since then. Triptolide exerted antiproliferative and proapoptotic effects on tumor cell lines in vitro, 14–16 restricted tumor growth or shrank tumor in vivo. 17,18 Further- more, triptolide sensitized tumor cells to other anticancer agents 19 and had synergistic effect with other chemotherapeu- tic agents in preclinical animal models. 15,18 Although tumor growth and metastasis depend upon angiogenesis, there are only preliminary studies on the effect of triptolide on angiogenesis. 20,21 Antiangiogenic therapy has been increasingly recognized as a promising therapy for cancer treatment. We have previ- ously identified that triptolide from T. wilfordii had potent antiangiogenic activity in zebrafish angiogenic assay through targeting angpt2-tie2 signaling pathway in both time- and dose-dependent manners. 22 To further confirm the antiangio- genic activity of triptolide in mammalian systems, we used human umbilical vein endothelial cells (HUVECs) and mouse Matrigel plug model to test its antiangiogenic effect in vitro Key words: antiangiogenesis, Tripterygium wilfordii, triptolide, Tie2, VEGFR-2 Additional supporting information may be found in the online version of this article. Grant sponsor: Chinese University of Hong Kong Direct Grant; Grant sponsor: National Science Council; Grant numbers: NSC-96- 2320-B006-046, NSC-95-2752-B006-003-PAE, NSC 94-3112-B-001- 003 and 94-3112-B-001-018-Y; Grant sponsor: Department of Health; Grant number: DOH-TD-B-111-004 DOI: 10.1002/ijc.24694 History: Received 30 Dec 2008; Accepted 18 Jun 2009; Online 30 Jun 2009 Correspondence to: Li-Wha Wu, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of China, Fax: þ852-26035646, E-mail: liwhawu@mail.ncku.edu.tw; or Paul Pui-Hay But, Institutes of Molecular Medicine and Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China, Fax: 1886-6-209-5845, E-mail: paulbut@hotmail.com Cancer Therapy Int. J. Cancer: 126, 266–278 (2010) V C 2009 UICC International Journal of Cancer IJC