Leukemia (2002) 16, 1490–1499  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Anti-angiogenic activity of the purine analog 6-thioguanine M Presta 1 , M Belleri 1 ,AVacca 2 and D Ribatti 3 1 Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Brescia, Italy; 2 Department of Biomedical Sciences and Human Oncology, University of Bari, Bari, Italy; and 3 Department of Human Anatomy and Histology, University of Bari, Bari, Italy The antimetabolite 6-thioguanine (6-TG) is utilized in the man- agement of acute myelogenous leukemia (AML). Angiogenesis is a possible therapeutic target in hematologic tumors. Thus, we addressed the possibility that 6-TG may also act as an anti- angiogenic molecule. 6-TG inhibited endothelial cell prolifer- ation triggered by fibroblast growth factor-2 (FGF2) and vascu- lar endothelial growth factor (VEGF) and delayed the repair of a mechanically wounded endothelial cell monolayer. Also, 6- TG inhibited sprouting within fibrin gel, morphogenesis on Matrigel, and collagen gel invasion by endothelial cells. 2-Ami- nopurine was ineffective. In vivo, 6-TG inhibited basal, VEGF- induced, and FGF2-induced vascularization in the chick embryo chorioallantoic membrane and prevented neovascu- larization triggered by leukemia LIK cells or their conditioned medium. Finally, bone marrow vascularization in AML patients was decreased to control values in the early remission phase and persisted unvaried after 8–12 months of maintenance ther- apy with 6-TG. Thus, 6-TG inhibits different steps of the angiog- enesis process in vitro and exerts a potent anti-angiogenic activity in vivo. Its anti-angiogenic activity, together with its antimetabolite activity towards tumor cells, may contribute to its action during maintenance therapy in AML. These results suggest a new rationale for the use of purine analogs in the management of AML. Leukemia (2002) 16, 1490–1499. doi:10.1038/sj.leu.2402646 Keywords: angiogenesis; purine analog; FGF2; VEGF Introduction In the adult, the proliferation rate of endothelial cells is very low compared to many other cell types in the body. Uncon- trolledendothelialcellproliferationisobservedintumorneo- vascularization, angioproliferative diseases like Kaposi’s sar- coma, and angiogenesis-dependent diseases like rheumatoid arthritis, psoriasis, and a number of eye diseases. 1 Various angiogenesis inhibitors have been developed so far, their effi- cacy has been evaluated in different in vitro and in vivo assays, 2 and their clinical evaluation in cancer patients is in progress (for further information about angiogenesis inhibitors in clinical trials see the NCI web site: http://cancertrials.nci.nih.gov). Recently, the hypothesis that anti-angiogenic compounds can be used in combination with cytotoxicdrugsfortumortherapyhasbeenadvanced(seeRef. 3andreferencestherein).Also,chemotherapeuticagentshave shown anti-angiogenic properties in vitro and in vivo, 4,5 lead- ing to the concept of anti-angiogenic scheduling of chemo- therapy. 5,6 Purine analogs were developed in the early 1950s as anti- neoplastic chemotherapeutic agents. 7 These antimetabolites Correspondence: M Presta, Unit of General Pathology and Immu- nology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, via Valsabbina 19, 25123 Brescia, Italy; Fax: +39-0303701157 Received 2 April 2002; accepted 14 May 2002 inhibit de novo purine synthesis and purine interconversion reactions and their metabolites can be incorporated into nucleic acids. 7 6-Thioguanine (6-TG) and 6-methylmercapto- purine riboside (6-MMPR) also alter membrane glycoprotein synthesis. 8 Purineanalogscanactasproteinkinaseinhibitors, 6-MMPR showing a high potency and selectivity for nerve growth factor-activated protein kinase N. 9 2-Aminopurine (2- AP) inhibits proto-oncogene and interferon gene transcrip- tion. 10 Combination chemotherapy regimens for the manage- ment of solid tumors have been proposed in which purine analogs are administered in association with cytotoxic drugs. 11,12 At present, the purine analog 6-TG is used in the management of acute myelogenous leukemia (AML) both in remission induction and in maintenance therapy. 7 An increasing body of evidence points to a role for bone marrow angiogenesis in hematologic tumors. 13–15 For instance,bonemarrowvascularizationisincreasedinpatients with AML. 15–17 Also, AML cells produce angiogenesis factors, includingfibroblastgrowthfactor-2(FGF2)andvascularendo- thelial growth factor (VEGF), 15 whose levels are an inde- pendent predictor of outcome. 18 Recently,wedemonstratedthatthepurineanalog6-MMPR modulates the angiogenic activity of FGF2 in vitro and affects blood vessel formation in vivo. 19 Also, continuous systemic administration of 6-mercaptopurine ribose phosphate inhibits angiogenesis in the rabbit cornea. 5 In contrast, 6-methylmer- captopurine,2-AP,andadeninearedevoidofanti-angiogenic activity, 19 thusindicatingthatsubtlestructuraldifferencesmay determinetheabilityofpurineanalogstoaffectneovasculariz- ation. On this basis, we addressed the possibility that 6-TG mayactasananti-angiogenicmolecule,thisactivitycontribu- ting to its efficacy in AML therapy. 6-TG was evaluated for the capacity to affect various steps of the angiogenesis process (ie cell proliferation, motility, endothelial cell sprouting, collagen invasion, and formation of capillary-like structures) induced by FGF2 and/or VEGF in cultured endothelial cells of different origin. The in vitro observations were compared to the effect of 6-TG on in vivo neovascularizationinthechickembryochorioallantoicmem- brane (CAM) under basal conditions or during neovasculariz- ation induced by FGF2 or VEGF or by human leukemia LIK cells grafted on to the CAM. Finally, we evaluated bone mar- row vascularization in AML patients given maintenance ther- apywith6-TG.Theresultsdemonstratethat6-TGinhibitsdif- ferent steps of the angiogenesis process in vitro and exerts a potentanti-angiogenicactivityintheCAM.Itsanti-angiogenic capacity, together with its antimetabolite activity, may con- tribute to its action during maintenance therapy in AML patients.