0023-6837/03/8310-1385$03.00/0 LABORATORY INVESTIGATION Vol. 83, No. 10, p. 1385, 2003 Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. COX-2/VEGF-Dependent Facilitation of Tumor-Associated Angiogenesis and Tumor Growth In Vivo Satoko Yoshida, Hideki Amano, Izumi Hayashi, Hidero Kitasato, Mariko Kamata, Madoka Inukai, Hirokuni Yoshimura, and Masataka Majima Departments of Pharmacology (SY, HA, IH, MK, MI, MM), Thoracic Surgery (SY, HA, HY), and Microbiology (HK), Kitasato University School of Medicine, and Department of Molecular Pharmacology (SY, HA, IH, MM), Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan SUMMARY: Nonsteroidal anti-inflammatory drugs are known to suppress the occurrence and progression of malignancies such as colorectal cancers. However, the precise mechanism of these actions remains unknown. We have evaluated the role of an inducible cyclo-oxygenase (COX-2) in tumor-associated angiogenesis and tumor growth, and identified the downstream molecules involved using a ddy mouse model of sponge angiogenesis, which mimics tumor angiogenesis and is COX-2 and vascular endothelial growth factor (VEGF) dependent. In this model, VEGF expression was down-regulated by selective COX-2 inhibition with NS-398. To find out the involvement of COX-2/VEGF pathway in tumor-associated angiogenesis, we estimated angiogenesis occurring around implanted Millipore chambers containing sarcoma-180 (S-180) cells or Lewis lung carcinoma cells. Daily oral administration of NS-398 or of aspirin, a nonselective COX inhibitor, suppressed angiogenesis seen around the Millipore chambers. S-180 cells implanted in ddy mice formed substantial tumors with extensive angiogenesis markedly suppressed by aspirin and COX-2 inhibitors NS-398 and JTE522, but not by mofezolac, an inhibitor of constitutive COX-1. Tumor-associated angiogenesis was also significantly suppressed by a neutralizing antibody against VEGF. S-180 tumor growth in the subcutaneous tissues was also suppressed by aspirin, COX-2 selective inhibitors, and the VEGF antibody, but not by the COX-1 inhibitor. These results demonstrate that the inhibition of the COX-2/VEGF-dependent pathway was effective in tumor-associated angiogenesis, tumor growth, and tumor metastasis. (Lab Invest 2003, 83:1385–1394). onsteroidal anti-inflammatory drugs (NSAIDs), actions of COX and specific PG synthases. Two COX N which block the enzymatic activity of cyclo- isoforms have been identified: COX-1 is constitutively oxygenase (COX), have been widely used for anti- expressed in various tissues, whereas COX-2 is in- inflammatory and analgesic purposes. Early in the duced by mitogens, growth factors, cytokines, and 1990s, it was reported that a significant reduction in tumor promoters. Using COX-2 knockout mice, it was mortality from colorectal cancer occurred depending revealed that a COX-2–dependent pathway increased on the cumulative doses of an NSAID, and further the number and size of intestinal polyps generated by evidence suggested that NSAIDs also affect the inci- a mutation in the adenomatous polyposis coli (APC) dence and progression of other types of cancer, gene. That report emphasized strongly the importance pointing to a possible role of COX in other tumor of COX-2 in colon tumor development (Ohshima et al, formations (Smally and Dubois, 1997). Prostaglandins 1996). In spite of the efficacy of NSAIDs as anticancer (PGs) belong to a group of oxygenated metabolites of agents, the precise mechanism or mechanisms of arachidonic acid and are produced by the sequential their antitumor action are not fully clarified and have given rise to wide-ranging discussion, in which some opinions are surprisingly unrelated to COX inhibition DOI: 10.1097/01.LAB.0000090159.53224.B9 (Marx, 2001; Prescott et al, 1996; Shiff and Rigas, Received April 7, 2003. 1997). This work was supported by grants from an Integrative Research Program of Angiogenesis is an important factor in tumor devel- the Graduate School of Medical Science, Kitasato University, and from opment, and tumor-associated angiogenesis is medi- Parents’ Association Grant of Kitasato University School of Medicine. This ated by the migration and proliferation of host endo- work was also supported by a research grant (#15390084), by a “High-tech thelial cells. With the use of an in vitro coculture Research Center” grant, and by a grant from The 21st Century COE system comprising endothelial cells and colon carci- Program, of Ministry of Education, Culture, Sports, Science and Technol- noma cells that overexpress COX-2, it was shown that ogy (MEXT). the increased production of PGs by the carcinoma Address reprint requests to: Dr. Masataka Majima, Department of Phar- macology, Kitasato University School of Medicine, Kitasato 1–15–1, cells was associated with endothelial cell migration Sagamihara, Kanagawa 228–8555, Japan. E-mail: en3m-mjm@ and tube formation (Tsujii et al, 1998). This suggested asahi-net.or.jp a role of COX-2 in tumor-induced angiogenesis. How- Laboratory Investigation • October 2003 • Volume 83 • Number 10 1385 Yoshida et al ever, the relevance of these in vitro observations to Roles of VEGF in Angiogenesis and Induction Factors of tumorigenesis in vivo remains to be established. For a VEGF in Sponge Implantation Model mechanistic analysis of angiogenesis in vivo, we have To examine the COX-2–dependent downstream mol- developed a sponge implant model, in which a poly- ecules, we first tested the effects of topical injections urethane sponge disk implanted subcutaneously in of the neutralizing antibodies against several growth rats induces extensive angiogenesis in surrounding factors and against a cytokine into the sponge im- proliferative granuloma tissues (Hayashi et al, 2002; plants. The antibodies against epithelial growth factor Majima et al, 1997, 2000; Muramatsu et al, 2000). With (EGF), transforming growth factor (TGF)-␤, and IL-1 the use of this model, we have previously shown that did not reduce the angiogenesis at all, but by contrast, angiogenesis occurs both concomitantly with the in- the VEGF antibody markedly suppressed the angio- duction of COX-2 mRNA and is inhibited by adminis- genic reaction (Fig. 2A). tration either of a nonselective NSAID (indomethacin) The expression of VEGF mRNA was not detectable or of selective COX-2 inhibitors (Majima et al, 2000). in normal subcutaneous tissues in mice without We further demonstrated that angiogenesis induced sponge implantation (data not shown). The expression by either endogenous COX-2 or exogenous PGs was was certainly up-regulated in the sponge granulation accompanied by increased expression of vascular tissue stimulated with bFGF (Fig. 2B). The up- endothelial growth factor (VEGF) and that angiogene- regulation of VEGF expression was suppressed with sis was abolished by administration of an antisense daily oral administration of NS398 (Fig. 2B). oligonucleotide specific for VEGF mRNA in that model Immunoreactive VEGF protein was also detectable (Majima et al, 2000). These results suggest that COX- by a specific ELISA in the exudates in the sponge 2–catalyzed PG formation may mediate the tumor- implants stimulated with bFGF (Fig. 2C). This VEGF associated angiogenesis and tumor growth in vivo in level was strongly reduced with topical injections of relation to VEGF expression. either an adenylate cyclase inhibitor, SQ22,536, and In the present study, with the use of the compounds the protein kinase A inhibitor H-89 (Fig. 2C). These selective of COX isoforms and growth factor neutral- results suggested that VEGF had a crucial role in the izing antibodies, we have now identified the COX facilitation of angiogenesis in this model and that its isoforms responsible for tumor-associated angiogen- induction was dependent on the COX-2 and adenylate esis and tumor growth. Furthermore, we have charac- cyclase–protein kinase A signaling pathway. terized the downstream molecules responsible for COX-2–dependent PG formation. The present results will address the effectiveness of inhibition in COX-2/ Roles of COX-2 and VEGF in Tumor-Associated VEGF pathway in tumor-associated angiogenesis to- Angiogenesis gether with lung metastasis. Seeking to identify the roles of COX-2 in tumor- associated angiogenesis, we first tested the Millipore Results chamber model, in which angiogenesis was deter- mined by the hemoglobin content of the granulation Roles of COX-2 in Angiogenesis in Sponge Implant Model tissues formed around the tumor-cell–containing Mil- lipore chambers. Implantation of the chambers con- To ascertain the roles of inducible COX-2 and its taining sarcoma 180 (S-180) cells enhanced angio- downstream molecules responsible for the generation genesis, compared with that seen in mice in which the of prostaglandins in tumor-associated angiogenesis, chambers were implanted without tumor cells. Daily we adopted a sponge implant model in the present oral administration of aspirin or NS-398 strongly re- study. This model is characterized by chronic hyper- duced angiogenesis (Fig. 3, A and B). The same was plastic inflammation and is designed to reflect tumor- true in the granulation tissues formed around the associated angiogenesis by mimicking the angiogenic chambers containing cells of another tumor cell line, responses in the granulation tissues formed around Lewis lung carcinoma (LLC) (Fig. 3, C and D). the implants. When the circular sponge discs were We next implanted the tumor cells into the subcuta- implanted