[CANCER RESEARCH 64, 2328 –2332, April 1, 2004] Advances in Brief The Matrix Metalloproteinase Inhibitor Prinomastat Enhances Photodynamic Therapy Responsiveness in a Mouse Tumor Model Angela Ferrario, 1 Christophe F. Chantrain, 1,2 Karl von Tiehl, 1 Sue Buckley, 5 Natalie Rucker, 1 David R. Shalinsky, 6 Hiroyuki Shimada, 3 Yves A. DeClerck, 1,2 and Charles J. Gomer 1,4 Departments of 1 Pediatrics, 2 Biochemistry and Molecular Biology, 3 Pathology, 4 Radiation Oncology, and 5 Surgery, Keck School of Medicine, University of Southern California and the Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California, and 6 Department of Pharmacology, Agouron Pharmaceuticals, Inc., a Pfizer Company, La Jolla, California Abstract Photodynamic therapy (PDT) clinical results are promising; however, tumor recurrences can occur and, therefore, methods for improving treatment efficacy are needed. PDT elicits direct tumor cell death and microvascular injury as well as expression of angiogenic, inflammatory, and prosurvival molecules. Preclinical studies combining antiangiogenic drugs or cyclooxygenase-2 inhibitors with PDT show improved treatment responsiveness (A. Ferrario et al., Cancer Res 2000;60:4066 –9; A. Fer- rario et al., Cancer Res 2002;62:3956 – 61). In the present study, we evaluated the role of Photofrin-mediated PDT in eliciting expression of matrix metalloproteinases (MMPs) and modulators of MMP activity. We also examined the efficacy of a synthetic MMP inhibitor, Prinomastat, to enhance tumoricidal activity after PDT, using a mouse mammary tumor model. Immunoblot analysis of extracts from PDT-treated tumors dem- onstrated strong expression of MMPs and extracellular MMP inducer along with a concomitant decrease in expression of tissue inhibitor of metalloproteinase-1. Gelatin zymography and enzyme activity assays per- formed on protein extracts from treated tumors confirmed the induction of both latent and enzymatically active forms of MMP-9. Immunohisto- chemical analysis indicated that infiltrating inflammatory cells and endo- thelial cells were primary sources of MMP-9 expression after PDT, whereas negligible expression was observed in tumor cells. Administration of Prinomastat significantly improved PDT-mediated tumor response (P  0.02) without affecting normal skin photosensitization. Our results indicate that PDT induces MMPs and that the adjunctive use of an MMP inhibitor can improve PDT tumor responsiveness. Introduction Photodynamic therapy (PDT) is used clinically for treating a variety of solid malignancies as well as nononcological disorders such as age-related macular degeneration and psoriasis (1, 2). The treatment involves the systemic administration of a photosensitizer followed by irradiation of the targeted lesion with visible light. This multistep procedure initiates the photochemical generation of cytotoxic reactive oxygen species such as singlet oxygen within the treatment field and leads to direct tumor tissue destruction and microvascular disruption (1). The Food and Drug Administration-approved photosensitizer Photofrin (PH), as well as a variety of second generation photosen- sitizers, is currently used in PDT clinical trials. Therapeutic results are encouraging; however, recurrences can occur and, therefore, methods to improve long-term PDT responsiveness are needed. PDT induces oxidative stress, localized inflammation, and vascular injury within treatment fields, and each of these responses can lead to increased expression of angiogenic factors, cytokines, and survival molecules (1, 3). Overexpression of these molecules can activate pathways associated with tumor recurrence. We recently documented increased expression of vascular endothelial growth factor and prostaglandin E 2 in murine tumors treated with PH-mediated PDT and demonstrated that combination procedures using inhibitors of vascular endothelial growth factor or cyclooxygenase-2 improved the therapeutic efficacy of PDT (4, 5). Growth of solid tumors depends on the formation and development of new blood vessels concomitant with the degradation of the extra- cellular matrix (6). Information obtained by numerous laboratories directly links the expression of matrix metalloproteinases (MMPs) with these processes (7, 8). MMPs are members of a multigene family of zinc-containing enzymes that function under both physiological and pathological conditions (9). These proteinases share sequence homology and are grouped into different subfamilies according to structure, substrate specificity, and cellular localization (9). Cyto- kines, growth factors, oncogenes, and reactive oxygen species are among the stimuli that activate MMP transcription (8 –10). The trans- lated proteins are usually expressed in an inactive or latent proenzy- matic form that requires the proteolytic cleavage of an NH 2 terminus peptide domain to be converted to a biologically active proteinase. A positive correlation exists between expression/activation of MMPs and tumor angiogenesis, growth, invasion, and metastatic potential (8, 9). In solid tumors, MMPs are often expressed by stromal cells and macrophages rather than by tumor cells (8, 11). The in vivo activity of MMPs is regulated in part by endogenous tissue inhibitors of MMPs or TIMPs (12). An imbalance in the expression and activation of TIMPs and MMPs leads to modifications in tumor growth. In the present study, we evaluated expression patterns, biological activity, and cellular sources of MMPs in a murine tumor model after PH-mediated PDT. We also examined the role of MMP activity in modulating tumor responses after PDT using the synthetic MMP inhibitor Prinomastat. Our results indicate that PDT-treated tumors have increased expression of MMPs and that pharmacological inhi- bition of MMPs using Prinomastat can selectively increase in vivo PDT tumoricidal activity. Materials and Methods Drugs. The photosensitizer PH was a gift from Axcan Scandipharma Inc., (Birmingham, AL) and was dissolved in 5% dextrose in water to make a 2.5 mg/ml stock solution. The MMP inhibitor Prinomastat (AG3340) was a gift from Agouron Pharmaceuticals Inc., a Pfizer Company (La Jolla, CA) and was dissolved in acidified water (pH 2.3) at a final concentration of 20 mg/ml. The solution was sterile filtered, stored at 4°C, and used within 2 weeks. Phorbol 12-myristate 13-acetate was purchased from Sigma (St. Louis, MO) and Received 1/0/04; revised 2/6/04; accepted 2/19/04. Grant support: Supported in part by NIH Grants RO-1 CA-31230 (to C. J. Gomer) CA-09897 (to C. J. Gomer), and PO-1 CA-81403 (to H. Shimada and Y. A. DeClerck), the Neil Bogart Memorial Fund of the Martell Foundation for Leukemia, Cancer and AIDS Research (to Y. A. DeClerck and C. J. Gomer), and the Las Madrinas Endowment for Experimental Therapeutics (to C. J. Gomer). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Charles J. Gomer, Childrens Hospital Los Angeles, Mail Stop 67, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: (323) 669-2335; Fax: (323) 669-0742; E-mail: cgomer@chla.usc.edu. 2328 Research. on October 21, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from