Figure S1. In vitro beta-casein degradation analysis (MMP-9); Figure S2. AARPalpha induces endothelial apoptosis; Figure S3. RK5, AARPalpha/AARPbeta exerted no inhibitory effects on non-endothelial cells; Figure S4. Detection of Serum AST, ALT, BUN and creatine; Figure S5. Body weights of mice xenografted with LLC and treated with RK5, AARPalpha, AARPbeta (25 mg/kg), paclitaxel (10 mg/kg).
ARTICLE ABSTRACT
Angiogenesis is required for tumor growth and metastasis, and targeting angiogenesis is a novel anticancer strategy. However, cancer development is a complex multistep process, and single antiangiogenic agents have limited therapeutic efficacy. Here, we report a triple fusion protein, namely CTT peptide–endostatin mimic–kringle 5 (AARP), consisting of MMP-2/9–selective inhibitory peptide (CTT peptide) and well-known endogenous antiangiogenic agents (endostatin mimic and kringle 5), which can simultaneously target matrix metalloproteinases (MMP) and endothelial cells, blocking their actions. AARP was bacterially expressed, and biologic activity of purified AARP was assessed. AARP could significantly inhibit the enzymatic activity of MMP-2/9, proliferation, migration, and tube formation of endothelial cells in vitro. The antitumor activity of AARP was shown in a concentration-dependent manner when injected i.p. into immunodeficient mice bearing multidrug-resistant human epidermoid carcinomas (KB), and AARP is superior to clinical grade endostatin in inhibiting KB xenograft growth. In mouse models of Lewis lung carcinoma (LLC) and hepatoma H22, when given as a single dose, AARP is highly effective for reducing tumor growth, angiogenesis, and metastasis, and increasing survival time. AARP possessed significantly greater antiangiogenic activity than endostatin mimic, CTT peptide–kringle 5 (RK5) both in vitro and in vivo. Compared with conventional chemotherapeutic agents (cyclophosphamide and paclitaxel), AARP is also effective. More importantly, AARP is cytocompatible and no tissue toxicity could be observed after large dose administration. Taken together, our findings suggest AARP is a highly effective, safe, and more potent antiangiogenic agent for blocking tumor angiogenesis and metastasis, and warrants further testing for clinical applications. Mol Cancer Ther; 13(11); 2674–87. ©2014 AACR.
