Antiangiogenic and antitumoral effects mediated by a vascular endothelial growth factor receptor 1 (VEGFR-1)-targeted DNAzyme

Abstract

Antiangiogenesis is a promising antitumor strategy that inhibits tumor vascular formation to suppress tumor growth. DNAzymes are synthetic single-strand deoxyribonucleic acid (DNA) molecules that can cleave ribonucleic acids (RNAs). Here, we conducted a comprehensive in vitro selection of active DNAzymes for their activity to cleave the vascular endothelial growth factor receptor (VEGFR-1) mRNA and screened for their biological activity in a matrigel tube-formation assay. Among the selected DNAzymes, DT18 was defined as a lead molecule that was further investigated in several model systems. In a rat corneal vascularization model, DT18 demonstrated significant and specific antiangiogenic activity, as evidenced by the reduced area and vessel number in VEGF-induced corneal angiogenesis. In a mouse melanoma model, DT18 was shown to inhibit B16 tumor growth, whereas it did not affect B16 cell proliferation. We further assessed the DT18 effect in mice with established human nasopharyngeal carcinoma (NPC). A significant inhibition of tumor growth was observed, which accompanied downregulation of VEGFR-1 expression in NPC tumor tissues. To evaluate DT18 effect on vasculature, we performed dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) on the human NPC xenograft mice treated with DT18 and showed a reduction of the parameter of K(trans) (volume constant for transfer of contrast agent), which reflects the condition of tumor microvascular permeability. When examining the safety and tolerability of DT18, intravenous administration of Dz18 to healthy mice caused no substantial toxicities, as shown by parameters such as body weight, liver/kidney function, and histological and biochemical analyses. Taken together, our data suggest that the anti-VEGFR-1 DNAzyme may be used as a therapeutic agent for the treatment of cancer, such as NPC.

Figure 1

In vitro cleavage of VEGFR-1 mRNA by DNAzymes. In vitro transcribed RNA with ³²P-labeling was incubated with individual DNAzyme in the cleavage buffer (50 mmol/L Tris-Cl, 10 mmol/L MgCl₂, pH 7.5). The samples were run on a 6% denaturing polyacrylamide gel and exposed on a PhosphorImager.

Figure 2

Molecular and cellular assays for DT18. (A) DT18 was assayed for its kinetic activity under a single turnover condition. Control was made with the same arms and an inverted catalytic core. The first order rate constant (k_obs ) was used to determine the cleavage efficiency. (B) Transfection efficiency of DT18 was measured using FITC-labeled DT18 in HUVECs by FACS. TMP was used as a transfection reagent. (C) Effect of DT18 on VEGFR-1 expression in HUVECs was examined by Western blot. (D) Matrigel tube formation assay was performed to assess the antiangiogenic activity of DT18.

Figure 3

Effect of DT18 on neovascularization in rat cornea. The corneas of 7-wk-old male Sprague Dawley rats were implanted with a 30 μmol/L VEGF-soaked nitrocellulose filter disk. A total of 100 μg DT18, or its control oligonucleotides, was administered to the conjunctiva adjacent to the implant site on the day of disk implantation. The corneas were removed to quantitate the area covered by vascularization (A, left) and the number of vessels growing within the cornea (A, right). The representative photographs of the rat corneas from each group were shown in (B). Values are the means ± SE of three replicates. *P < 0.05 compared with the control.

Figure 4

Antitumor effect of DT18. (A) DT18 was coinjected with B16 tumor cells into mice, and tumor volume was measured (n = 8). (B) Effect of Dt18 on B16 cell proliferation was measured by using MTT assay. (C) Human nasopharyngeal carcinoma was grown in nude mice, and DT18 mixed with Fugene6 was injected intratumorally when the tumor reached about 60–100 mm³. Injections were made twice a week and tumor volumes were measured on alternate days (n = 5). (D) Immunohistochemical staining of the tumor tissues with the VEGFR-1-specific antibody in DT18-, INV-Ctrl– or saline-treated mice.

Figure 5

Effect of DT18 on tumor vasculature assayed by DCE-MRI. MRI was performed 3 d after the DT18 treatment on superconducting magnetic resonance imaging scanners (GE Signa HDx 3.0T) with 2.0 inches coil (FT-AN). Gd-DTPA, a blood-pool contrast agent, was injected via the tail-vein catheter. The representative images were presented (A). K^trans was generated from raw data of DCE-MRI by using NordicICE software (version 2.3.6) (B). Values are the means ± SE of three replicates. *P < 0.05 compared with the control.

Figure 6

In vivo assessment of DT18 toxicity. (A) Effect of intravenously injected DT18 on body weight (n = 6). (B) Representative hematoxylin and eosin–stained sections of liver, lung, spleen and kidney from mice treated with DT18, INV-Ctrl or saline.