DNAzymes Targeting VEGFR-1Inhibit Angiogenesis And Tumor Growth

The contemporary anti-tumor therapy is a multimodality treatment including surgery, radiotherapy and chemotherapy. The molecularly targeted drugs have atracted more and more clinical attention. One of the most promising therapeutic strategies is to inhibit the tumor angiogenesis process by angiogenesis inhibitor in order to suppress the tumor proliferation. DNAzyme is a new molecular tool in gene therapy field, which can manipulate gene expression by cutting the RNA molecule at the site between the paired pyrimidines and unpaired purines. This study designed the type10-23DNAzyme targeting VEGFR-1mRNA and observed the effect of angiogenesis inhibition in vivo and in vitro. Furthermore, we assessed the biological effect of10-23DNAzyme DTI8in the melanoma and nasopharyngeal carcinoma models. This study demenstrated the feasibility of targeting VEGFR1by DNAzymes as a novel therapeutic strategy for cancer treatment.Part ⅠPurpose:The design and biochemical analysis of10-23DNAzyme targeting at VEGFR-1mRNA.Method:This study designed and synthesized the type10-23DNAzymes based on the homologus sequences of VEGFR-1mRNA of mouse and human and modified the DNAzymes with phosporothioate linkages..Through in vitro selection assay we chosed the most active DNAzyme and transfected the active DNAzyme into HUVEC by TMP. We observed the uptake of the FITC-labeled DNAzyme in HUVEC and assessed the transfection efficiency and gene suppression activity by flow cytometry and immunoblotting.Results:Eleven10-23DNAzymes targeting at different sites in the VEGFR-1mRNA were designed and synthesized. Among them, one of the most active DNAzymes called DT18was selected through in vitro selection assays. Kinetic analysis showed a favorable kinetic profile in a single turnover model. When transfected into HUVECs, we observed the green fluorescence in50%cells under microscope24hours after TMP transfection of DT18and confirmed the80%transfection efficiency through flow cytometry. The results of immunoblotting confirmed that DT18could significantly suppress VEGFR-1expression. Compared with control group, the vessel formation capability of DT18tranfected cells significantly decreased.Conclusion:We successfully selected the active DNAzymes targeting at VEGFR-1mRNA.Part ⅡPurpose:Assessment of anti-angiogenesis effects of the10-23DNAzyme DT18both in vitro and in vivo.Method:Rat corneal pocket assay was used to examine the anti-angiogenesis activity of DT18. Three groups of animal including DT18, INV-Ctrl and saline groups were used in the assay. Neovesculature was monitored by counting the vessel number and area affected under microscopy. The mouse model with the transplanted melanoma B16cells was established to observe the impact of DT18on the tumor vasculture and tumor growth in vivo. Effect of DT18on B16cell proliferation was examined using MTT assay.Results:DT18significantly inhibited the formation of new corneal vessels. Compared with controls, the area of neovasculature decreased by72%and the number of vessels decreased by61%respectively in DT18group. The differences between the three groups are statistically significant (P<0.05). In the established melanoma model, the enriched vessels and high VEGFR1expression were observed. Using intratumoral injection of DT18, B16tumor growth was significantly inhibited.The differences of the tumor growth between the three groups were statistically significant(P<0.05). When FITC-labeled DT18was transfected into B16cells by the aid of Fugene, we observed the green fluorescence in80%cells48h post transfection under microscope. MTT asaay showed that the DT18had no impact on cell proliferation (P>0.05).Conclusion:The cornea vessels formation model and melanoma transplanted model in mice were succefully established. In these models, we proved that the DT18, the10-23DNAzymes with phosphorothioate modifications, could significantly suppress the angiogenesis, whereby the tumor growth was inhibited.Part ⅢPurpose:Evaluation of DT18effect on tumor vessel permeability in vivo by molecular imaging technique (Dynamic contrast-enhanced magnetic resonance imaging, DCE MRI). Methods:For the human nasopharyngeal carcinoma xenograft model, six-week-old female athymic nude mice were injected subcutaneously with5×106CNE1-LMP1cells (five mice per group). Tumors were measured on alternate days using a calliper, and tumor volumes were calculated. When the tumor volume reached60-100mm3, the animals were injected intra-tumourally with100g of DT18or INV-Control with FuGene6, or saline alone. MRI was performed3days after the DT18treatment. All data was acquired on a Superconducting magnetic resonance imaging scanners with a coil of2.0inche diameter for animal experiments. The raw data of DCE-MRI were analysed using NordicICE software (version2.3.6) without knowing animal grouping information. The tumor specimen was subjected to VEGFR IHC staining.Results:The differences in Ktrans between three groups were statistically significant (P<0.05). The P value between DT18group and INV-Ctrl group was0.028, and the P value between DT18group and saline group was0.026. The expression level of VEGFR1in tumor tissues of the DT18group was markedky reduced, compared with the controls (P<0.05).Conclusion:Based on the data collected in IHC and Ktrans data, we demonstrated that DT18could affect the vessel formation, decrease the vessel permeability and reduce the tumor volume. The Ktrans data could be used as an indicator to evaluate the therapeutic efficacy of anti-angiogensis agents.Part ⅣPurpose:Assessment of the pharmacokinetics and safety of DT18. Methods:1. Pharmacokinetics analysis:To determine pharmacokinetics of DT18,32P end-labelled DT18(2001at1mg/ml) was intravenously administrated into mice (10mg/kg,3mice per group). The blood samples (0.5ml) were taken at different times. Plasma was collected by centrifugation and DT18was extracted by phenol/chloroform. Samples were assayed by Southern blots and quantitated as a function of absolute concentration derived from a standard curve. Pharmacokinetic parameters were calculated using PK Solution software.2. Toxicological analysis:Three groups of ten Balb/C mice (6-8weeks, male/female even) were injected with DT18(20mg/kg), INV-Ctrl (20mg/kg) or saline via i.v. route. Mice in each group were observed and weighed daily. Blood was collected via retro-orbital sinus puncture at various time points, and tissues from euthanized animals were collected. Specimens were processed for clinical pathology, including hematological and serum chemistry, immunological and histopathological evaluations.Results:Toxicology studies are essential for the evaluation of candidate drugs. After a single intravenous administration of DT18(20mg/kg), DT18safety and tolerability was demonstrated by several criteria. DT18did not adversely affect body weight, food intake and tissue index when administered intravenously in mice. No significant clinical haematology, clinical biochemistry, necropsy, or organ histological treatment-related changes were observed. Moreover, DT18did not impact on the proportion of T cells, indicating that the antitumor effect of DT18was not caused by activating immune system. Pharmacokinetics of DT18following i.v. administration in mice showed rapid elimination from the plasma compartment. The distribution half-life is0.1hr and the elimination half-life is46.7hr. The maximal blood drug concentration is84.4mg/ml. The AUC of drug concentration-time curve is97mg/ml hr. And the apparent distribution volume is890ml/kg.Conclusion:The tolerance and safety of DT18are excellent and the DT18has a promising clinic application value.