The Suppressing Effect Of Ad-Flk1-Fc On Bladder Tumor Growth And Angiogenesis

In China, bladder tumor is the commonest malignant tumor of the urinary system, which is characterized by multiple tumors and high relapse rates. In addition, recurrent tumors tend to be increasingly malignant and, finally, tumor infiltration and metastasis may threaten life. How to increase the efficacy of bladder tumor treatment is a challenging topic in urology. With research advances in tumorigenesis, it has been known that tumor growth, infiltration and metastasis depend on neovascularization. It was shown that a tumor of 1~2mm in diameter requires blood supplies by new vessels to prevent tumor cell death from ischemia and hypoxia. Based on these findings, a new anti-angiogenesis treatment has been proposed to treat solid tumors. Compared to conventional tumor treatments, the anti-angiogenesis strategy has many advantages, such as high efficacy, easy access to target vascular cells, and no resistance or obvious toxic and side effects. Although great research advances in anti-angiogenesis treatment have been achieved over recent years and many anti-angiogenesis drugs under clinical trials and experimental studies have shown sound effects, such drugs have obvious drawbacks:①these treatment molecules are all administered, and must be administered continually due to their short action durations;②these molecules need to be administered intravenously at high doses due to their limited solubility, thus increasing drug toxicity substantially.Vascular endothelial growth factor (VEGF) is the most important pro-angiogenesis factor yet known so far. Most tumor tissues and tumor cell strains overexpress VEGF. VEGF exerts its biological action primarily through binding to high-affinity tyrosine kinase receptor VEGFR-2, and the VEGF/VEGFR-2 signal transduction pathway plays a key role in tumor angiogenesis. Blocking this pathway may suppress tumor angiogenesis and, subsequently, suppress tumor growth and metastasis. Based on this, we designed a new treatment to block VEGF binding to its receptors: adenovirus vector (Ad-Flk1-Fc) carrying a fusion gene comprising the extracellular domain of VEGF R-2 (Flk1) and IgG2a Fc was constructed, and used to transfect bladder tumor cells and endothelial cells. The transfected cells expressed and secreted free Flk1-Fc, which can capture blood VEGF in the microenvironment in which tumor cells and new endothelial cells grow, thus blocking autocrine/paracrine VEGF binding to VEGF R-2 and suppressing angiogenesis and tumor growth. This method may serve as an effective, safe treatment for bladder tumor. The study and main results are described below.Part 1 Construction and expression of adenovirus vector Ad-Flk1-Fc1. Construction of adenovirus vector Ad-Flk1-Fc: pCD-Flk1-Fc plasmid containing Flk1-Fc fragment was amplified and extracted, and digested with Xba I and BamH I. Agarose gel electrophoresis of the digestion products resulted in a band of approximately 5.1kb in length, which was consistent with the expected length of the target gene. Adenovirus shuttle plasmid pshuttle-CMV was linearized by digestion with the two endonucleases and inserted into downstream of pshuttle-cytomegalovirus promoter to construct the adenovirus shuttle plasmid vector pShuttle-CMV-Flk1-Fc. The obtained vector was linearized by digestion with Pme I, and transformed into competent bacteria containing AdEasy-1BJ5183 to perform homologous combination with AdEasy-1. Finally, recombinant adenovirus plasmid pAd--Flk1-Fc was obtained.2. Packaging of adenovirus vector Ad-Flk1-Fc: Recombinant adenovirus plasmid pAd-Flk1-Fc was used to transfect human embryonic kidney 293 cells. At 48h after transfection, green fluorescence was observed under fluorescence microscopy in the cells, suggesting successful plasmid transfection and expression of the exogenous gene. After one day of culture, the supernatants were collected, and replication-defective adenovirus Ad--Flk1-Fc was obtained. HEK293A cells were infected with the obtained plasmid repeatedly to amplify adenovirus Ad-Flk1-Fc. Recombinant adenoviruses were purified by cesium chloride gradient centrifugation, and the final titer of recombinant adenovirus was determined by the half culture tissue infection method to be 2×109PFU /ml.3. Expression and identification of adenovirus vector Ad-Flk1-Fc: The packaged adenoviruses were used to transfect bladder tumor cells BTT739, and total cellular protein was extracted for Western blotting using Flk1 antibody. The results indicated expression of an 165kD protein, which was in agreement with the theoretical molecular weight of Flk1-Fc fusion protein. After transfection of COS-7 cells with Ad-Flk1-Fc, Flk-1 expression was detected by immunocytochemical staining. The results indicated that Ad-Flk1-Fc transfected cells were stained positive and the control group negtive, suggesting Flk1-Fc expression by COS-7 cells.These results indicated the successful construction of adenovirus vector Ad-Flk1-Fc and expression of fusion protein Flk1-Fc in eukaryotic cells such as bladder tumor cells BTT739.Part 2 Effect of Ad-Flk1-Fc fusion protein on experimental in vitro angiogenesis of HUVECs1. Suppression of HUVEC growth by Ad-Flk1-Fc: Cultured HUVECs were treated with cellular culture supernatant of Ad-Flk1-Fc, or not treated with it(control). Control groups were set up with Ad-Fc,pAd and PBS. Endothelial cell proliferation suppression evaluation (MTT assay) indicated that after 48h culture, cells proliferated quickly in the control group, and the cell number decreased in the Ad-Flk1-Fc group cells, when compared to that in the control group. Compared with the blank group, HUVEC growth suppression rate was 56.3% in the Ad-Flk1-Fc group, 6.68% in the Ad-Fc group, and 4.23% in the pAd group. The results indicated significant suppression of HUVEC growth by Ad-Flk1-Fc, while no suppression in the other three groups (p<0.01).2. Induction of HUVEC apoptosis by Ad-Flk1-Fc: The effect of cellular culture supernatant of Ad-Flk1-Fc on endothelial cell apoptosis was investigated by flow cytometry. The groups were set as describe above. The results indicated that the apoptotic rate was significantly higher in the Ad-Flk1-Fc group than the Ad-Fc group (45.33% vs 21.49%), the pAd group (45.33% vs 17.4%) and the PBS group (45.33% vs 18.32%). The results suggest that Ad-Flk1-Fc induced HUVEC apoptosis significantly (p<0.01).3. Suppression of HUVEC migration by Ad-Flk1-Fc: The effect of cellular culture supernatant of Ad-Flk1-Fc on HUVEC migration in Transwell was observed, The groups were set as describe above. The suppression rate of HUVEC migration was 72.23% in the Ad-Flk1-Fc group, 13.70% in the Ad-Fc group, and 17.13% in the pAd group, suggesting significant suppression of HUVEC migration (p<0.01).4. Suppression of formation of HUVEC tubules by Ad-Flk1-Fc: It was found that cellular culture supernatant of Ad-Flk1-Fc significantly suppressed the formation of HUVECs tubules in Matrigel.These results suggest that Ad-Flk1-Fc suppresses the growth, migration and tubule formation of HUVECs significantly in vitro, and induces HUVEC apoptosis significantly, which provides a theoretical basis for future development and application of Ad-Flk1-Fc.Part 3 Therapeutic effect of recombinant adenovirus plasmid pAd- Flk1-Fc on transplantable mouse bladder tumor1. Tumor-suppressing effect of Ad-Flk1-Fc: BTT739 bearing mice models were established successfully. 48 BTT739 bearing mice were randomized to 4 groups (n=12). Each group was divided into two subgroups, i.e., the dynamic group and the pathology group. The observations of the dynamic group included①Changes in tumor size: The tumor size was measured twice weekly until 50% of mice in a group died;②survival rate: The survival of tumor-bearing mice was observed for 2 months. Mice of the pathology group were sacrificed on day 21, and the tumors were isolated, and determined for size and weight. After calculation of the tumor weight and size suppression rates, the tumors were subject to pathological examination. Mice were implanted with homologous tumor cells BTT739 subcutaneously; meanwhile, Ad-Flk1-Fc (2×109 PFU) was injected twice per week for 2 weeks. Control groups were set using Ad-Fc, pAd and PBS. The average tumor weight and volume were significantly lower in the Ad-Flk1-Fc treatment group than the three control groups, and the tumor suppression rate and survival time of tumor-bearing mice were significantly higher in the Ad-Flk1-Fc treatment group than the three control groups.2. Suppression of tumor angiogenesis by Ad-Flk1-Fc: After Ad-Flk1-Fc treatment, pathological examination revealed obvious necrosis of bladder tumor cells. MVD was calculated according to CD31 antigen-labeled tumor endothelial cells. MVD was obtained in all bladder tumor tissues, and positive stains were mostly located between tumor cells. Microvessels were polymorphous, being asterisk, slit-shaped or branched. Microvessels were patchy or focal in tumor nests. MVD was significantly lower in the Ad-Flk1-Fc treatment group than the three control groups (P<0.01). Tumor cell apoptosis assay by TUNEL indicated an apoptotic rate higher in the Ad-Flk1-Fc treatment group than the three control groups (P<0.01) 3. Suppression of non-tumor angiogenesis by Ad-Flk1-Fc: 24 BTT739 bearing mice were randomized as above to 4 groups, and were injected subcutaneously with 0.4ug/ml VEGF Matrigel, Ad-Flk1-Fc, Ad-Fc, pAd and PBS, respectively. Then, endothelial cells were stained and quantified with fluorescein-carrying Isolectin B4, and the fluorescence intensity was significantly lower in the Ad-Flk1-Fc treatment group than the three control groups.The above results show that Ad-Flk1-Fc suppresses transplanted mice bladder tumor and tumor angiogenesis and prolongs the survival of tumor-bearing mice.Conclusions:1. Adenovirus vector Ad-Flk1-Fc was constructed successfully and the fusion protein Flk1-Fc was expression in eukaryotic cells such as bladder tumor cells BTT739.2. Flk1-Fc fusion protein suppressed the in vitro growth, migration and tubule formation of HUVECs significantly and induced HUVEC apoptosis significantly.3. Ad-Flk1-Fc suppressed transplanted mice bladder tumor and prolonged the survival of tumor-bearing mice mainly through suppressing tumor angiogenesis.This study provides a good basis for developing and applying the novel drug Ad-Flk1-Fc for bladder tumor treatment.