The Study Of Specific Cytotoxicity Against Tumor-derived Endothelial Cells Induced By DNp73α Recombinant Adenovirus And Endostar^? In Vitro

Background:Lung cancer is one of the most aggressive carcinoma.,it becomes the leading cause of cancer-related deaths. Tumorigenesis involves a complicated multi-step process and it is made clear by molecular biology's researchs that tumorigenesis are in connections with activations of proto-oncogenes and inactivation of various antioncogenes at different stages. These gene changes will eventually lead to limitlessness of tumor cell growth. In this process, interaction based on one correlated gene has not significant efficiency. Tumor microvessels are essential for the growth and migration of malignant tumors. Targeting tumor microvessels by inhibiting formation or destructing existing vessels can block the nutrition supplies and migration path. Therefore, antio-angiogenesis therapies have become an important strategy in cancer therapy【1】. The main methods of specific cytotoxicity against tumor-derived endothelial cells at present included: 1. Chemical medicines that have specific inhibitory effect on endothelial cells; 2. Though specific cytotoxicity of T cells, which is the main way to immune response against cancer cells.Endostatin was originally isolated from the culture medium of murine endothelioma (EOMA) as an endogenous angiogenesis inhibitor by Reilly et al.【2】. It is the c-terminal fragment of collegen X VIIIC, and it demonstrated a nearly complete inhibition of tumor-induced angiogenesis, conveying a high anti-tumor activity. Numerous studies have shown that endostatin can specifically inhibit the proliferation of blood vessel endothelial cells【3】, block their migration, and induce apoptosis and cell cycle arrest【4-5】, while having no inhibitory effect on smooth muscle cells or fibroblasts. It is interesting that long-term use of endostatin does not induce drug resistance in cancer cells. In addition, it has low cellular toxicity. In 1999, a phase I trial of Pichia pastoris expressed human recombinant endostatin as cancer therapeutic was approved by FDA. In 2001, a phase II trial was conducted. However, human recombinant endostatin has a short half life; its refolding and large-scale production is relatively difficult. Therefore, the clinical application of endostatin has been limited. In 2005, Chinese scientists invented a new protein named endostar, which is endostatin with an addition of a 9-amino acid peptide (MGGSHHHHH). The stability and purity of endostar are greatly enhanced, and the half life is prolonged. Most importantly, the biological activity is increased【6】. However, the effect and mechanism of endostar on tumor endothelial cells remains unknown, and whether this effect is specific to tumor-derived endothelial cells also needs to investigate.Among varied biotherapy patterns, immunotherapy is the most attractive one. It was confirmed that specific anti-tumor immune response was done by activated T lymphocyte, and exotic antigens had to be processed and presented to helper t cells. So it is the key point to activate the specific cytotoxic T lymphocyte (CTL). Dendritic cells (DC) originated from hemopoieticstemcell,and are the most potent professional antigen-presenting cells and only ones initiating primary T lymphocyte. It is capable of uptaking (endocytosis and phagocytosis), presenting tumor associated antigen in the context of both MHC classⅠand classⅡin association with costimulatory molecules. Much attention has been directed to the problem of how and what antigens should be pulsed to DC in their clinical application as immunotherapy.The DNp73αprotein, a protein of the p53 family, was recently identified to be generated from the p73 gene under an alternative promoter in intron 3, and thus DNp73αprotein lacks a transactivation domain presenting in p73. DNp73αis not expressed in normal tissues but overexpressed in lung cancer, ovarian cancer, vulval cancer, neuroblastoma and breast cancer cell lines; it acts as a potent transdominant inhibitor of the wild-type p53 and the transactivation-competent TAp73. Several studies found that positive expression of DNp73αwas a significant independent factor for predicting a poor prognosis, and considered it an important molecular target for effective therapy. However, few studies have reported experimental manipulations targeting DNp73α.This study was aimed to investigate the effects of dendritic cells transfected with recombined adenovirus vector encoding DNp73αon induction of immunity against DNp73α-overexpressing tumor cells, simultaneously,we studied wheather endostar has specific cytotoxicity against tumor-derived endothelial cells and its antiangiogenesis'mechanism,which provide evidences for subsequent animal and clinical research.Methods:1. A recombinant replication-deficient adenovirus vector carrying a HA tagged full-length human DNp73αcDNA driven by a CMV promoter, was prepared using the AdEasy system and referred to as Adv-DNp73α. In brief, human DNp73αcDNA was cloned into adenovirus transfer vector pAdTrack-CMV. The resulting plasmid, pDC315-EGFP Vector-DNp73α, was then linearized with PmeI and transformed into E. coli strain BJ5183 containing pAdEasy-1, the viral DNA plasmid, for homologous recombination. The recombinant adenoviral construct was then cleaved with PacI and transfected into 293T cells to produce viral particles. The same adenoviral vector backbone carrying the marker gene EGFP, Adv-EGFP, was used as the control vector. Titers of the viral stocks were determined with a plaque-forming assay.2. We obtained HUVEC by trypsinization and centrifugation method and cultured Human umbilical vein endothelial cells (HUVEC) and differentiated them into Tumor-derived Endothelial Cells(Td-ECs) after co-cultured with supernatants of A549 cells,and examined the anti-angiogenesis effect of endostar on Td-ECs in vitro with the methods of MTT, cell migration assay, flow cytometry and TUNEL assay.For futher study of Endostar's effect,3. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from human umbilical cord blood were transfected with the adenovirus vector with centrifugal force method. Following up expression of GFP, we detected the infective efficiency of varied M.O.I. recombined adenovirus. Combined with viability of DC evaluated through FCM, we then ascertained the optimal infection programmer and M.O.I..4. RT-PCR and Western Blot were use to detect the level of DNp73αmRNA and protein in the transfected DC to ensure the overexpression of DNp73α.5. Autologous T cells were isolated and purified using nylon wool columns. The purified cells were cultured in 96-well U-bottom plates in the presence of cytomycin-C-pretreated transfected or untransfected DC as stimulators for 5 days. During the last 8 h of incubation, 1μCi/well of [3H] thymidine was added. Assays were performed in triplicate. And then the cells were harvested and [3H] thymidine incorporated into T cells was measured by a beta counter.6. T cells (1×106) were co-cultured with Adv-DNp73αmodified DC (5×104) for 72 h to induce cytotoxic T lymphocytes (CTLs). Then the CTLs were collected and used as the effector cells in CTL assays. TDEC/DNp73αcells, HUVEC cells, and TDEC cells, as the target cells with different DNp73αespression levels determined by real time PCR, were placed in 96-well tissue culture plates at 1×104 cells per well respectively, and co-cultured with effector cells (CTLs) at varied E/T (effect cells : target cells) ratio of 1:10, 1:20, 1:40 and 1:80. The cytotoxic activities were determined by CytoTox non-radioactive cytotoxicity assay. Normal DC and Adv-EGFP transfected DC were used as the control.7. Cultured Human umbilical vein endothelial cells (HUVEC) and differentiated them into Tumor-derived Endothelial Cells(Td-ECs) after co-cultured with supernatants of A549 cells,and examined the anti-angiogenesis effect of endostar on Td-ECs in vitro .8. Established human-xenograft-nude mouse model, Endostar was injected directly to tumors as local treatment and conservative management of tumor growth was close observed in the future days.9. For a single comparison of two groups, Student's t test was used to evaluate the significance of differences. If the data distribution was not normal, the Mann-Whitney rank-sum test was employed for the nonparametric analysis. For all analyses, the level of significance was set at p<0.05. All statistical calculations were performed using the SigmaStat statistical software package SPSS10.0. Data are presented as the mean±SD.Results:1. Via directed cloning and homologous recombination, infection-competent adenovirus was generated in 293T package cell line. Following repeatedly infecting, high titer adenovirus particles yielded.2. Human umbilical vein endothelial cells (HUVEC) were cultured and differentiated to tumor-derived endothelial cells (TDECs) after co-culturing with conditioned medium from A549 cells, a lung cancer cell line. we could detect the mRNA of TEM1 and TEM8 in the Td-Ec with RT-PCR. Td-EC had more active cellular proliferation and cell migration capacity, G0/G1 and S phase proportion of Td-Ec was higher than HUVEC.3. After co-culturing with conditioned medium from A549 cells, a lung cancer cell line, HUVEC can be induced to TDEC which has the characteristics of tumor vessels endothelial cells.4. Endostar has a significant inhibition effect in time-and concentration-dependent for TDEC in vitro, at the same time, though Endostar at a certain concentration can inhibit cell migration process significantly, and induced G0/G1 and S phase arrest and cell apoptosis, under the same conditions, Endostar has little impact on HUVEC. In addition, a certain concentration of Endostar after local injection, further study details was provided that concentration of Ca2+ of the tumor be significantly higher than the contral group's, and had significant anti- angiogenesis effect and tumor growth inhibition.5. The addition of purified Adv-DNp73αvector to the DC culture with centrifugal force method, at an M.O.I. of 200, resulted in a transduction efficiency of nearly 80% without significant cytotoxic effects.6.DC/Adv-DNp73αstimulated effector T cells could effectively lyse DNp73α-overexpressing TDEC/DNp73αcells but not the DNp73α-null TDEC and HUVEC cells. The untreated T cells had minimal lysis of all the tumor cells. LAK cells showed comparable lytic activity against TDEC/DNp73αcells with that of DC/Adv-DNp73αstimulated T cells, but demonstrated much more effective killing of TDEC cells than the DC/Adv-DNp73αstimulated T cells.Conclusions:After co-culturing with conditioned medium from A549 cells, a lung cancer cell line, HUVEC can be induced to TDEC which has the characteristics of tumor vessels endothelial cells,and its had more active properties of proliferation and cell migration.Endostar has a significant inhibition effect in time-and concentration-dependent for TDEC in vitro, at the same time, endostar has little impact on HUVEC, so, this inhibitory effects are some kind of specific cytotoxicity to tumor, and have significant efficacy and minimal side effects in clinical treatment. DNp73α-engineered DC can induce DNp73α-specific CTL against tumor cells over-expressing DNp73α.