| Lung caner is one of the most common malignant tumor whose growth and metastasis are critically dependent on angiogenesis. Antiangiogenic therapy, therefore, is emerging as a potentially promising anti-cancer strategy. Vascular endothelial growth factor (VEGF), which is secreted by a variety of tumor cells and exerts its biological effects on endothelial cells by binding to endothelial cells transmembrane receptors(VEGFR-1 and VEGFR-2), plays an important role in neovascularization in a variety of tumor types. Therefore, blocking the VEGF signaling transduction pathway by preventing VEGF from binding to its receptors is most important and direct target to antitumor angiogenesis therapy. Soluble VEGFR-1(sVEGFR-1) and soluble VEGFR-2(sVEGFR-2), which are truncated soluble form of VEGFR-1 and VEGFR-2, respectively, retain their high affinity binding to VEGF, but cannot function as signal transduction to induce endothelial proliferation for lack of the tyrosine kinase domain. The two soluble receptors have been introduced for inhibition of VEGF-mediated angiogenesis. In the present study, we investigated whether gene transfer of either human sVEGFR-1 or sVEGFR-2 could inhibit angiogenesis and growth of lung cancer formed by transgenic A549 cells themselves in vivo, and whether the combined gene transfer of these two soluble receptors would produce a greater inhibition of tumor growth than that produced by either one alone, with an investigation into their possible mechanisms.Methods1. A sVEGFR-1 and a sVEGFR-2 cDNA coding sequence fragments were amplified by RT-PCR from total RNA extracted from human placenta tissue and then were ligated respectively into eukaryotic expression vector pCMV-Script to become recombinant pCMV-Script-sVEGFR-1(named psR1)and pCMV-Script-sVEGFR-2 (named psR2), which were identified by double digestion with restriction endonucleases and sequencing.2. These eukaryotic expression vectors were transfected into A549 cells derived from human lung adencarcinoma by the cationic liposome. The A549 cells transfected with pCMV-Script, psR1 and psR2 were named respectively A549-pCMV, A549-psR1 and A549-psR2, and the A549 cells co-transfected with both psR1 and psR2 were named A549-psR1R2, while un-transfected A549 cells were named parental A549.3. The stably transfected A549 clones were screened with G418. RT-PCR, Western blot and immunocytochemical stain were used to measure the transgene expression at both mRNA and protein level in transfected A549, and ELISA was employed to quantitate sVEGFR-1 and sVEGFR-2 secreted by A549 cells in supernatants in culture.4. The proliferation of A549 cells and human umbilical vein endothelial cells (HUVEC) cocultured with the A549 cells were assayed by cell counting and growth curve, MTT, flow cytometry(FCM).5. In vivo the antiangiogenic activity of the recombinant human sVEGFR-1 and sVEGFR-2 secreted by A549 cells was evaluated by the chicken embryo chorioallantoic membrane (CAM).6. A549 lung cancer xenograft models were established: Fifteen 4-week-old male athymic nude mice were divided randomly into five groups: A549-psR1, A549-psR2, A549-psR1R2, A549-pCMV and parental A549 groups, with three mice in each group. For generation of nude mice subcutaneous tumors, corresponding 2×107cells/200ul of each group were injected subcutaneously into nude mice at a dorsal flank site, respectively.7. The tumorigenesis was monitored and the tumor volumes were measured weekly. The routine histopathologic examination of tumor was performed. The intratumoral microvessel density(MVD) was determined by immunohistochemical stain with CD31 as the first antibody. Tumor metastasis and the survival time of tumor-bearing mice were observed.Results1. The sizes of PCR products amplified from human placenta tissue, which were confirmed by electrophorese with 1.0% agarose gel, were identical with those of the expected sVEGFR-1 and sVEGFR-2 fragments. The DNA sequence analysis by BLAST showed that the sVEGFR-1 and the sVEGFR-2 fragments cloned into pCMV-Script vectors were in coincidence with those in GenBank. 2. After transfection of recombination or control vectors into A549 cells, the stable transgenic cell clones were screened successfully with G418. The transgene expression at both mRNA and protein level in A549-psR1, A549-psR2 and A549-psR1R2 groups could be detected by RT-PCR, Western blot and immunocytochemical stain, respectively, and the sVEGFR-1 and the sVEGFR-2 proteins in cultured cell supernatants of above groups could be measured by ELISA. Whereas, the negative outcomes for the A549-pCMV and parental A549 groups were found under the same as condition.3. After stable transfection, the A549 cells were subcultured. Cell counting, MTT, FCM showed that the proliferation of transgenic A549 cells in culture was not affected compared with parental A549 cells in culture under the same as condition. However, the proliferation of HUVEC treated with either A549-psR1 or A549-psR2 or A549-psR1R2 demonstrated a significant growth inhibition, as compared with the untreated HUVEC. But there were no significant differences between the HUVEC treated with either A549-pCMV or parental A549 and the untreated HUVEC.4. The number of newly formed blood vessels in chick embryo CAM intervened with the cultured cell supernatants from A549-psR1, A549-psR2 and A549-psR1R2 groups (7.33±1.53, 7.67±1.53 and 4.67±1.15, respectively) was significantly fewer than that in un-intervened CAM(12.33±1.53)(all P<0.01). There were also significant differences between A549-psR1R2 intervened group and either A549-psR1 or A549-psR2 intervened group(all P<0.05).5. After s.c. transplantation of A549 cells, All the mice developed subcutaneous tumor nodules at injection site. But the tumors in all recombinant vectors transfected A549 groups grew significantly slower than those in either parental A549 or A549-pCMV groups. The tumors in A549-psR1R2 group grew even slower. At experimental endpoint, the tumor volume in A549-psR1, A549-psR2 and A549-psR1R2 groups (1338.3±78.8, 1443.2±90.9 and 1171.5±40.8, respectively) was significantly smaller than those in parental A549 group (1620.0±27.8, all P<0.01) and A549-pCMVgroup(1626.7±10.4, all P<0.01). There were also significant differences in tumor volumes between A549-psR1R2 and either A549-psR1 or A549-psR2 group(all P<0.01). However, no significantly differences were observed between parental A549 group and A549-pCMVgroup(P>0.05). 6. Immunohistochemical stain showed that the MVD in tumors from A549-psR1, A549-psR2 and A549-psR1R2 groups (15.8±2.6, 18.6±2.3 and 10.8±1.3, respectively) were significantly lower than those in tumors from either parental A549 group (23.6±3.0, P<0.01) or A549-pCMV group(25.2±1.3, P<0.01) which were not significantly different from each other. Furthermore, the MVD in the tumors from A549-psR1R2 group was also significantly lower than those in tumors from either A549-psR1 or A549-psR2 group(P<0.01).7. Survival analysis showed that the median survival time of tumor-bearing mice in A549-psR1, A549-psR2 and A549-psR1R2 groups(68, 74 and 79 days, respectively) was significantly longer than those in either parental A549 group(41 days) or A549-pCMV group (37 days)(all P<0.01).Conclusions1. The recombinant eukaryotic expression vectors of human sVEGFR-1 and sVEGFR-2 gene were successfully cloned and can express at both mRNA and protein level in transgenic A549 cells, and these proteins expressed in transgenic A549 cells can be secreted effectively to the extracellular region.2. The proliferation of these transgenic A549 themselves in vitro was not affected, but the sVEGFR-1 and sVEGFR-2 proteins expressed in transgenic A549 cells can inhibit the proliferation of HUVEC and the neovascularization of chicken embryo CAM.3. Gene transfer of either sVEGFR-1 or sVEGFR-2 into A549 cells can inhibit the intratumoral angiogenesis and the growth of tumor derived from these transgenic A549 cells themselves. The ability of sVEGFR-1 or sVEGFR-2 to inhibit tumor growth is presumably attributable to their paracrine inhibition of tumor angiogenesis in vivo, but no direct target at cancer cells.4. The combined gene therapy of sVEGFR-1 and sVEGFR-2 can produce an enhanced inhibitory effect compared with their individual effects.
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