The Establishment Of Prokaryotic Expression Vector Of Soluble Vascular Endothelial Growth Factor Receptor2(sKDR) And Its Inhibitory Effect On Bladder Cancer T24Cell Line

Aims: Just like all of other malignant carcinomas, the proliferation and metastasis ofbladder cancer rely on the production of neovessel. The production of tumor vessel isregulated by kinds of angiogenesis factors. Vascular endothelial growth factor (VEGF)family and relative receptors are the most powerful known growth factors, which working onendothelial cells specifically. It has been reported that vascular endothelial cells and kinds oftumor cells express kinase domain receptor (KDR). Our former study has also confirmed thatthe positive expression rate of VEGF and KDR was88%(53/60) and85%(51/60) in bladdertransitional cell carcinoma, and increasing with the elevation of pathological stages and cellgrades. In current study, we will explore the regulation of soluble kinase domain receptor(sKDR), by constructing the prokaryotic expression vector of sKDR.Methods: sKDR cDNA fragment was obtained by human umbilical vein endothelialcells (HUVEC) which was taken as the template and the recombinant pQE40-sKDR plasmidwas constructed. After transformating the recombinant pQE40-sKDR plasmid intocompetence E. Coli M15to induce and purify the expression of sKDR, the depression effectof sKDR protein on the proliferation of HUVEC was analyzed by cell culture in vitro andchick embryo cultivation.Results: The2238bp fragment of sKDR cDNA was obtained by RT-PCR techniquefrom HUVEC cDNA which was taken as the template and the recombinant pQE40-sKDRplasmid was constructed. Transformating the constructed recombinant pQE40-sKDRplasmid into competence E. Coli M15to induce and purify the expression of sKDR, andconfirmed by Western Blot. The inhibition experiment of proliferation was carried out withsKDR protein and HUVEC of different concentrations with MTT method. It was showedthat sKDR protein had inhibition effects on the proliferation of vascular endothelial cells,which enhancing with the increasing of sKDR's concentration. The proliferation of HUVECwas inhibited when adding VEGF and sKDR protein into the HUVEC culture solution. Inchick embryo cultivation, the adding of sKDR protein could significantly inhibit theangiogenesis of chick chorioallantoic membrane. Comparing to the PBS and VEGF groups, the vessel density decreased obviously.Conclusions: The prokaryotic expression vector of sKDR was constructed according tothe first to the forth coding sequence of the extracellular domains of KDR, which using KDRas the target. It has been proved that sKDR protein could inhibit HUVEC's proliferationobviously in vitro cell cultivation. The depression effect would reinforce with the increasingof the concentration of sKDR protein. sKDR could inhibit the VEGF induced proliferation ofHUVEC by competitively combination with VEGF. In chick embryo cultivation, sKDRprotein could inhibit the angiogenesis of chick chorioallantoic membrane, which wasstimulated by VEGF. Aims: Basing on the construction of prokaryotic expression vector of sKDR andconfirming its depression effect to human umbilical vein endothelial cells (HUVEC) byexperiments in vivo and in vitro, exploring the depressing effect of eukaryotic secretingexpression vector of sKDR to VEGF and human bladder cancer cell line T24. With the highaffinity of sKDR with VEGF, inhibiting the angiogenesis and the growth of tumor byblocking the signal transduction pathway of VEGF/KDR to provide research evidence to thebiological targeting treatment of bladder cancer.Methods: Basing on the construction of prokaryotic expression vector of sKDR,transforming its eukaryotic secreting expression vector pCI-sKDR into human bladdercancer cell line T24, with pCI-neo T24and T24as control groups. In sKDR transformedhuman bladder cancer T24cell line, evaluating the affinity to rhVEGF by ELISA; analyzingthe expressing status of KDR mRNA by semiquantitative RT-PCR; detecting the cell cycleand apoptosis by flow cytometry; inspecting the mRNA and protein expression of VEGF andMMPs(MMP2and MMP9),BAX,Bcl-2, which are in the downstream of VEGF/KDRsignaling pathway, by RT-PCR and Western Blot; evaluating the proliferative ability and theregulating ability to HUVEC's proliferation with MTT. Implanting sKDR transformedhuman bladder cancer T24cell line to nude mice's subcutaneous tissue in back and recording the tumor size on day7,14,21and28. Sacrificing mice on day28and comparing tumormass and volume.Results: The binding activity between the supernatant of cell culture medium ofpCI-sKDR T24cells and rhVEGF165protein was positively correlated with the dilutionfactor (dilution range of1:1to1:16) while there is no binding activity between the twocontrol groups and rhVEGF165protein. The ratio of KDR mRNA level to-actin inpCI-sKDR T24cell (0.665) was4.16times of that in pCI-neo T24cell and non-transformedT24cell (both0.160). The proliferation of the pCI-sKDR T24cells decreased (61.2%and55.1%respectively)(P<0.01) when comparing with pCI-neo T24cell and non-transformedT24cell. The cell cycle arrested in G0/G1phase while the cells in S phase reduced (P<0.01).Apoptotic cells accounted for12.7%of the tested pCI-sKDR T24cells, shown as ahypodiploid apoptosis peak before G1phase. The genetic expression of MMP2,MMP9andBcl-2significantly decreased and the expression of BAX significantly enhanced at both themRNA level and protein level. The adding of cultural supernatant solution of pCI-sKDR T24cell inhibited the proliferation of HUVEC (53.6%), which decreased by46.4%and41.6%(P<0.01). The experiment of cancer-inducing in vivo with nude mice showed that tumorcould be found on day13in pCI-sKDR T24cell after implantation, comparing to on day7innon-transformed T24cell and pCI-neo T24cell. The tumor growth speed in pCI-sKDR T24cell group was slower than those in non-transformed T24cell group and pCI-neo T24cellgroup. On day28, the tumor mass and volume was1.07±0.21g and1.589±0.181cm3innon-transformed T24cell,0.91±0.19g and1.283±0.05cm3in pCI-neo T24cell,0.42±0.16gand0.344±0.167cm3in pCI-sKDR T24cell, respectively. Both of tumor mass and volumewere significantly smaller in pCI-sKDR group than in non-transformed T24group andpCI-neo T24group (P<0.01).Conclusions: sKDR transformed human bladder cancer cell line T24could stablyexpress the mRNA and protein of KDR, which had high affinity to rhVEGF. Althoughnormal T24cell and pCI-neo T24cell could express KDR production in some degree, therewas no affinity with VEGF. Comparing to that of the normal T24cell and pCI-neo T24cell,the proliferation ability decreased and the apoptotic speed increased significantly inpCI-sKDR T24cell. By analyzing the angiogenesis/apoptosis gene and protein inVEGF/KDR related pathway, it has been shown that the sKDR, which was transformed andexpressed stably in T24cell, could block the expression of angiogenesis/apoptosis signal indownstream conductive pathway by biding to VEGF. Human bladder cancer T24cell line, which stably expressing sKDR, has good tumor-inhibition effect in nude mice.