Gene Therapy With Tumor Necrosis Factor-related Apoptosis Inducing Ligand Against Glioma

Gliomas are the most common primary tumors in the brain. Primary brain malignancies tend to spread locally, infiltrating the adjacent brain parenchyma, potentially causing edema and mass effect. GBM is highly aggressive with the average patient surviving no more than 12-18 months. Most patients with high-grade gliomas (GBMs) receive radiation and chemotherapy regardless of the extent to which the visible tumor has been removed. Radiotherapy can increase this period by only several months, and additional chemotherapy does not lead to a substantial improvement of this dismal prognosis. The overall survival rate of high-grade gliomas was 40% at 1 year and only slightly higher (46%) after combined radiotherapy and chemotherapy.Current treatment of GBM involves surgery, chemotherapy and radiotherapy; however, these therapies are only marginally effective in altering the ultimate progression of this disease. Consequently, new effective therapies are urgently needed to improve survival and the quality of life in patients diagnosed with GBM.TRAIL was identified in 1995 based on its sequence homology to FasL/APO1L and TNF. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells. However, TRAIL is not toxic against most normal cells.TRAIL has apoptosis-inducing capacity in a variety of tumor cells in culture and in tumor implants in mice. TRAIL showed no toxicity when systemically administered in rodents and nonhuman primates. It was shown recently that subtoxic concentrations of cytotoxic drugs could sensitize tumor cells from various origins to TRAIL-mediated cell deathTRAIL protein is expensive and half life is short . In order to improve the survival of patients with a glioblastoma multiforme tumor (GBM), my experiment constructed eukaryotic expression vector with TRAIL (114-281) for glioma therapy.We constructed a eukaryotic expression vector (pEGFP/sTRAIL), which can secrete TRAIL protein . After administrating vector to the U251 in vitro and in vivo , we studied the effect and machenism of apoptosis.Engineering of a eukaryotic experssion vector pEGFP-TRAIL1 Construction of a eukaryotic expression vector (pEGFP-TRAIL):Using human TRAIL(114-281) gene sequences from pEGFP-TRAIL, we selected suitable target site, and synthesized oligonucleotides as DNA template encoding TRAIL(114-281), then annealed and ligated into pEGFP-C1 eukaryotic expression vector.We analyzed of C-terminus and N-terminus of EGFP, and approved that the protein possess the fluorescencing property and ligand biological function. EGFP did not affect the biological function of destination protein. Consequently, We select EGFP as a reporter, which construct expressing a fusion between the protein of interest and enhanced green fluorescent protein (EGFP). Then we can observe and assess the transfection of vector and gene expression.2 Construction a secreting eukaryotic expression vector.Because TRAIL combine with cell surface DRs and result in apoptosis of tumor cell extraceller, we synthesised human insulin signal peptide sequence: 5′-ctagcatggccctgtggatgcgcctcctgcccctgctggcgctgctggccctctggggacctgacccagccgcagcc-3′5′-ctagggctgcggctgggtcaggtccccagagggccagcagcgccagcaggggcaggaggcgcatccacagggccatg-3′Then we connected signal peptide with EGFP 5′and constructed a eukaryotic expression vector which can secrete TRAIL protein.Study in vitro1 Transfection of U251After having successfully cultivated U251 glioma cells to its logarithmic growth phase, we use liposome to transfect with a eukaryotic expression vector pEGFP/sTRAIL and eukaryotic expression vector pEGFP-C1 individually. Then we use G418 to select stable transformants.After transfected 48h, the cells were assessed by PCR and observed by fluorescence microscope. Then we can analyze the transfection and expression of aim gene. We observed that the expression of EGFP by fluorescent microscope. About 500bp fragment appeared in recombinent vector by PCR. It was identical with objective gene length. There was no fragment in control and pEGFP-C1 groups. So we confirmed that TRAIL gene had transfected in U251 cell . 2 Inhibition of glioma cell in vitroWe set untransfecting group, pEGFP-C1 group and recombinent vector group. After transfected 48h, the cells morphological change were assessed by microscope. The cell proliferation was measured with MTT assay and the cell cycle progression and apoptosis was detected by flow cytometry.We observed the cell morphological change by microscope. The result of the control group and pEGFP-C1 group cell growth was in good condition. The recombinent vector group morphology changed. Cell became circled, and cytoplasm restracted. Cell border was not complete and cytoplasm appear particular material. Alone with the time extending, cell morphological changed heavyly. After tresfection 72h, the recombinent vector group cell death and floted in culture medium. Cell membrane was not smoothing. Some cell ruptured.MTT colorimetric result: the proliferation of U251 glioma cell was significantly inhibited by pEGFP/sTRAIL vector. The inhibitory ratio was 28.66%,while there was no distinctively difference between pEGFP-C1 group and control group.Compared with control group, the recombinent vector group cell apoptosis (P<0.01) after tresfected 48h. The flow cytometry showed the hypodiploid peak before G1 phase. The apoptosis ratio is 34.46%.Above results authenticated that pEGFP/sTRAIL vector can inhibite glioma cell growth and induce it apoptosis.Study in vivo We builded animal model by inoculating glioblastoma cell strain in Nude mice. When the transplant tumor diameter was 3.5mm, nude mice were divided into 4 groups stochasticly. The groups were administered pEGFP/sTRAIL vector, CDDP and pEGFP/sTRAIL +CDDP. Control group was administered sterile solution .The condition of drinking and eating of all groups nude mice is normal, and there was no diarrhea in the entire experiment course. There was no distinction between control group and recombinant vector group.While nude mice appear worry and delaying activation in CDDP group and CDDP+vector group. All cases were free of neurological symptoms. No significant difference was found among the groups in body weight and food intake(P>0.05). No case was found to have cachexia syndrome and no death was found in all groups.The nude mice were executed after 30 days therapy. We collect the neoplasms whice were routinly progressed by histopathology and electrical microscope.1 Nude mice neoplasm volume and weightIn comparison with that in control group , the neoplasm weight and volume of pEGFP/sTRAIL vector, CDDP group and CDDP+vector group were obviously lower. There was a significant difference among groups (P﹤0.05, P﹤0.05, P﹤0.05).The therapeutic effects of CDDP+vector group was best. Compared with the pEGFP/sTRAIL vector group and CDDP group, there was a significant difference in neoplasm weight and volume (P﹤0.05, P﹤0.05). No significant difference was found between pEGFP/sTRAIL vector and CDDP group. 2 Histopathological change of neoplasmThe results showed that raryopyknosis, raryorrhexis, cytoplasm was light red in color(apoptosis cell). Common cell nucleus was light blue, necrosis cell nucleus was light blue or achromaticity. Liver and kidney were taken off, there was no morphology changes of liver and kidney. The result showed that pEGFP/sTRAIL vector is not toxic to liver and kidney.3 Ultrastructure changes of tumorThe apoptosis character was found among pEGFP/sTRAIL vector and CDDP group and CDDP+vector groups.We can conclude that TRAIL recombinant vector can induce glioma cell apoptosis and inhibit it growth. There was no toxic to liver and kidney. When combined with CDDP, there was synergistic effect.Research of mechanism of apoptosis1 RT-PCR of TRAIL-R2We design TRAIL-R2 nucleotide sequencing. The target gene was identified respectively by PCR analysis. Result:there was anticipate band in relevant position among all groups. No significant difference was found betweenβ-actin and TRAIL-R2. We can conclude that TRAIL-R2 did not increased after applied pEGFP/sTRAIL vector.2 Western blot of caspase-8We extract protein and analyzed western blot results. Specific band was found in relevant position. There was no difference between control group and pEGFP-C1 group. The level of expression of pEGFP/sTRAIL vector group increased.3 Analyse of immunohistochemical of Bcl-2The brown positive expression was found in cytoplasm of cell observed by the immunohistochemical staining method. Compared with control group, the expression decreased among pEGFP/sTRAIL vector and CDDP group and CDDP+vector groups.The conclusions are as followed: TRAIL combined with CDDP may induce cancer cell apoptosis using common intracellular signal path. Although the mechanism was unclear, caspase-8 and Bcl-2 play the role when inducing apoptosis.We construct successfully secretory recombinant vector (pEGFP/sTRAIL), and study the effct of inhibitory glioma cell growth. The results approved that pEGFP/sTRAIL can induce glioma cell apoptosis and inhibit it growth. The experiment offered a new method for glioma therapy. We explored initially the relevant elements of apoptosis, and offer a potential approach for glioma gene therapy for clinical application.