Treatment of brain glioblastoma multiforme with pcDNA3.1-Egr. 1p-p16 combined with gamma knife radiation: An experimental study on nude mice
Correspondence Address: Source of Support: Supported by the basic research funds of
Science & Technology Department of Sichuan province, Conflict of Interest: None DOI: 10.4103/0028-3886.121917
Source of Support: Supported by the basic research funds of Science & Technology Department of Sichuan province, Conflict of Interest: None
Background: High post-operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM). Objectives: The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio-surgery (GKRS) and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. Materials and Methods: The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G) was carried out to establish the animal models. Various doses of liposome-mediated pcDNA3.1-Egr. 1p-p16 recombinant plasmid were transfected through intra-tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal-deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription-polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. Results: Rates of tumor growth were significantly lower in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1-Egr. 1p-p16 plasmid group and the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with marginal-dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with the marginal-dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1-Egr. 1p-p16 plasmid group. Conclusions: In animal studies, pcDNA3.1-Egr. 1p-p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene therapy. It may be a novel approach for the treatment of human patient with GBM.
Keywords: Gamma knife, gene therapy, glioblastoma multiforme, recombinant plasmid pcDNA3.1-Egr. 1p-p16
The annual incidence rate of glioblastoma is about 5/100,000 persons, 60-70% of which are glioblastoma multiforme (GBM).  Integrated treatment of surgical resection combined with radiotherapy and chemotherapy is the mainstream therapeutic solution in GBM. However, high postoperative recurrence and poor prognosis, which is likely due to the infiltrative growth of the tumor and it also effects the quality of life the patients. Combined usage of tumor suppressor gene p16 with radiation-induced promoter Egr-1p is new approach of gene and radiation therapy for GBM. In our experimental study, pcDNA3.1-Egr. 1p-p16 plasmid is innovatively combined with gamma knife radio-surgery (GKRS) for the treatment of brain GBM, which has not been reported before. Our study is to investigate the possible synergistic effect of the combined treatment of GKRS and gene therapy for GBM and to explore the role of GKRS for the temporal and spatial regulation of the gene expression.
Reagents and apparatus
Cell thawing and cultivation
Frozen cells were removed from liquid nitrogen and quickly placed in 37°C water bath for rewarming and thawing. The cells were washed once with Dulbecco's modified eagle medium (DMEM) and then centrifuged at 1500 rpm for 5 min. The supernatant was removed and the cells were then cultivated on the complete DMEM at 37°C in a 5% CO 2 incubator.
The medium was removed and the cells were washed twice with pre-warmed serum-free DMEM and digested by 1% trypsin-ethylene diamine tetraacetic acid. The digestion was terminated by adding complete DMEM when the cells became smaller and round under the microscope. After the cells were blown scattered, the liquid was collected and centrifuged at 1500 rpm for 5 min. The cells were re-suspended in the complete medium after precipitation and divided into flasks for continuous cultivation. When grew to a certain quantity, the well grown cells were collected and washed with sterile phosphate buffer solution (PBS). The cells were counted on a counting chamber under microscope to adjust the concentration to 5 × 10 7 cells/ml.
Preparation of plasmid
The bacterial strain for converting the pcDNA3.1-Egr. 1p-p16 recombinant plasmid was inoculated in the Luria-Bertani medium and then cultivated at 37°C on a shaker overnight. The plasmid was extracted by the alkaline lysis method with modification. Restriction enzyme digestion was performed on collected samples for confirmation.
Animal model establishment
A total of 70, 4-6 weeks aged BALB/c-nu/nu nude mice, weighing approximately 20 g each, were used for the experimental study. Each nude mouse was inoculated with 5 × 10 6 T98G cells (0.1 ml) subcutaneously under the right axilla. An equal volume of PBS was subcutaneously injected under the right axilla of the control nude mice. The animals were treated i when the tumors in the mice of the experimental group grew up to 5 mm in diameter. The grouping and treatments are detailed in [Table 1].
The liposome-encapsulated pcDNA3.1-Egr. 1p-p16 recombinant plasmid was transfected to the tumor cells by intra-tumor injection. The nude mice were anesthetized by intra-peritoneal injection of 3% pentobarbital sodium (40 mg/kg) 48 h after transfection. The animal was fixed on a previously prepared polymethyl methacrylate made fixation device. Then the device was fixed onto the Leksell stereotactic head frame [Figure 1]. The radiation procedure was finished using Leksell Gamma Knife Model C under the guidance of magnetic resonance imaging (Siemens sonata 1.5T). According to the grouping rules, the tumor was covered with the established isodose line [Figure 2]. The radiation doses are detailed in [Table 1].
Observation of anti-tumor effect
Tumor volume was recorded by measuring its length (a) and width (b) using a vernier caliper every 4 days after the GKRS. Tumor growth rate and volume were calculated based on the formulas: tumor growth rate f = post-radiation tumor volume/pre-radiation tumor volume; tumor volume V (mm 3 ) = (a × b 2 )/2. The data were presented in x ± s. To compare the tumor volume changes, a t-test was used. Statistical signiﬁcance was considered at a P≤ 0.05.
One nude mouse from each group was sacrificed 1 week after GKRS radiation. The subcutaneous tumor tissues were collected and were then prepared for pathological analysis after dehydration and paraffin embedding. The TUNEL apoptosis testing kit from Roche was used to study the cell apoptosis. The total apoptotic cells from ten high power fields (×400) of each specimen were counted under-microscopy. The percentage of apoptotic cells in all cells was calculated.
Reverse transcription-polymerase chain reaction (RT-PCR) test of p16 messenger ribonucleic acid (mRNA) expression
RT-PCR testing was used to determine the mRNA expression of p16 gene. The procedure of RT-PCR test (TaKaRa Kit) is as follows:
Electrophoresis of the PCR products was performed with 1% ago-gel and photographed under a digital image analysis system.
Anti-tumor effect of pcDNA3.1-Egr. 1p-p16 plasmid combined with gamma knife
Tumor growth rate of the nude mice, which were treated by intra-tumor injection of 20 μg pcDNA3.1-Egr. 1p-p16 encapsulated in 50 μl liposome + GKRS with the marginal-dose of 20 Gy radiation, was significantly lower (P < 0.05) than that in other groups 28 days after treatment. Tumors in other parallel groups showed marked growth, which was even more significant in the null controls (P < 0.05). The differences were not significant among the R, palone + R, P1 and P2 groups [Figure 3] and [Table 1].
Effect of pcDNA3.1-Egr. 1p-p16 plasmid combined with gamma knife upon tumor cell apoptosis
The in situ TUNEL kit was used to determine the apoptosis level of the tumor tissue. The apoptotic cells were stained as dark brown. The apoptotic cells were not notably seen in the null controls while markedly increased in the P2 + R group. The apoptosis index of the P2 + R group was significantly higher than that of other groups [Figure 4].
Effect of Gamma Knife on p16 gene expression
The reference amplifiable segment of reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) was 225 bp. And the amplifiable segment of the p16 sequence was 471 bp. GAPDH expression was observed in the tumor tissue of each group. The p16 gene expression was observed in the tumor tissue of P1, P2, P1 + R and P2 + R group. Significantly elevated p16 gene expression of the P2 + R group was observed [Figure 5].
The infiltrative growth of the GBM may be the results of abnormal regulation of cell cycle. In case of deoxyribonucleic acid (DNA) abnormality, the corresponding part of the cell cycle can be blocked via gene regulation, which subsequently results in cell apoptosis. However, due to abnormal gene regulation, tumor cells can continue to survive and proliferate. Radiotherapy and gene therapy are a trend in the treatment of GBM in addition to surgical interventions. During the different phases of cell cycle, sensitivity of tumor cells to radiation is different. The cells in the G1 phase are fairly sensitive to radiation, but are resistance to radiation in the S phase. Recently, p16 gene has been introduced as a gene of anti-oncogene for generation cycle regulation. The p16 protein, coding by p16 gene, is the inhibitor of a cyclin-dependent kinase 4 (CDK4). In this way, it inhibits the tumor cell generation cycle cloning from G1 phase to S phase. Early growth response-1 (Egr-1) promoter, which is a radiation-inducible promoter, is the cis-element (−550-0 bp) of the Egr-1 gene. The activity of Egr-1 promoter may be induced by radiation. The activation of Egr-1 promoter can enhance the expression of downstream genes.  We attached the p16 gene as the downstream gene of the Egr-1 promoter to construct the recombinant plasmid pcDNA3.1-Egr. 1p-p16 and employed this recombinant plasmid for treating GBM in combination with GKRS.
Although few evidence counts for the advantages of using GKRS in these patients with gliomas, several studies reported acceptable benefits in tumor control and patients' survival. , The radiation doses to the tumor foci in this study were based on the evidence of the clinical studies on human patients. , As we discovered in the study, when GKRS alone was used, tumor growth rate was significantly lower than that in the controls. And a higher apoptosis index was observed in the group compared with the controls. The results added the evidence of the anti-tumor effect of GKRS therapy for GBM in vitro. The results, which the tumor growth rate was significantly lower in the P1 group with obvious cell apoptosis and marked p16 gene expression than that in the control group, suggested that p16 gene therapy have significant tumor suppression effect for GBM.
Significant elevated apoptosis index and better tumor control were recorded in the combination therapy group, which suggested that the combined treatment of p16 gene and GKRS may further improve the therapeutic effect comparing to the gene therapy or GKRS alone. There reasons for the mechanism of the enhanced treatment effects may be: (1) the irradiation reinforced transfection and integration of p16 gene and improved its anti-tumor effect; (2) the effect of p16 gene on cell cycle was strengthened which induced increasing in cell grouth in the non-S phase, which was more sensitive to radiation. When Egr. 1p promoter was introduced to construct the recombinant pcDNA3.1-Egr. 1p-p16 plasmid (P2 group), the results did not show any significant difference in tumor growth between P2 group and P1 group. However, significant improvement in tumor control effects was observed when GKRS was added for the management (P1 + R and P2 + R). The tumor growth rate was considerably lower in the P2 + R group and the apoptosis index was higher. The cause of this result may be that Egr-1 promoter in the P2 + R group enhanced p16 gene expression.
In summary, the results of this study showed notable synergistic effects in the treatment of GBM undergone the combined usage of pcDNA3.1-Egr. 1p-p16 plasmid and GKRS, which helped to improve the efficacy for tumor control. The results of this study suggested that the combined approach may be a novel and effective treatment option for the management of GBM. The mechanism of this treatment may be based on the following factors: (1) GKRS causes immediate damage to cell DNA, changes the potential of the mitochondrial membrane, causes release of cytochrome C and triggers the procedure of cell apoptosis; ,, (2) the direct damage to cell membrane by the radiation causes release of ceramide, which leads to the damage of mitochondrial membrane and triggers apoptosis with cytochrome C; , (3) the product of p16 gene expression may compete with cyclin in binding with CDK4, which helps to inhibit CDK4's activity and to prevent the cells in the G1 phase from entering the S phase;  (4) the radiation may induce Egr. 1p promoter to elevate the expression of p16 gene for a better tumor suppression effect. Due to the significant differences in the structure and function between the human and nude mice, the benefits of the combinational treatment found in this study may not be the same. T. Yet further studies concerning on the safety and long-term outcome of the therapy are needed.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]