Study On Adenovirus-mediated IL-24 Gene Killing U251 Glioma Cells And Correlate Mechanism

Malignant gliomas are the most common brain tumors in humans. It has become the second death inducing factor in the group under 15 among people suffered from the diease. The results are fourth for men in 35-54 ages'group and women in 15-34 ages'group. Current treatments fail to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy. It clearly requires development of more effective therapeutic strategies that will improve long-term control and survival. To date, gene therapy has provided a new way to effectively therapy of glioma along with the development of molecular biology and molecular genetics in the past few years.Interleukin-24(IL-24) is a new member of interleukin-10 family. It is also called melanoma differentiation-associated gene-7(mda-7), since it was discovered overexpression in human melanoma during terminal differentiation. Now, several groups have demonstrated that Ad-mda7 (using Ad-mda7) induces apoptosis in a wide range of cancer cells (lung, prostate, breast, ovary, pancreas, hepatoma). In addition, there are some researches about combination of IL-24 and radiotherapy or chemotherapy. For example, it was found that IL-24 can enhance the radiosensitizing effect of non-small cell lung cancer (NSCLC) cell lines. Because of its characteristic killing cancer cell without harm normal cell, IL-24 has become candidate gene and successful carried out in Phase 1 clinic trail. But, the effects of IL-24 gene on glioma cell and its molecular events have been not clearly understood.In the present study, we construct a new type recombine adenovirus included IL-24 gene (Ad5F35-IL24). After transfected human glioma cell line U251 cells with this new type recombined adenovirus, we use MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit, Single cell gel electrophoresis(SCGE) assays to observe the effects of Ad5F35-IL24 on U251 cells. The transwell assay and Cell scratch assay were used to detect the invasion and migration ability of the cells after transfected by Ad5F35-IL24. And then, we use Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expression induced by Ad5F35-IL24. And also, the MTT, flow cytometry, double fluorochrome stain methods were used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells. The expression of bcl-2, caspase-3 and TopoⅡα. proteins were examined with Western blot, RT-PCR, flow cytometry, immuocytochemistry assays.Part 1 Construction of recombinant adenovirus Ad5F35-IL24 vectorObjective: to construct recombinant adenovirus Ad5F35-IL24 vector.Methods: When plasmids of ATCC were cut by both HindⅢand SalⅠenzymes, the goal bands were obtained. Plasmids of pDC316 were cut by both HindⅢand SalⅠenzymes. Subsequently, after some fragments were connected, transformed and filtered, reorganized plasmids of pDC316 were obtained. Thereafter,it was identified with PCR and enzymes cut analysis. Then the plasmids were carried out sequence determination in whole automatic nucleic acid analyzer. Subsequently competent cells were prepared and transformed. After large-scale preparation of plasmid DNA, recombined adenoviruses Ad5F35-IL24 were generated with homologous recombination by cotransfection of 293 cells with a couple plasmids. After identified with PCR, Ad5F35-IL24 recombinant adenovirus species was amplified and purified. Then the titre was determined according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL.Results: Reconstructed plasmids of pDC316-IL24 containing IL-24 gene was successfully constructed and identified by PCR, enzymes cut and sequence determination. Escherichia coli strains containing IL-24 gene was obtained. Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, amplified, purified and identified with PCR. Its titre was assayed according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL, and the titer was 2.8×108 PFU/mL.Conclusions: Reconstructed adenovirus vector Ad5F35-IL24 was successfully constructed.Part 2 Ad5F35-IL24 induces apoptosis and its influence on cell invasion and migration of glioma U251 cellsObjective: Observe the effects of Ad5F35-IL24 on the apoptosis, invasion and migration of U251 cells. Methods: MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit were used to detect Ad5F35-IL24 inducing U251 cell apoptosis, and observed its influnces on cell invasion and migration with the transwell assay and Cell scratch assay.Results: Ad5F35-IL24 induces growth suppression and apoptosis in human glioma cells U251 cells. The apoptosis was increased obviously along with Ad5F35-IL24 drug concertration increase. Ad5F35-IL24 could suppress U251 cell cycle; the cells were blocked at G2/M phase. Ad5F35-IL24 could effective decrese the invasion and migration of U251 cells.Conclusion: Ad5F35-IL24 could inhibit the growth, invasion and migration of human glioma U251 cell, and induces apoptosis.Part 3 The effects of Ad5F35-IL24 on expression of ERK and p38 MAPK cell signel transduction pathwaysObjective: to investigate mechanism of Ad5F35-IL24 effect on ERK and p38 MAPK protein expression.Methods: After administration of Ad5F35-IL24 for 24 hour. We used Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expressions. And the MTT, flow cytometry, double fluorochrome stain were also used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells. Results: Western Blot analyses found that IL-24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK and p-p38 MAPK protein expression(0.46±0.07, 0.60±0.07)(P<0.05). After combined with p38 MAPK inhibitor SB203580,it was showed that the inhibition rate was decreased(20.83±1.34)contrasted with the single Ad5F35-IL24 group(36.15±2.61)(P<0.05). On the other hand, ERK and p-ERK protein expression have not significant changed in contrast with control group(P>0.05). After blocked ERK signal transduction pathway with dministration of ERK inhibitor PD98059, the results showed that it could increase apoptosis of U251 cells(P<0.05).Conclusions: Ad5F35-IL24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK pathway. Blocking ERK pathway could increase Ad5F35-IL24 to induce cell apoptosis.Part 4 Ad5F35-IL24 influence of TopoⅡα,bcl-2 and caspase-3 protein expressionObjective: To observe Ad5F35-IL24 influence of bcl-2,caspase-3 and TopoⅡαexpressionMethods: After transfected U251 cell with Ad5F35-IL24 for 24 hours, the bcl-2, caspase-3 and TopoⅡαprotein expression were detected with Western blot, RT-PCR, immuocytochemistry assays.Results: After Ad5F35-IL24 transfection, the related proteins were detacted by Western blot. We found that caspase-3 significantly raised up (0.71±0.09)contrasted with that of control group(0.43±0.04), and bcl-2 expression was significantly cut down(0.02±0.01)compared to the control group(0.17±0.02)(P<0.05). TopoⅡαexpression were found significantly decreased ( 0.48±0.03, 0.31±0.02 ) in contrast with the control group(1.35±0.07)(P<0.05). Immuocytochemistry method also showed that TopoⅡαexpression was decreased down after Ad5F35-IL24 transfected(P<0.05).Conclusions: IL-24 could inhibit TopoⅡαand bcl-2 expression, and increase caspase-3 expression in U251 glioma cell, which provided refrences for effectively therapy of glioma in future.